WO2006095559A1

WO2006095559A1 - USE OF Fgf21 AS HEMATOPOIETIC FACTOR

Info

Publication number: WO2006095559A1
Application number: PCT/JP2006/302921
Authority: WO
Inventors: Nobuyuki Itoh; Hajime Yamauchi
Original assignee: Kyoto University
Priority date: 2005-03-11
Filing date: 2006-02-14
Publication date: 2006-09-14
Also published as: JP2006246823A

Abstract

It is intended to provide novel drugs and reagents relating to Fgf21, means useful in developing them and so on. More specifically speaking, it is intended to provide: an agent for controlling the differentiation of hematopoietic stem cells which contains a substance capable of controlling the expression or function of Fgf21; a method of controlling the differentiation of hematopoietic cells and a method of evaluating the differentiation efficiency thereof by using Fgf21; a method of screening a substance capable of controlling the differentiation of hematopoietic stem cells which comprises evaluating whether or not a test substance can control the expression or function of Fgf21; a method of identifying a polymorphism in Fgf21 capable of altering the ability to control the differentiation of hematopoietic stem cells; a method of evaluating the body conditions of an animal under the differentiation of hematopoietic stem cells and a diagnostic therefor; a kit which contains a substance capable of controlling the expression or function of Fgf21; and so on.

Description

Specification

Use of F g f 2 1 as a hematopoietic factor

The present invention provides an agent for regulating differentiation of hematopoietic stem cells, a method for regulating differentiation of hematopoietic stem cells, a method for screening a substance capable of regulating the differentiation of hematopoietic stem cells, and the like. Background art

Blood cells are indispensable for various life activities such as oxygen transport and antibody production. Red blood cells are the most abundant blood cells in the blood and differentiate from mesoderm to red blood cells via hemangioblast, hematopoietic stem cells, and erythroid progenitor cells. The search for hematopoietic factors that contribute to this developmental process and the analysis of their functions are thought to contribute to the elucidation of the pathogenesis, prevention, and treatment of various hematopoietic diseases.

Zebrafish are fertilized and developed outside of the mother's body, and their development is fast and the embryo is transparent throughout the development period. In addition, experimental developmental analysis by introduction of foreign genes and cell transplantation can be easily performed. Even if blood circulation is completely lost, it can survive for several days by passive diffusion of oxygen from the body surface. Furthermore, it has been shown that the cell differentiation process is well preserved from zebrafish to humans. Therefore, zebrafish is an excellent model animal for examining human blood cell differentiation (Thisse, C. and Zon, LI (2002) Science 295, 457-462; Davidson, AJ and Zon, LI (2004) Oncogene 23, 7233-7246).

It has been clarified that fibroblast growth factor (FGF s) currently exists in humans based on amino acid sequence homology. It is known that the effect is not only proliferative activity on fibroblasts but also deeply involved in various life phenomena such as morphogenesis, angiogenesis, tumor formation, wound healing, nerve survival, and metabolic regulation. (Ornitz, DM and Itoh, N. (2001) Genome Biol. 21, REVIEW3005; Itoh, N. and Ornitz, DM (2004) Trends Genet. 20, 563-569). We have previously described human F gf 2 1 (Nishimura, T. et al. (2000) Biochim. Biophys. Acta 1492, 203-206). However, its function is still unknown. Disclosure of the invention

Functional analysis of genes leads to the development of drugs or reagents with new mechanisms of action for various diseases. The present invention is based on the knowledge obtained by functional analysis of the F gf 21 gene, to provide a drug or a reagent having a new action mechanism for various diseases, and to develop a drug or a reagent. Its purpose is to provide useful means.

The present inventors suppressed the function of F gf 21 in zebrafish and analyzed its characteristics. It was found that the differentiation into one myeloid lineage cell was suppressed. The present inventors have also found that differentiation of hematopoietic stem cells into lymphocyte lineage cells can be promoted by suppressing the function of F gf 21 in zebrafish. Thus, F gf 2 1 is the differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells (eg, erythrocytes, megakaryocytes, eosinophils, neutrophils, basophils, monocytes, rod cells), or It is thought that the differentiation of hematopoietic stem cells into lymphocyte lineage cells (eg, T cells, B cells, NK cells) can be controlled. In addition, substances that regulate the expression or function of F gf 21 have the ability to regulate the differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphocyte lineage cells and / or diseases caused by abnormal blood cells ( For example, it may be useful for the development of medicines and research reagents for hematopoietic diseases, immune diseases, allergic diseases).

Based on the above, the present inventors have completed the present invention. That is, the present invention is as follows:

[1] A hematopoietic stem cell differentiation regulator comprising a substance that regulates the expression or function of F g f 21;

[2] A substance that regulates the expression or function of F gf 21 The agent of [1] above, which is a promoting substance;

[3] The agent according to [2] above, wherein the substance that promotes the expression or function of F g f 21 is F g f 21 or an expression vector thereof;

[4] The agent according to [1] above, wherein the substance that regulates the expression or function of F g f 21 is a substance that suppresses the expression or function of F g f 21.

(5) The substance that suppresses the expression or function of F gf 21 is selected from the group consisting of an antisense nucleic acid, a ribozyme, an RNAi-inducible nucleic acid, a targeting vector, an antibody, and a dominant negative mutant. Agent of [4] above;

[6] The agent of [2] above, which is an agent for promoting differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells, or an agent for suppressing differentiation of hematopoietic stem cells into lymphocyte lineage cells;

[7] The agent according to [2] above, which is an agent for promoting differentiation of hematopoietic stem cells into erythroid lineage cells; [8] an inhibitor of differentiation of hematopoietic stem cells into erythrocyte-myelocyte lineage cells, or lymphocyte lineage cells of hematopoietic stem cells The agent of [4] above, which is a differentiation promoting agent into

[9] The agent of [1] above, which is a preventive or therapeutic agent for hematopoietic disease, immune disease or allergic disease;

[10] a method for regulating differentiation of hematopoietic stem cells, comprising culturing hematopoietic stem cells in a medium in the presence of a substance that regulates the expression or function of F g f21;

[1 1] The method according to [1 0] above, wherein the substance that regulates the expression or function of F g f 2 1 is F g f 2 1;

[12] The method according to [10] above, which further comprises isolating erythrocyte-myeloid lineage cells or lymphocyte lineage cells induced to differentiate from hematopoietic stem cells, ;

[13] The method according to [10] above, further comprising further differentiating erythrocyte-myelocyte lineage cells differentiated from hematopoietic stem cells;

[1 4] comprising culturing hematopoietic stem cells in a medium in the presence of F gf 21 and evaluating the differentiation efficiency of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphocyte lineage cells, Method for determining the differentiation efficiency of hematopoietic stem cells by the method; I

Four

[15] a screening method for a substance capable of regulating the differentiation of hematopoietic stem cells, comprising evaluating whether or not the test substance can regulate the expression of F g f 21;

[16] The method according to [15] above, comprising the following steps (a) to (c):

(a) contacting the test substance with a cell capable of measuring the expression of F gf 21; (b) measuring the expression level of F gf 21 in the cell contacted with the test substance; Comparison with the expression level of F gf 21 in the non-contacted control cells;

(c) selecting a test substance that regulates the expression level of F g f 21 based on the comparison result of (b) above;

[17] The method of [15] above, comprising the following steps (a) to (c):

(a) administering a test substance to an animal;

(b) a step of measuring the expression level of F gf 21 in an animal administered with the test substance and comparing the expression level with the expression level of F gf 21 in a control animal not administered with the test substance; ) Selecting a test substance that regulates the expression level of F gf 21 based on the comparison result of

[18] Methods for identifying F gf 2 1 polymorphisms that change the ability to regulate differentiation of hematopoietic stem cells, including analyzing the effects of specific polymorphisms of F gf 2 1 on the ability to regulate differentiation of hematopoietic stem cells ;

[19] including measuring the expression level and / or polymorphism of F gf 21 using a biological sample collected from an animal, and evaluating the biological state of the animal against hematopoietic stem cell differentiation based on the measurement result. A method for determining the biological state of an animal for hematopoietic stem cell differentiation;

[20] A diagnostic agent for the biological state of an animal against hematopoietic stem cell differentiation, which contains a reagent for measuring the expression level of F g f 21 or polymorphism;

[2 1] Kit including the following (a) and (b):

(a) a substance that modulates the expression or function of F g f 2 1; and

(b) a reagent for identifying hematopoietic stem cells or differentiated cells thereof, and a reagent for regulating differentiation of differentiated cells of hematopoietic or stem cells; [22] A method for regulating differentiation of hematopoietic stem cells, comprising administering to a subject an effective amount of a substance that regulates the expression or function of F gf 21;

[23] Use of a substance that regulates the expression or function of F g f 21 in the production of a hematopoietic stem cell differentiation regulator. Brief Description of Drawings

FIG. 1 shows the amino acid sequence of zebrafish F g f 21 and the position of the gene on the chromosome. (A) Comparison of the amino acid sequences of zebrafish F g f 21 and human F g f 21. The amino acid sequence homology is 33.3%. (B) Location of the zebrafish F g f 21 gene and human F g f 21 gene on the chromosome. Both exist adjacent to D h rs 1 0 and C a 1 1. Mb, raegabase

FIG. 2 shows erythropoiesis in a zebrafish fetus with suppressed F g f 21 function. (A) F gf 21 MO (l Ong) was introduced into a fertilized egg, and the fetus was observed 24 hours after fertilization. Wild-type (left) and F g f 21 function-suppressed fetus (right). (B) To observe erythropoiesis, the area enclosed by the square in Figure A was enlarged. Wild-type (left) and Fgf2 1 function-suppressed fetus (right). Arrows indicate red blood cells.

FIG. 3 shows angiogenesis in a zebrafish fetus with suppressed F g f 21 function. (A) F g f 21 MO (l Ong) was introduced into a fertilized egg, dextran labeled with FITC was introduced into the fetal vein 48 hours after fertilization, and blood vessels were observed with a fluorescence microscope. Wild-type (left) and Fg f 2 1 function-suppressed fetus (right). Arrows indicate blood vessels. (B) F g f 21 MO (l Ong) was introduced into a fertilized egg, and the expression of the vascular endothelium gene marker f 1 k 1 in the fetus 48 hours after fertilization was observed by whole mount in situ hybridization. Dextran labeled with FITC was introduced into the vein, and blood vessels were observed with a fluorescence microscope. Arrows indicate blood vessels.

Figure 4 shows a model diagram of the differentiation process of hematopoietic cells. Bold letters indicate a marker gene for hematopoietic cells.

Fig. 5 shows marker of hematopoietic cells in zebrafish fetuses with suppressed Fgf21 function. The expression of the gene is shown. F gf 21 MO (l Ong) was introduced into fertilized eggs, and the expression of hematopoietic cell gene markers in the fetus was observed by whole mount in situ hybridization. The arrows indicate the disappearance or decrease of expression in F gf 2 1 function-suppressed fetuses.

(A) Expression of s c 1, a marker of hemangioblast, in the lateral mesoderm of fetus lateral plate of 5 som i tes. (B) Expression of the hematopoietic stem cell gene, g a t a 2, in fetal ICM 18 hours after fertilization. (C) Expression of erythroid-myeloid progenitor cell marker gene, ga t a 1, in fetal ICM 24 hours after fertilization. (D) Expression of mpx, a myeloid cell marker gene, in fetal ICM 24 hours after fertilization. (E) Expression of a lymphocyte progenitor marker gene, i k a r o s, in fetal ICM 24 hours after fertilization.

Fig. 6 shows the action model of F g f21. F g f 21 acts on the differentiation process from hematopoietic stem cells to erythroid-myeloid progenitor cells. On the other hand, erythropoietin acts on the differentiation process from erythroid-myeloid progenitor cells to erythrocytes. BEST MODE FOR CARRYING OUT THE INVENTION

(1. Hematopoietic stem cell differentiation regulator)

The present invention provides a hematopoietic stem cell differentiation regulator comprising a substance that regulates the expression or function of F g f 21.

Hematopoietic stem cells differentiate into common myeloid progenitor cells (CMP cells) or lymphocyte progenitor cells (CLP cells). CMP cells are divided into granulocyte / macrophage progenitor cells (GMP cells) or megakaryocyte Z red; 3¾ precursor progenitor cells (megaparyocyte / erythroid progenitor cells). GMP cells further differentiate into eosinophils, neutrophils, basophils, monocytes, etc. via myelocytic cells or monocytic cells. Differentiate into spheres or megakaryocytes. On the other hand, CLP cells are common T cell progenitor cells (T-lymphocyte progenitor cells: CTP cells) and B cell common progenitor cells. Differentiate into B-lymphocyte progenitor cells (CBP cells) or NK progenitor cells (NKP cells). The present inventors have found that a substance that promotes / suppresses the expression or function of F gf 21 is capable of differentiating hematopoietic stem cells into CMP cells or differentiated cells thereof (eg, Gata 1 positive cells, mp x positive cells). It has been found that it can be promoted / suppressed and that the differentiation of hematopoietic stem cells into CLP cells or differentiated cells thereof (for example, I karos positive cells) can be inhibited / promoted.

As used herein, the following terminology “lineage cells” is used where appropriate.

The term “erythrocyte-myelocyte lineage cell” comprehensively means CMP cells and differentiated cells thereof, and includes erythrocyte lineage cells, megakaryocyte lineage cells, and myeloid lineage cells. Here, “erythroid lineage cells” comprehensively mean CMP cells, MEP cells, erythroblasts, and erythrocytes. The term “megakaryocyte lineage cell” means CMP cells, MEP cells, and megakaryocytes comprehensively. “Myeloid lineage cell” is a generic term for CMP cells, GMP cells, myeloid cells, monocytic cells, eosinophils, neutrophils, basophils, and monocytes.

“Lymphocyte lineage cell” means CLP cells and differentiated cells comprehensively,

Includes T cell lineage cells, B cell lineage cells, and NK cell lineage cells. Here, “T cell lineage cell” comprehensively means CLP cells, CTP cells, and sputum cells. The “spider cell lineage cell” comprehensively means CLP cells, CBP cells, and sputum cells. The “spider cell lineage cell” comprehensively means CLP cells, sputum cells, and sputum cells.

F gf 21 refers to human F gf 21 (see, for example, GenBank accession number: AB021975) or orthologs thereof, or mutants thereof (including SNPs and haplotypes). The ortholog of F gf 21 is not particularly limited, for example, any animal, for example, fish, birds, mammals (eg, urushi, hidge, pig, goat, monkey, usagi, rat, hamster, guinea pig, mouse) It can be derived from. F gf 21 is a secreted protein, and a signal sequence (for example, corresponding to amino acid residues 1 to 28 in the amino acid sequence shown in SEQ ID NO: 1) can be removed by processing. In the method of the present invention, the signal sequence is removed as F gf 2 1. I

Any of those obtained without the signal sequence can be used, but those from which the signal sequence has been removed are preferred.

In one embodiment, the substance that modulates the expression or function of F g f 21 may be a substance that promotes the expression of F g f 21.

The expression of F g f 2 1 means that a translation product (ie, protein) from F g f 21 is produced and functionally localized at the site of action. Therefore, the substance that promotes the expression of F gf 2 1 acts at any stage including F gf 2 1 transcription, post-transcriptional regulation, translation, post-translational modification, localization and protein folding. Also good. As used herein, promotion of F g f 21 expression includes supplementation with F g f 21 (protein).

An example of a substance that promotes the expression of F g f 21 may be an expression vector containing a nucleic acid encoding F g f 21 or F g f 21.

F g f 21 can be a natural protein or a recombinant protein. F gf 21 can be prepared by a method known per se, for example, a) F gf 21 may be recovered from a biological sample (eg, blood) containing F gf 21, and b) a host cell (eg, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells) are introduced into F gf 2 1 expression vectors (described later), and transformed transformants are produced and produced by the transformants. C) F gf 2 1 may be synthesized by a cell-free system using rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate or the like. F gf 2 1 is a method that uses solubility such as salting-out solvent precipitation method; mainly the difference in molecular weight such as dialysis method, ultrafiltration method, gel filtration method, and SDS-polyacrylamide gel electrophoresis method. Method that utilizes the difference in charge such as ion exchange chromatography, method that utilizes specific affinity such as affinity chromatography, use of F gf 2 1 antibody, etc. It is appropriately purified by a method using a difference in hydrophobicity such as a method using a difference in isoelectric point such as isoelectric focusing, or a method combining these.

In another embodiment, the substance that modulates the expression or function of F gf 21 is F gf 2 It may be a substance that suppresses the expression of 1. Substances that suppress the expression of F gf 21 may act at any stage such as F gf 21 transcription, post-transcriptional regulation, translation, post-translational modification, localization, and protein folding. .

An example of a substance that suppresses the expression of F g f 2 1 is an antisense nucleic acid against a transcription product of F g f 2 1, in particular, mRNA or an initial transcription product. Antisense nucleic acid consists of a base sequence that can hybridize with the target mRNA (early transcript) under physiological conditions of a cell that expresses the target mRNA (early transcript), and the target mRNA in a hybridized state. A nucleic acid capable of inhibiting the translation of a polypeptide encoded by (early transcript). The type of the antisense nucleic acid may be DNA or RNA, or may be a DNAZRNA chimera. In addition, since a natural antisense nucleic acid is easily degraded by its phosphodiester bond by a nucleolytic enzyme present in the cell, the antisense nucleic acid of the present invention is a thiophosphate type ( It can also be synthesized using a modified nucleotide such as a phosphate bond (P = 0 is replaced with P = S) or 2'-O-methyl type. Other important factors in the design of antisense nucleic acids include improving water solubility and cell membrane permeability, but these can be overcome by devising dosage forms such as the use of ribosomes and microspheres. it can. The length of the antisense nucleic acid is not particularly limited as long as it can specifically hybridize with the transcription product of F gf 21. The short one is about 15 bases, and the long one is in the entire mRNA (initial transcript) sequence. The sequence may include a complementary sequence. From the viewpoint of ease of synthesis, antigenicity problems, etc., for example, oligonucleotides comprising about 15 bases or more, preferably about 15 to about 30 bases are exemplified. In addition, antisense nucleic acids not only hybridize with F gf 21 transcripts to inhibit translation, but also bind to double-stranded DNA to form triplex, It may be capable of inhibiting transcription.

Another example of a substance that suppresses the expression of F g, f 2 1 is the transcription product of F gf 2 1, specifically mRNA or the initial transcription product, and the inside of the coding region (intron part in the case of the initial transcription product) And a liposome that can be cleaved specifically. With lipozyme Refers to RNA having an enzyme activity that cleaves nucleic acid, but recently it has been clarified that oligo DNA having the base sequence of the enzyme active site also has a nucleic acid cleaving activity. As long as it has a sufficient nucleic acid cleavage activity, it is used as a concept including DNA. The most versatile ribozyme is self-splicing RNA found in infectious RNAs such as viroid and virusoid, and the hammerhead type and hairpin type are known. In addition, when the ribozyme is used in the form of an expression vector containing the DNA that encodes it, a hybrid ribozyme in which tRNA-modified sequences are further linked to promote cytoplasmic translocation. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

Yet another example of a substance that suppresses the expression of F g f 21 is an RNA i-inducible nucleic acid. An RNA i-inducible nucleic acid refers to a nucleic acid that can induce an RNA i effect when introduced into a cell, and is preferably RNA. The RNAi effect is a phenomenon in which RNA having a double-stranded structure containing the same nucleotide sequence (or a partial sequence thereof) as mRNA suppresses the expression of the mRNA. In order to obtain the R N Ai effect, for example, it is preferable to use RNA having a double-stranded structure having at least 20 or more consecutive target mRNAs and the same nucleotide sequence (or a partial sequence thereof). The double-stranded structure may be composed of different strands, or may be a double-stranded structure provided by a single RNA stem-loop structure. Examples of RNA i-inducible nucleic acids include si RNA, st RNA, mi RNA, and the like.

Another example of a substance that suppresses the expression of F gf 21 is a targeting vector. The targeting vector used in the present invention comprises a first polynucleotide and a second polynucleotide that are homologous to the F gf21 gene capable of inducing homologous recombination of the Fgf21 gene, and a selection marker. The first and second polynucleotides are polynucleotides having sufficient sequence identity and length to cause homologous recombination with genomic DNA containing F gf, 21. The first and second polynucleotides are the first and second polynucleotides in the genomic DNA containing the F gf 21 gene. ί

11 Selection of a deletion of a partial region of genomic DNA between two regions homologous to the polynucleotide results in a functional defect in the F g f21 gene. Selectable markers include positive selectable markers (eg, neomycin resistance gene, hygromycin B phosphotransferase (BPH) gene, plasticidin S deaminase gene, puromycin resistance gene), negative selectable markers (eg, simple hell) Examples include pessimidal virus (HSV) thymidine jelly "one (tk) gene, diphtheria toxin A fragment (DTA) gene", etc. Targeting vectors are either a positive selection marker, a negative selection marker, or both. A targeting vector can also include two or more recombinase target sequences (eg, 1 ox P sequence used in the Cre / 1 ox P system from Batateriophage P 1, FLP ZFRT system from yeast FRT sequences used in It may contain a.

In another embodiment, the substance that modulates the expression or function of F g f 21 may be a substance that suppresses the function of F g f 21. The substance that suppresses the function of F gf 21 is not particularly limited as long as it is a substance that can interfere with the action of F gf 21, but an antibody against F gf 21, a dominant negative mutant of F gf 21, and these codes An expression vector containing a nuclear acid is exemplified.

The antibody against F gf 21 may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a well-known immunological technique. The antibody may be an antibody fragment (eg, Fab, F (ab ′) ₂ ) or a recombinant antibody (eg, a single chain antibody). Furthermore, a nucleic acid encoding the antibody (that is operably linked to a nucleic acid having promoter activity) is also preferable as a substance that suppresses the expression of F gf21.

For example, a polyclonal antibody is commercially available using F gf 21 or a fragment thereof (if necessary, a complex cross-linked to carrier protein such as ushi serum albumin or KLH (Keyhole Limpet Heraocyanin)) as an antigen. Animal adjuvants (eg, complete or incomplete Freund's adjuvant) I

12 Administered subcutaneously or intraperitoneally about 2 to 4 times every 2 to 3 weeks (by measuring the antibody titer of the partially collected serum by a known antigen-antibody reaction and confirming its rise), from the final immunization It can be obtained by collecting whole blood after about 3 to about 10 days and purifying the antiserum. Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

Monoclonal antibodies can also be produced by cell fusion methods (for example, Takeshi Watanabe, principles of cell fusion methods and creation of monoclonal antibodies, Akira Taniuchi, Toshitada Takahashi, “Monoclonal antibodies and basics and clinical one”, 2- 14 pp., Science forum publication, can be Seisuru created by 198 ⁵ years). For example, the factor is administered to a mouse subcutaneously or intraperitoneally 2-4 times with a commercially available adjuvant, and the spleen or lymph node is collected about 3 days after the final administration, and white blood cells are collected. The leukocytes and myeloma cells (for example, NS_1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the factor. Cell fusion can be performed using the PEG method [J. Immunol. Methods, 81 (2): 223-228 (1985)] or the voltage pulse method [Hybridoraa, 7 (6): 627-633 (1988)]. . A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a well-known EIA or RIA method. The culture of the hybridoma producing the monoclonal antibody can be carried out in vitro, or in vivo, such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively.

However, considering the therapeutic effect and safety in humans, the antibodies of the present invention may be chimeric antibodies, humanized or human antibodies. Examples of chimeric antibodies include “Experimental Medicine (Special Issue), Vol. 6, No. 10, 1988”, Japanese Patent Publication No. 3-73280, and human antibodies include, for example, Japanese Patent Publication No. 4-506458. According to Japanese Patent Laid-Open No. 62-296890, etc., human antibodies include, for example, “Nature Genetics, Vol. 15, p. 146-156, 1997”, “Nature Genetics, Vol. 7, p. 13- No. 21, 1994, Special Publication No. 4-504365, International Application Publication No. TO94 / 25585, Nikkei Science, June, 40 to 50, 1995, Nature, Vol. 368, I

13 p. 856-859, 1994 ”, JP-T 6-500233, and the like.

A dominant negative mutant of F g f 21 refers to one whose activity has been reduced by introducing a mutation into F g f 21. The dominant negative mutant can indirectly inhibit its activity by competing with natural F g f 21. The dominant negative mutant can be prepared by introducing a mutation into a nucleic acid encoding F g f 21. Examples of the mutation include an amino acid mutation (for example, deletion, substitution or addition of one or more amino acids) that causes a decrease in the function of the site at the functional site. The dominant negative mutant can be prepared by a method known per se using PCR or a known kit.

In the following description, antisense nucleic acids, liposomes, RNAi-inducible nucleic acids, targeting vectors, antibodies, and dominant negative mutants may be abbreviated as “inhibitory substances”.

When the substance that regulates the expression or function of F gf 21 is a nucleic acid molecule or a protein molecule, the agent of the present invention comprises an expression vector containing a nucleic acid molecule encoding the nucleic acid molecule or protein molecule as an active ingredient. You can also. In the expression vector, the oligonucleotide or polynucleotide encoding the above-mentioned nucleic acid molecule must be operably linked to a promoter capable of exerting a promoter activity in mammalian cells to be administered. The promoter used is not particularly limited as long as it can function in the mammal to be administered. For example, SV 40-derived early promoter, cytomegalovirus LTR, rous sarcoma virus LTR, Mo M u LV origin Examples include viral promoters such as LTR and adenovirus-derived early promoters, and mammalian constituent protein gene promoters such as the / 3-actin gene promoter, PGK gene promoter, and transferrin gene promoter.

The expression vector preferably contains a transcription termination signal, ie, a terminator region, downstream of the oligo (poly) nucleotide encoding the nucleic acid molecule. further, I

14 Further selectable marker genes for selection of transformed cells (such as genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, and genes that complement auxotrophic mutations) It can also be contained.

Vectors suitable for administration to mammals, such as S, humans, etc., which are basic skeletal vectors used as expression vectors, plasmids or viral vectors, include adenowinoles, retrowinoles, adeno-associated viruses, herpes. Virus vectors such as S. virus, ヮ Cincinoainoles, Box Wizores, Poliouinoles, Sindbisuinoles, Sendai Wineles, Epstein.

The agent of the present invention can contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to the substance that modulates the expression or function of F gf 21. Examples of pharmaceutically acceptable carriers include sucrose, starch, mannitol, sorbit, lactose, dalcoose, cellulose, talc, calcium phosphate, calcium carbonate, etc., cellulose, methylcellulose, hydride Roxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, canoleoxymethyl cenorelose, hydroxypropizole starch, sodium monoglycol starch H, sodium hydrogen carbonate, calcium phosphate, calcium kennate and other disintegrants, magnesium stearate, air mouth gill, tanolec, sodium lauryl sulfate and other lubricants, citrate, menthol, glycyrrhizin, ammonium salt, glycine , Fragrances such as orange powder, Preservatives such as sodium benzoate, sodium bisulfite, methylparaben, propylparaben, stabilizers such as succinic acid, sodium succinate, acetic acid, etc. Dispersing agents such as turbidity agents, surfactants, water, raw foods; brine, diluents such as orange juice, base waxes such as cacao butter, polyethylene glycol, white kerosene, etc. It is not a thing. I

15 Preparations suitable for oral administration include solutions in which an effective amount of substance is dissolved in a diluent such as water or physiological saline, capsules containing an effective amount of the substance as solids or granules, sachets or Examples thereof include tablets, suspensions in which an effective amount of a substance is suspended in an appropriate dispersion medium, and emulsions in which a solution in which an effective amount of a substance is dissolved is dispersed in an appropriate dispersion medium and emulsified. Formulations suitable for parenteral administration (eg, intravenous injection, subcutaneous injection, intramuscular injection, local injection, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Buffer solution, antibacterial agent, tonicity agent and the like may be contained. Aqueous and non-aqueous sterile suspensions can also be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. In addition, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.

The dose of the agent of the present invention varies depending on the activity and type of the active ingredient, the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age, etc. In general, the amount of active ingredient per day for an adult is about 0.01 to about 500 mg Z kg.

The agent of the present invention is useful, for example, as a pharmaceutical or research reagent. For example, when the agent of the present invention contains a substance that promotes the expression or function of F gf 21, it is useful as an agent for promoting differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells. The present inventors have found that F gf 21 is a factor responsible for promoting differentiation of hematopoietic stem cells (for example, gata 2 positive) into CMP cells (for example, gata 1 positive). Therefore, it is considered that a substance that promotes the expression or function of F gf 21 can promote the differentiation of hematopoietic stem cells into CMP cells, and thereby increase the number of cells such as erythrocytes, megakaryocytes, and myeloid cells. Since the agent of the present invention increases the number of cells such as erythrocytes more efficiently, a substance that promotes differentiation into erythrocytes (for example, erythropoietin), a substance that promotes differentiation into megakaryocytes (for example, thrombopoietin), It may further contain a substance that promotes differentiation into myeloid cells (eg granulocyte-macrophage colony-stimulating factor). In this case, ί

16 Clarifying agents can be used for diseases where an increase in the number of cells such as red blood cells, platelets, and myeloid cells is desired. Examples of diseases in which an increase in the number of red blood cells is desired include anemia and erythrocytopenia (for example, after cancer chemotherapy and radiation therapy). Examples of diseases for which an increase in the number of platelets is desired include thrombocytopenia (for example, after cancer chemotherapy and radiation therapy). Examples of the disease for which an increase in the number of myeloid cells is desired include myelocytopenia (for example, cancer chemotherapy, after radiation therapy). In addition, the agent of the present invention can also be used for the purpose of improving a biological function (for example, motor function) related to oxygen transport.

In addition, when the agent of the present invention contains a substance that promotes the expression or function of F g f 21, it is useful as an inhibitor of differentiation of hematopoietic stem cells into lymphocyte lineage cells. A substance that promotes the expression or function of F gf 21 indirectly suppresses the differentiation of hematopoietic stem cells into CLP cells (eg, ikaros positive) as a result of promoting differentiation of hematopoietic stem cells into CMP cells. It is believed that the number of lymphocyte lineage cells can be reduced. In this case, the agent of the present invention can be used for diseases in which a decrease in the number of lymphocyte lineage cells (eg, T cells, B cells) is desired. Examples of diseases for which the number of lymphocyte lineage cells is desired include autoimmune diseases and allergic diseases.

On the other hand, when the agent of the present invention contains a substance that suppresses the expression or function of F g f 21, it is useful as an agent for suppressing the differentiation of hematopoietic stem cells into erythrocyte-one myeloid lineage cells. The present inventors have found that suppression of the function of F g f 21 suppresses differentiation of hematopoietic stem cells into CMP cells. Therefore, a substance that suppresses the expression or function of F gf 21 suppresses the differentiation of hematopoietic stem cells into erythrocyte-one myeloid lineage cells, thus reducing the number of red blood cells, megakaryocytes, myelocytes, etc. It is thought to get.

In addition, when the agent of the present invention contains a substance that suppresses the expression or function of F gf 21, it is useful as an agent for promoting differentiation of hematopoietic stem cells into lymphocyte lineage cells. Substances that suppress the expression or function of F gf 21 indirectly promote the differentiation of hematopoietic stem cells into CLP cells as a result of inhibiting the differentiation of hematopoietic stem cells into CMP cells, thereby increasing the number of lymphocyte lineage cells. It can be increased. The agent of the present invention is more efficient I

17 In order to increase the number of lymphocyte lineage cells, it may further contain a substance that promotes differentiation into lymphocyte lineage cells. Another substance that promotes differentiation into lymphocyte lineage cells is, for example, interleukin-1. In this case, the agent of the present invention can be used for diseases in which an increase in the number of lymphocyte lineage cells (for example, T cells, B cells) is desired. Examples of diseases for which an increase in the number of lymphocyte lineage cells is desired include immunodeficiency diseases.

If the modulator of the present invention contains other substances in addition to substances that promote the expression or function of F gf 21, they are in a combined form (for example, stored as a mixture in the same container) Alternatively, they can be provided in isolated form (eg, stored in different containers). The modulator of the present invention can be used in vitro or in vitro. (2. Methods for regulating differentiation of hematopoietic stem cells)

The present invention also provides a method for regulating differentiation of hematopoietic stem cells. The method includes, for example, culturing hematopoietic stem cells in a medium in the presence of a substance that modulates the expression or function of F gf21.

Hematopoietic stem cells can be used in any animal, such as fish, birds, mammals (eg, ushi, sheep, pigs, goats, monkeys, humans, rabbits, rats, hamsters, guinea pigs, mice). It can be a derived cell. Hematopoietic stem cells can also be cells derived from any tissue, but can be cells present in tissues such as bone marrow and fetal liver. Hematopoietic stem cells can be cells induced to differentiate from pluripotent stem cells such as embryonic stem cells and somatic stem cells. In addition, when transplantation of cells obtained by the differentiation regulation method of the present invention is intended, cells suitable for allogeneic transplantation can be obtained by using cells derived from the same species as the animal intended for transplantation. By using a cell derived from an individual intended to be transplanted, a cell suitable for allogeneic transplantation can be obtained. Hematopoietic stem cells can be obtained by a method known per se (for example, F A C S) using a cell surface marker. Examples of cell surface markers for hematopoietic stem cells include S e a-1 and c kit.

The medium used for culturing hematopoietic stem cells is not particularly limited as long as it is suitable for the differentiation of hematopoietic stem cells, and is appropriately selected. For example, the minimum essential medium (MEM), Dulbecco modified I

18 Modified minimal essential medium (D M E M), F 12 medium or R P M I 16 40 medium, or a mixture thereof. Examples of additives to the medium include various amino acids, various inorganic salts, various vitamins, various antibiotics, and buffering agents. The culture conditions are also appropriately determined. For example, the pH of the medium is about 6 to about 8, and the culture temperature is usually about 30 to about 40 ° C. The medium may or may not contain serum, but a serum-free medium is preferable from the viewpoint of preventing contamination of unidentified components and reducing the risk of infection.

In one embodiment, the differentiation-regulating method of the present invention comprises culturing hematopoietic stem cells in a medium in the presence of F gf21. The culture of hematopoietic stem cells in the presence of F g f 21 is not particularly limited as long as it results in culturing hematopoietic stem cells in a medium containing F g f 21. Therefore, F g f 21 may not be present in the medium at the start of culture. Examples of the culture of hematopoietic stem cells in the presence of F gf 21 include culture of hematopoietic cells in a medium supplemented with F gf 21, culture of hematopoietic stem cells into which an F gf 21 expression vector has been introduced, F gf 21 Examples include co-culture of hematopoietic stem cells with expression cells, but culture of hematopoietic stem cells in a medium supplemented with F gf 21 is preferred.

When hematopoietic stem cells are cultured in a medium supplemented with F g f 21, F g f 21 added to the medium can be a natural protein or a recombinant protein. F g f 21 can be prepared by a method known per se as described above.

When culturing hematopoietic stem cells into which an F gf 21 expression vector has been introduced, the expression vector introduced into the hematopoietic stem cells can be the same as the F gf 21 expression vector described above. The expression vector can be introduced into hematopoietic stem cells by a method known per se, for example, an electroporation method, a calcium phosphate precipitation method, a microinjection method, a liposome, a method using a lipid such as a cationic lipid. Further, a part or all of the expression vector may or may not be integrated into the hematopoietic stem cell genome. For integration of the expression vector into the intracellular genome, methods known per se, such as methods using retroviruses, A method using a getting vector or the like can be used.

When co-culturing F gf21-expressing cells and hematopoietic stem cells, the cells to coexist with hematopoietic stem cells in the medium are not particularly limited as long as Fgf21 can be expressed. The expression cell can be, for example, a cell obtained by introducing an F gf21 expression vector into a host cell, or a natural cell that expresses Fgf21. As natural cells that express F g f 21, cells derived from tissues expressing F g f 21 (eg, bone marrow, liver, thymus) can be used. The F gf21-expressing cells can also be primary cultured cells, cell lines derived from primary cultured cells, cells obtained by culturing undifferentiated cells such as stem cells (eg, differentiated cells), and the like. The F g f21 expressing cells can also be cells from the same animal or individual as the hematopoietic stem cells. Co-culture can be performed when hematopoietic stem cells and F gf 21-expressing cells are in physical contact with each other, or when these cells are present in the same culture system but separated by a septum that allows passage of substances. This includes cases where physical contact is not possible. The case where hematopoietic stem cells and F gf 2 1-expressing cells are present in the same culture system, but separated by a partition wall that allows the passage of substances, is not possible for physical contact of the cells themselves.For example, it is used for normal cell culture One example is a case where both cells are cultured separately using a filter (for example, a cartridge).

In another embodiment, the method for regulating differentiation of the present invention comprises culturing hematopoietic stem cells in a culture medium so as to suppress the expression or function of Fg 21. Examples of such hematopoietic stem cell culture include culture of hematopoietic stem cells in a medium supplemented with an F gf 21 inhibitor (described above).

The differentiation regulation method of the present invention can further include isolating cells induced to differentiate from hematopoietic stem cells (for example, erythroid-one myeloid lineage cells, lymphocyte lineage cells). Isolation of differentiated cells can be performed by a method known per se using a cell surface marker.

The differentiation control method of the present invention can also include further differentiation of cells induced to differentiate from hematopoietic stem cells. The method for further differentiation of the cells induced to differentiate from hematopoietic stem cells is not particularly limited. For example, F gf 21 expression or Is a method of culturing hematopoietic stem cells in the presence of a substance that regulates function and other differentiation regulators, or after culturing hematopoietic stem cells in the presence of a substance that regulates the expression or function of F g ί 21 And a method of using a differentiation regulator. Examples of other differentiation regulating substances include those mentioned above. A preferred example of a method for further differentiation of cells induced to differentiate from hematopoietic stem cells is a method for promoting differentiation into erythrocytes using erythropoietin.

The differentiation regulation method of the present invention is useful for producing predetermined differentiated cells from hematopoietic stem cells. For example, the obtained differentiated cells can be used for cell therapy (transplantation). In this case, from the viewpoint of allogeneic transplantation, it is preferable to unify the substances and materials used in the culture with substances and materials derived from the same animal as the subject to receive cell therapy. In addition, from the viewpoint of allogeneic transplantation, the cells used in the culture are preferably cells derived from the same species as the subject receiving cell therapy, but from the viewpoint of allogeneic transplantation, cells derived from the subject are used. More preferred.

(3. Method for determining differentiation efficiency of hematopoietic stem cells)

The present invention also provides a method for determining the differentiation efficiency of hematopoietic stem cells by F g f 21. The determination method of the present invention includes, for example, culturing hematopoietic stem cells in a medium in the presence of F gf 21 and evaluating the efficiency of differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphocyte lineage cells. .

The hematopoietic stem cells used in this determination method may be the same as those used in the differentiation regulation method of the present invention, but it is desirable to determine the differentiation efficiency of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphocyte lineage cells. Hematopoietic stem cells taken from a subject to be tested (eg, a subject being considered for treatment with a substance that modulates F gf 21 expression or function) can be used.

Culture of hematopoietic stem cells in a medium in the presence of F g f 21 can be carried out in the same manner as the differentiation regulating method described above, but culture of hematopoietic stem cells in a medium supplemented with F g f 21 is preferred.

Evaluation of differentiation efficiency of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphocyte lineage cells The value can be determined by a method known per se. For example, the differentiation efficiency of hematopoietic stem cells into red blood cells, myeloid lineage cells or lymphocyte lineage cells is sufficient to affirm the usefulness of treatment with substances that modulate the expression or function of F gf 21. The power can be evaluated.

This determination method is useful, for example, in predicting the effect of treatment using a substance that modulates the expression or function of F gf 21 in a given patient and determining whether or not to adopt the treatment in the patient. It can be.

(4. Screening method for substances that can regulate the differentiation of hematopoietic stem cells)

The present invention also includes a screening method for a substance capable of regulating the differentiation of hematopoietic stem cells, comprising evaluating whether the test substance can regulate the expression or function of F gf 21, and the screening method. And a hematopoietic stem cell differentiation regulator comprising the substance.

The test substance to be used for the screening method may be any known compound and novel compound, for example, nucleic acid, carbohydrate, lipid, protein, peptide, low-molecular-weight organic compound, and yunpina chemistry technology Compound libraries, random peptide libraries prepared by solid phase synthesis or phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.

In one embodiment, the screening method of the present invention can be performed using cells. In this case, the screening method of the present invention includes the following steps (a) to (c):

(a) contacting the test substance with a cell capable of measuring the expression of F g f 21;

(b) measuring the expression level of F g f 21 in cells contacted with the test substance and comparing the expression level with the expression level of F g f 21 in control cells not contacted with the test substance;

(c) A step of selecting a test substance that regulates the expression level of F g f 21 based on the comparison result of (b).

In step (a) of the above method, the test substance and cells capable of measuring the expression of F gf 21 Place under contact conditions. Contact of the test substance with a cell capable of measuring the expression of F gf 21 can be performed in a culture medium.

A cell capable of measuring the expression of F g f 21 refers to a cell capable of directly or indirectly evaluating the expression level of a product of F g f 21, for example, a transcription product or a translation product. Cells that can directly evaluate the expression level of the F gf 2 1 product may be cells that can naturally express F gf 2 1, while indirectly evaluating the expression level of the F gf 2 1 product. A possible cell may be a cell that allows reporter assembly for the F gf21 transcriptional regulatory region. The cell capable of measuring the expression of F g f 21 can be an animal cell, for example, a mammalian cell such as a mouse, a rat, a hamster, a guinea pig, a rabbit, an inu, a monkey, or a human.

The cell capable of naturally expressing F gf21 is not particularly limited as long as it can potentially express Fgf21. Such cells can be easily identified by those skilled in the art, and primary cultured cells, cell lines derived from the primary cultured cells, commercially available cell lines, cell lines available from cell banks, and the like can be used.

A cell that enables reporter assembly for the F gf21 transcriptional regulatory region is a cell that contains a Fgf21 transcriptional regulatory region and a reporter gene operably linked to the region. The F g f21 transcriptional regulatory region, reporter gene can be inserted into an expression vector. The F gf 21 transcriptional regulatory region is not particularly limited as long as it is a region capable of controlling the expression of F gf 21, but for example, in the region from the transcription start point to about 2 kbp upstream, or in the base sequence of the region Examples include a region consisting of a base sequence in which one or more bases have been deleted, substituted or added, and having the ability to control transcription of F gf 21. The reporter gene may be any gene that encodes a detectable protein or an enzyme that produces a detectable substance. For example, a GFP (green fluorescent protein) gene, a GUS (one-darc mouth nidase) gene, an LUC (luciferase) ) Gene, CAT (chloramphee-colacetyltransferase) gene, and the like.

F gf 21 transcriptional regulatory region, reporter gene operably linked to the region The cell into which is introduced is not particularly limited as long as the F gf 21 transcriptional regulatory function can be evaluated, that is, as long as the expression level of the reporter gene can be quantitatively analyzed. However, since it expresses a physiological transcriptional regulatory factor for F gf 21 and is considered to be more appropriate for the evaluation of the expression regulation of F gf 21, F gf 21 is naturally introduced as the introduced cell. Cells that can be expressed in are preferred.

The medium in which the test substance is contacted with the cells capable of measuring the expression of F gf 21 is appropriately selected according to the type of cells used, for example, about 5 to 20% Examples include minimal essential medium (MEM) containing serum, Dulbecco's modified minimal essential medium (DMEM), RPM I 1640 medium, and 1999 medium. The culture conditions are also appropriately determined according to the type of cells used, etc.For example, the pH of the medium is about 6 to about 8, and the culture temperature is usually about 30 to about 40 ° C. The incubation time should be about 12 to 72 hours.

In step (b) of the above method, first, the expression level of F g f 21 in the cell contacted with the test substance is measured. The expression level can be measured by a method known per se in consideration of the type of cells used. For example, when cells that can express F gf 21 naturally are used as cells capable of measuring F gf 21 expression, the expression level is for the product of F gf 21, for example, a transcription product or a translation product. It can be measured by a method known per se. For example, the expression level of the transcript can be measured by preparing total RNA from cells and performing RT_PCR, Northern blotting, or the like. In addition, the expression level of the translation product can be measured by preparing an extract from the cells and immunologically. Examples of immunological methods include radioisotope immunoassay (RIA method), EL ISA method (Methods in Enzymol. 70: 419-439 (1980)), and fluorescent antibody method. On the other hand, when a cell capable of reporter assembly for the F gf21 transcriptional regulatory region is used as a cell capable of measuring Fgf21 expression, the expression level can be measured based on the signal intensity of the reporter.

Next, the expression level of F gf 2 1 in the cells contacted with the test substance is compared with the expression level of F gf 21 in the control cells not contacted with the test substance. Ratio of expression level i

The comparison is preferably based on the presence or absence of a significant difference. The expression level of F gf 2 1 in the control cells not contacted with the test substance may be the expression level measured in advance compared to the measurement of the expression level of F gf 2 1 in the cells contacted with the test substance. Although the expression level measured at the same time may be used, the expression level measured at the same time is preferable from the viewpoint of the accuracy and reproducibility of the experiment.

In step (c) of the above method, a test substance that regulates the expression level of F g f 21 is selected. The regulation of the expression level of F g f21 can be an increase or decrease in the expression level. For example, the test substance that increases the expression level of F gf 2 1 (promotes the expression) can be used to promote differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells, or hematopoietic stem cells into lymphocyte lineage cells. It can have a differentiation-inhibiting action, and can be used as a preventive or therapeutic agent for diseases such as anemia, autoimmune diseases, and allergic diseases. On the other hand, the test substance that decreases the expression level of F gf 2 1 (suppresses the expression) is an inhibitory effect on the differentiation of hematopoietic stem cells into erythrocyte-one myeloid lineage cells or promotes differentiation of hematopoietic stem cells into lymphocyte lineage cells It can have an effect and can be a preventive or therapeutic agent for diseases such as immunodeficiency diseases. Accordingly, it is possible to select a pharmaceutical agent such as a preventive / therapeutic agent for a predetermined disease or a complement for a research reagent, using the expression level of F g f 21 as an index.

The screening method of the present invention can also be performed using animals. In this case, the screening method of the present invention includes the following steps (a) to (c):

(a) administering a test substance to an animal;

(b) measuring the expression level of F gf 21 in an animal administered with the test substance, and comparing the expression level with the expression level of F gf 21 in a control animal not administered with the test substance; (c) b) A step of selecting a test substance that regulates the expression level of F gf 21 based on the comparison result of (b).

In step (a) of the above method, mammals such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys and the like are used as animals. Administration of the test substance to the animal can be performed by a method known per se.

In step (b) of the above method, the expression level of F gf 21 is measured by a method known per se. I

25 can be determined. For example, the blood concentration of F g f 21 is measured as the expression level of F g f 21. The comparison of the expression level in this step (b) and the step (c) of the above method can be carried out in the same manner as in the screening method using cells capable of measuring the expression of F g f 21.

The screening method of the present invention enables screening of a substance that can regulate the differentiation of hematopoietic stem cells, or a prophylactic / therapeutic agent for a given disease. Therefore, the screening method of the present invention is useful for the development of the above-mentioned pharmaceuticals or research reagents.

(5. Identification method of F gf 2 1 polymorphism that causes a change in the ability to regulate differentiation of hematopoietic stem cells) The present invention also analyzes the influence of a specific polymorphism of F gf 2 1 on the ability to regulate differentiation of hematopoietic stem cells. A method for identifying an F gf 2 1 polymorphism that causes a change in the ability to regulate the differentiation of hematopoietic stem cells, and a method for causing an alteration in the ability to regulate the differentiation of hematopoietic stem cells identified by the method. Containing proteins ■ Nucleic acid molecules are provided.

An F gf 2 1 polymorphism means a nucleotide sequence variation found in a certain frequency in genomic DNA containing the F gf 2 1 gene in a population, and in the genomic DNA containing the F gf 2 1 gene. One or more DNA substitutions, deletions, additions (eg, SNPs, haplotypes), as well as repeats, inversions, translocations, etc. of partial regions in the genomic DNA. The type of polymorphism of F gf 21 identified by the method of the present invention is that among all types of polymorphisms in F gf 21, hematopoietic stem cells are transformed into erythrocyte-myeloid lineage cells or lymphocyte lineage cells. It may be a nucleotide sequence variation that causes a change in differentiation efficiency, or a nucleotide sequence variation that differs in frequency between an animal suffering from a predetermined disease (eg, hematopoietic disease, immune disease) and an unaffected animal. The animal to be analyzed for the F g f 21 polymorphism is not particularly limited as described above, but humans are preferred.

The analysis can be performed by a method known per se. For example, when there is a significant difference in the frequency of occurrence of a specific polymorphism depending on the frequency and severity of a given disease (eg, hematopoietic disease, immune disease) as a result of an analysis method such as linkage analysis Can be determined as a polymorphism that causes a change in the ability to regulate the differentiation of hematopoietic stem cells. The analysis is It is also possible to do it in vitro. For example, hematopoietic stem cells are cultured in the presence of F gf 21 containing a specific polymorphism, and the differentiation efficiency of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphoid lineage cells is compared with that of control F gf 21 By comparing with differentiation efficiency, polymorphisms can be analyzed.

The identification method of the present invention may further include a step of subjecting a DNA sample prepared from a biological sample derived from a mammal to sequencing and determining a new type of the F gf 21 polymorphism. . Biological samples can be any tissue containing genomic DNA, such as hair, nails, skin, mucous membranes, as well as samples (eg, blood) containing F gf 21-expressing tissues or cells (eg, B cells). . Considering availability, burden on the human body, etc., the biological sample is preferably hair, nails, skin, mucous membrane, blood, plasma, serum, saliva or the like. Polymorphism can be determined by analyzing a large number of nucleotide sequences of genomes or transcripts contained in biological samples of different origins and identifying mutations found at a certain frequency in the determined nucleotide sequence.

The identification method of the present invention and the F g f21 polymorphism determined by the method are useful for determining the risk of developing a predetermined disease.

(6. Judgment method and diagnostic agent of animal biological state for hematopoietic stem cell differentiation) (6. 1. Judgment method and diagnostic agent based on measurement of expression level)

The present invention provides a method for determining the biological state of an animal against hematopoietic stem cell differentiation based on the expression level of F g f21. This method can be useful, for example, as a method for determining the onset or risk of developing a predetermined disease (eg, hematopoietic disease, immune disease) in an animal.

In one embodiment, the determination method of the present invention includes the following steps (a) and (b):

(a) measuring the expression level of F g f 21 in a biological sample collected from an animal;

(b) Evaluating the biological state of the animal against hematopoietic stem cell differentiation based on the expression level of F g f21.

In step (a) of the above method, the expression level of F gf 21 is measured in a biological sample collected from an animal. The animal is not particularly limited as described above, but human is preferable. ί

27 The biological sample is not particularly limited as long as it can measure the expression level of F g f 21, and examples thereof include blood, bone marrow fluid, liver, and thymus, but less invasive blood is preferable. The measurement of the expression level of F g f 21 can be performed in the same manner as the screening method of the present invention.

In step (b) of the above method, the biological state of the animal with respect to hematopoietic stem cell differentiation can be evaluated based on the expression level of F g f 21. Specifically, first, the measured expression level of F g f 21 is compared with the expression level of F g f 21 in an animal without abnormal hematopoietic stem cell differentiation or in a normal animal. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference. The expression level of F g f 21 in animals without normal hematopoietic stem cell differentiation or in normal animals can be determined by a method known per se.

Next, based on the comparison of the expression level of F gf 21, whether the animal may have abnormal hematopoietic stem cell differentiation or may be affected by a predetermined disease Or whether it is more or less likely to be affected in the future. From the results of this example, when the expression level of F gf 21 is high, the differentiation of hematopoietic stem cells into erythrocyte-one myeloid lineage cells is further promoted, and the differentiation of hematopoietic stem cells into lymphocyte lineage cells is further suppressed. On the other hand, when the expression level of F gf 21 is low, the differentiation of hematopoietic stem cells into lymphocyte lineage cells is further promoted, and the differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells can be further suppressed. Conceivable. It is also known that in animals that develop a specific disease, changes in the expression level of genes related to the disease are often observed. Furthermore, it is known that changes in the expression level of specific genes are often observed before the onset of specific diseases. Therefore, by analyzing the expression level of F g f 21, it is considered possible to determine abnormal hematopoietic stem cell differentiation, the onset of a predetermined disease, or the risk of onset.

The present invention also provides a diagnostic agent for the biological state of an animal with respect to hematopoietic stem cell differentiation, comprising a reagent for measuring the expression level of F gf21. The diagnostic agent can be useful, for example, as a diagnostic agent for the onset or risk of developing a given disease in animals.

Reagents for measuring the expression level of F gf 21 can be used as long as the expression of F gf 21 can be quantified. I

28 Although not particularly limited, it may include, for example, an antibody to F gf 2 1 (described above), a nucleic acid probe to F gf 2 1 transcript, or multiple primers capable of amplifying F gf 2 1 transcript . These may or may not be labeled with a labeling substance. When not labeled with a labeling substance, the diagnostic agent of the present invention can further contain the labeling substance. Examples of labeling substances include fluorescent substances such as FITC and FAM, luminescent substances such as luminol, luciferin, and lucigenin, radioisotopes such as 3H, ¹⁴ C, ³² P, ³⁵ S, and ¹²³ I, piotin, and streptavidin. Examples thereof include affinity substances such as gin.

The nucleic acid probe for the F g f21 transcript may be either DNA or RNA, but DNA is preferable in consideration of stability and the like. The probe may be either single-stranded or double-stranded. The size of the probe is not particularly limited as long as a transcription product of F gf 21 can be detected, but is preferably about 15 to; L 2 O 2 O bp, more preferably about 50 to 500 bp. The probe may be provided in a fixed form on a substrate like a microarray.

A plurality of primers (eg, primer pairs) capable of amplifying F g f 21 are selected such that a detectable size nucleotide fragment is amplified. Detectable size nucleotide fragments can have a length of, for example, about 100 bp or more, preferably about 200 bp or more, more preferably about 400 bp or more. The size of the primer is not particularly limited as long as F gf 21 can be amplified, but it is preferably about 15 to 100 bp, more preferably about 18 to 50 bp, and even more preferably about 20 to 30 bp. possible. When the means capable of quantifying the F g f 21 transcript is a primer, the diagnostic agent of the present invention can further contain a reverse transcriptase.

The determination method and diagnostic agent of the present invention are useful because they enable determination of abnormal hematopoietic stem cell differentiation, the onset of a predetermined disease, or the risk of onset.

(6. 2. Determination method and diagnostic agent based on measurement of polymorphism)

The present invention provides a method for determining the biological state of an animal with respect to hematopoietic cell differentiation based on the F gf 21 polymorphism. The method can be used, for example, for certain diseases in animals (eg, 1

29 It may be useful as a method for determining the risk of developing hematopoietic diseases and immune diseases.

(a) measuring a polymorphism of F g f 2 1 in a biological sample collected from an animal;

(b) A step of evaluating the biological state of the animal for hematopoietic stem cell differentiation based on the type of polymorphism.

In step (a) of the above method, the polymorphic type of F g f 2 1 is measured in biological samples collected from animals. The animals are as described above. The biological sample may be the same as that described above in the identification method of the present invention.

The polymorphic type can be measured by a method known per se. For example, RF LP (Restriction Enzyme Fragment Length Polymorphism) method, PCR—SS CP (—Single-stranded DN A conformational polymorphism analysis) method, ASO (Allele Specific Oligonucleotide) hybridization method, Direct sequence method , ARMS (Amplification Refracting Mutation System) method, Denaturing Gradient Gel Electrophoresis (RNaturing) method, RNase A cleavage method, DOL (Dye-labeled Oligonucleotide Ligation) method, TaqMan PCR method, Invader method, etc. Can be used. In step (b) of the above method, the biological state of the animal with respect to hematopoietic stem cell differentiation can be evaluated based on the type of polymorphism. Specifically, it is determined whether an animal may have abnormal hematopoietic stem cell differentiation (excessive promotion or suppression), or whether it is more or less likely to suffer from a given disease in the future. obtain. From the results of this Example, when F gf 21 contains a polymorphism that enhances its function, the differentiation of hematopoietic stem cells into erythrocyte-one myeloid lineage cells is further promoted, and lymphocyte lineage cells of hematopoietic stem cells On the other hand, when F gf 21 contains a polymorphism that reduces its function, differentiation of hematopoietic stem cells into lymphocyte lineage cells is further promoted, and It is considered that differentiation into myeloid lineage cells can be further suppressed. In addition, it is known that animals that are likely to develop a specific disease often have a specific type of polymorphism in a gene associated with the disease. Therefore, an animal containing a polymorphism that reduces the function of F gf 21 has a disease such as anemia, autoimmune disease or allergic disease. I

30 The likelihood of developing is relatively high. Similarly, an animal containing a polymorphism that enhances the function of F gf21 is considered to have a relatively high possibility of developing a disease such as an immunodeficiency disease. Therefore, it is considered possible to determine the possibility of the development of a given disease by analyzing the polymorphism. The polymorphic type to be measured in this step can be obtained, for example, by the identification method of the present invention.

The present invention also provides a diagnostic agent for the biological state of an animal with respect to hematopoietic stem cell differentiation, comprising a reagent for measuring a polymorphism of F g f 21. This method can be useful, for example, as a diagnostic agent for the risk of developing a given disease in an animal.

The reagent for measuring the polymorphism of F g f 21 is not particularly limited as long as the polymorphism of F g f 21 can be determined. The reagent may be labeled with a labeling substance. Further, when the reagent is not labeled with a labeling substance, the labeling substance can be further included in the form of a kit.

Specifically, the reagent for measuring the polymorphism of F gf 2 1 is a nucleic acid probe capable of specifically measuring F gf 2 1 having a specific type of polymorphism, or F having a specific type of polymorphism. It may contain a plurality of primers capable of specifically amplifying gf21. Nucleic acid probes, primers can be for genomic DNA containing F g f 21 or F g f 21 transcripts. The nucleic acid probe and primer may be provided together with a transcription product or a reagent for extracting genomic DNA.

The nucleic acid probe capable of specifically measuring F gf 21 having a specific type of polymorphism is not particularly limited as long as F gf 21 having a specific type of polymorphism can be selected. The probe may be either DNA or RNA, but DNA is preferable in consideration of stability and the like. The probe may be either single-stranded or double-stranded. The size of the probe is preferably as short as possible so that F gf 2 1 having a specific type of polymorphism can be selected. For example, the size of the probe may be about 15 to 30 bp. The probe may be provided in a form fixed on a substrate like a microarray. The probe enables, for example, an ASO (Allele Specific Oligonucleotide) hybridization method. Multiple primers (eg, primer pairs) capable of specifically amplifying F gf 21 with a particular type of polymorphism are selected such that measurable size nucleotide fragments are amplified. . Such a plurality of primers is designed to include a polymorphic site at the 3 ′ end of one of the primers, for example. The measurable size nucleotide fragment may have a length of, for example, about 100 bp or more, preferably about 200 bp or more, more preferably about 400 bp or more. The size of the primer is not particularly limited as long as F gf 21 can be amplified, but is preferably about 15 to 10 O bp, more preferably about 18 to 50 bp, and even more preferably about 20 to 30 bp. It can be. When the means capable of measuring the polymorphism of F gf 21 is a primer pair for the F gf 21 transcript, the determination kit can further contain a reverse transcriptase.

In addition, as a reagent for measuring the polymorphism of F g f 21, a reagent containing a restriction enzyme that recognizes a specific type of polymorphic site can also be mentioned. According to such a reagent, polymorphism analysis by RF LP becomes possible.

The above-described determination method and diagnostic agent of the present invention enable determination of abnormal hematopoietic stem cell differentiation and the onset risk of a predetermined disease, and provide an opportunity for improving lifestyle habits for the purpose of preventing the predetermined disease. Because it is useful.

(7. Kit)

The present invention provides a kit comprising the following (a) and (b):

(a) a substance that modulates the expression or function of F g f 2 1; and

(b) A reagent for identifying hematopoietic stem cells or differentiated cells thereof, and / or a reagent for regulating differentiation of hematopoietic stem cells.

The substance that regulates the expression or function of Fgf21 in (a) above may be the same as described above.

The reagents for identifying hematopoietic stem cells in (b) above include hematopoietic stem cell-specific cell surface markers (eg, Sea-1, ckit), hematopoietic stem cell-specific cell non-surface markers (eg, Gata— l) a substance having a specific affinity for (for example, Antibody).

Examples of the reagent for identifying a differentiated cell of the hematopoietic stem cell (b) include a reagent for identifying an erythrocyte-myeloid lineage cell and a lymphocyte lineage cell. As a reagent for identification of erythrocyte-myeloid lineage cells, a substance having a specific affinity for a cell non-surface marker specific to erythrocyte-myeloid lineage cells (eg, G 1 obin, mp X) (for example, For example, antibodies). Reagents for identifying lymphocyte lineage cells include cell surface markers specific for lymphocyte lineage cells (eg, B cell receptor, T cell receptor), cell surface markers specific for lymphocyte lineage cells (eg, I karos). ) And a substance having a specific affinity (for example, an antibody).

Examples of the differentiation regulating reagent for differentiated cells of hematopoietic stem cells (b) above include reagents for regulating differentiation of erythrocyte-myeloid lineage cells and lymphocyte lineage cells. Examples of the differentiation regulator for erythrocyte-myeloid lineage cells include those containing thrombopoietin as a differentiation regulator for erythrocyte-one myeloid lineage cells. Examples of the differentiation regulator for lymphocyte lineage cells include those containing interleukin-17 as a differentiation regulator for lymphocyte lineage cells.

The kit of the present invention is useful for the preparation of the agent of the present invention and for simply carrying out the method of the present invention.

(8. Novel F gf 21 gene, protein, and accompanying invention) The present invention also provides a novel F gf 21 polypeptide, polynucleotide. The polypeptide of the present invention is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3 or a polypeptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3 is (a) 1 or 2 or more (for example, 1 to 50, preferably 1 to 3) in the amino acid sequence represented by SEQ ID NO: 3. An amino acid sequence in which 0, more preferably 1 to 20, even more preferably 1 to 10, most preferably 1 to 5 amino acids are substituted, deleted, inserted or added, (b ) Shown in SEQ ID NO: 3 Significant amino acid sequence identity (for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, even more preferably about 95% or more, most An amino acid sequence having preferably about 9 7%, 98%, or 99% or more amino acid sequence identity) is mentioned. The identity (%) can be determined using a program commonly used in this field (for example, BLAST, FASTA, etc.) by default. In another aspect, identity (%) is determined by any algorithm known in the art, such as Needleman et al. (1970) (J. Mol. Biol. 48: 444-453), Myers and Miller ( It can be determined using the algorithm of CABIOS, 1988, 4: 11-17). Needleman et al.'S algorithm is incorporated into the GAP program in the GCG software package (available at www.gcg.com) and the identity (%) is, for example, BL0SUM 62 matrix or PAM250 matrix, and gap weight: 16, It can be determined by using 14, 12, 10, 8, 6 or 4 and length weight: 1, 2, 3, 4, 5 or 6. The Myers and Miller algorithms are also incorporated into the ALIGN program, which is part of the GCG sequence alignment software package. When the ALIGN program is used for comparing amino acid sequences, for example, PAM120 weight residue table, gap length penalty 12, and gap penalty 4 can be used. The polypeptide of the present invention may also have activities such as hematopoietic stem cell differentiation-regulating activity. The polypeptide of the present invention may also lack or have a signal sequence portion. The signal sequence portion is a portion corresponding to the 1st to 19th amino acids of the amino acid sequence represented by SEQ ID NO: 3.

The polynucleotide of the present invention is a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2 or a polynucleotide having a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 2. The polynucleotide having the nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 2 includes: (a) High stringent conditions for a polynucleotide comprising a complementary sequence of the nucleotide sequence represented by SEQ ID NO: 2 Polynu that hyprid under (

34 nucleotides, (b) significant nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 2 (eg, about 70% or more, preferably about 80% or more, more preferably about 90% or more) Even more preferably about 95% or higher, most preferably about 9 7%, 98% or 99% or higher nucleotide sequence identity). The high crystallization conditions under high stringency conditions are: 6 XSSC (sodium chloride / sodium cirate) / 45 ° C after high hydration, 0.2 XSSC / 0.1% SDS / 50 ~ 6 5. One or more washings with C may be mentioned. Nucleotide sequence identity (%) can be determined in the same manner as amino acid sequence identity (%). The polynucleotide of the present invention may also lack or have a signal sequence portion. The signal sequence portion is a portion corresponding to the first to 57th nucleotides of the nucleotide sequence represented by SEQ ID NO: 2.

The present invention also provides various inventions related to the polypeptides and polynucleotides of the present invention. Such an invention includes an antibody against the polypeptide of the present invention (for example, a monoclonal antibody, a polyclonal antibody) and its producing cells (ie, a hybridoma), a polynucleotide encoding the polypeptide, and a polynucleotide. Expression vectors, transformants into which expression vectors have been introduced, novel F gf 2 1 inhibitors (eg, antisense nucleic acids, ribozymes, RNA i-inducible nuclear acids, dominant negative mutants), novel F gf 2 1 Reagents (eg, nucleic acid probes, primer pairs or 3 or more primer sets), novel F gf 2 1 transgenic animals and cells (eg, animals and cells overexpressing new F gf 2 1, and new Animals and cells in which the expression or function of F gf 21 is suppressed, ie knockout animals and cells) etc. And the like. These series of inventions can be made by the methods disclosed herein and other methods known in the art. The contents of all publications, including patents and patent application specifications mentioned in the detailed text, are incorporated herein by reference to the same extent as if all were explicitly stated. It is what f

The following examples further illustrate the present invention in more detail, but the present invention is not limited to the following examples. Example

Example 1: Isolation and structural analysis of zebrafish F g f 21c cNA

Using the amino acid sequence of human F gf 21 as an index, the zebrafish DNA database (genomic DNA, ESTDNA) (GenBank: http://www.ncbi.nlra.nih.gov/, Emsenble: http: / A homology search was performed using /ww.ensemble.org/) to find a zebrafish gene fragment expected to encode Fg 2. Furthermore, from these sequences, all translation sequences of F gf 2 1 (primers that can amplify I (5,-cttagtcagtcatgctttttg cc—3, (酉自歹 (J number 4), 5 '— tgctaatgtaaagcatgtctt-3' (sequence No. 5)) was prepared, and the F gf 21 cDNA was amplified using the zebrafish cDNA as a template by the PCR method, and then the amplified F gf 21 cDNA was cloned. The nucleotide sequence (SEQ ID NO: 2) of the entire translation region of 2 1 cDNA was determined.

As a result, it was revealed from the base sequence (SEQ ID NO: 2) of the isolated zebrafish F g 2 21 cDNA that F gf 2 1 consists of 194 amino acid residues (SEQ ID NO: 3). The amino acid sequence (SEQ ID NO: 3) was found to have the highest homology (3.3.3% amino acid sequence identity) with human F gf 21 among known human proteins ( Figure 1 A). In addition, the human F gf 2 1 gene has a force S that is close to carbonic anhydrase XI (CA 1 1) and dehydrogenase / reductase member 10 (DHR S 1 0) on the chromosome S (LocusLink: http: // www. ncbi. nlm. nih. gov / LocusLink /), the zebrafish F g ί 2 1 gene was also in close proximity to C a 1 1 and Dh rs 1 0 on the zebrafish chromosome (Enzemble Zebrafish Genome Brower: (http: // wiv. ensemble.org/) (Figure 1 J3.

Based on the above, this gene was confirmed to be the zebrafish F gf 21 gene. It was.

Example 2: Function suppression experiment of F g f 21 by introducing F g f 21 MO

It has been clarified that the target gene can be effectively and specifically inhibited by introducing the target gene MO into the fertilized egg of zebrafish (Nasevicius, A. and Ekker, SC (2000) Nat. Genet. 26, 153-60). Therefore, we introduced F g f 2 1 MO into fertilized eggs to suppress the function of F g f 2 1 and examined its phenotype. Specifically, a morpholino-modified antisense oligo having a sequence (5, 1 GGCAAAAAGCATGACTGACTAAGCT-3, (SEQ ID NO: 6), 25 bases) complementary to the 5 'untranslated sequence of F gf 2 1 mRNA and a part of the translated sequence. Nucleotide (MO) was introduced into a fertilized egg (10 ng g fertilized egg), and the phenotype of the embryo in which the function of F gf 21 was suppressed was analyzed. As a control experiment, control MO (5 '— GGAAATAAG CATCACTGAGTAACCT— 3' (SEQ ID NO: 7), 25 bases) in which 25 bases of F gf 21 MO were replaced with other bases was used. Phenotypes were similarly analyzed in the introduced embryos (1 Ong / fertilized egg). Furthermore, as a functional recovery experiment, F gf 21 MO and F gf 21 mRNA were simultaneously introduced into fertilized eggs (F gf 21 MO: 10 ng / fertilized egg, F gf 21 mRNA: 10 p gZ fertilized egg), and phenotype Was analyzed. F g f 21 mRNA was prepared by incorporating the translation region of F g f 21 cDNA into a pCS2 + vector and using it as a cage.

Red blood cells were observed for embryo phenotype analysis. The observation of erythrocytes was performed by removing the egg membranes of zebrafish embryos 24 hours after fertilization, immersing them in o-dianisidine solution for 15 minutes in the dark and adding H ₂ 0 ₂ to develop the color.

As a result, it was confirmed that the F gf 2 1-introduced fetus had curvature of the body axis (Fig. 2A) and disappearance or reduction of red blood cells (Fig. 2B, Table 1). As a control experiment, almost no abnormalities were observed in embryos introduced with control MO (Table 1). Furthermore, as a functional recovery experiment, embryos introduced with F gf 2 1 MO and F gf 2 1 mRNA simultaneously reduced the rate of disappearance or reduction of red blood cells seen when F gf 2 1 MO was introduced alone (Table). 1). I

37

(table 1 )

From the above, it was considered that the disappearance of erythrocytes confirmed by the introduction of F g f 21 M was specific to the function suppression of F g f 21.

Example 3: Examination of the role of F g f 21 in angiogenesis

There are two possible causes for the disappearance or decrease of red blood cells in F g f 2 1 function-suppressed embryos: 1) no blood flow due to abnormal angiogenesis, or 2) no red blood cells produced. Therefore, in order to clarify the cause of the disappearance or decrease of red blood cells, we first examined the angiogenesis of F g f 21 function-suppressed embryos.

Dextran labeled with the fluorescent dye FITC was introduced into the vein of a zebrafish embryo (48 hours after fertilization), and angiogenesis was observed with a fluorescence microscope. The expression of the vascular endothelial cell marker gene, f1k1, was observed by whole mount in situ hybridization (Koshida, S. et al. (1998) Dev. Biol. 244, 9-20). Embryos 24 hours after fertilization were fixed with 4% paraformaldehyde, and the digoxigenin-labeled flklcRNA probe was hyperpresed.

As a result, the angiogenesis of the F g f 2 1 function-suppressed embryo was normal (FIG. 3A). In addition, the expression of the vascular endothelial cell marker, flkl (Thisse, C. and Zon, L.I. (2002) Science 295, 457-462) was normal (Fig. 3B).

Based on the above, it was considered that the disappearance or decrease of red blood cells in F g f 2 1 function-suppressed embryos was not due to abnormal angiogenesis but to abnormal red blood cell production.

Example 4: Examination of the role of F g f 21 in the blood cell development process

Red blood cells such as zebrafish are nucleated, unlike mammalian red blood cells, but the basic process of blood cell development is well conserved among vertebrates (Thisse and Zon, 2002; Davidson and Zon, 2004). That is, pattern formation of the mesoderm occurs in the early gastrulation stage of development, and hemangioplast is a progenitor cell common to blood cells and vascular endothelial cells. The last is generated. Furthermore, as a blood cell lineage, it differentiates into hematopoietic stem cells and differentiates into various blood cells such as erythrocytes, myeloids, and lymphocytes (Fig. 4) (Thisse and Zon, 2002; Davidson and Zon, 2004). These hematopoietic processes progress in a tissue called the intermediate cell mass (ICM) in the zebrafish fetus. Therefore, we investigated which stage of F gf 2 1 is involved in blood cell development.

The expression of hematopoietic cell marker genes in zebrafish embryos at various developmental stages was observed by whole mount in situ hybridization. Embryos were fixed with 4% paraformaldehyde and hybridized with a digoxigenin labeled marker cRNA probe. Various hematopoietic cell markers include sc 1 (hemangioblast), gata 2 (hematopoietic stem cell), gata 1 (erythrocyte-one myeloid progenitor cell), mpx (myeloid), ikaros (lymphocyte progenitor cell) (Thisse , C. and Zon, LI (2002) • Science 295, 457-462).

As a result, in the lateral plate mesoderm that will form I CM in the future, the expression of hemangioblast's major gene, sc1, was normal in the Fgf21 function-suppressed embryo (Fig. 5A). In addition, the expression of the hematopoietic stem cell marker gene, gata2, was normal (Fig. 5B). The expression of the erythrocyte one myeloid progenitor cell marker gene, gatal, and also the myeloid marker gene, mpX, disappeared (Figures 5C and D). Therefore, it was clarified that F g f 21 plays an essential role in the differentiation from hematopoietic stem cells to erythrocyte monoeloid progenitor cells. On the other hand, the expression of the lymphocyte progenitor cell marker gene, ikarose, increased (Fig. 5E).

From the above, it was shown that suppression of the function of F g f 21 inhibits the differentiation of erythrocyte-myeloid precursor cells from hematopoietic stem cells and increases lymphocyte progenitor cells.

(Conclusion)

This study revealed that F gf 21 has an important function in erythropoiesis. F gf 21 acts on hematopoietic stem cells and was found to be a hematopoietic factor that plays an important role in the generation of erythrocyte myeloid progenitor cells (Fig. 6). Erythropoietin, well known as a hematopoietic factor, acts on erythroid progenitors and increases it. Promotes growth and differentiation into red blood cells (Figure 6). Therefore, F gf 21 is expected to be clinically applied as a new hematopoietic factor having a mechanism of action different from that of erythropoietin. Industrial applicability

The agent of the present invention may be useful for regulating differentiation of hematopoietic stem cells and preventing or treating certain diseases such as hematopoietic diseases and immune diseases. The differentiation regulation method of the present invention is useful for regulating differentiation of hematopoietic stem cells into CMP cells, CLP cells or their differentiated cells, particularly regulation of differentiation of hematopoietic stem cells into CMP cells. The method for determining differentiation efficiency according to the present invention comprises a method for regulating the expression or function of F gf 21 in the treatment of patients with diseases caused by abnormalities in blood cells (eg, hematopoietic diseases, immune diseases). This is useful because it allows prediction of treatment effects in patients. The screening method of the present invention is useful for development of medicines for diseases caused by abnormal blood cells. The determination method and diagnostic agent of the present invention are useful for evaluating the onset or risk of developing diseases caused by abnormal blood cells. This application is based on Japanese Patent Application No. 2 0 0 5-0 7 0 0 7 2 filed in Japan (Filing Date: March 1, 1, 2005), the contents of which are hereby incorporated by reference. It is included.

Claims

The scope of the claims

1. A hematopoietic stem cell differentiation regulator comprising a substance that regulates the expression or function of F g f 21.

2. The agent according to claim 1, wherein the substance that regulates the expression or function of F g f 21 is a substance that promotes the expression or function of F g f 21.

3. The agent according to claim 2, wherein the substance that promotes the expression or function of F g f 21 is F g f 21 or its expression vector.

4. The agent according to claim 1, wherein the substance that regulates the expression or function of F g f 21 is a substance that suppresses the expression or function of F g f 21.

5. The substance that suppresses the expression or function of F gf 21 is selected from the group consisting of an antisense nucleic acid, a ribozyme, an RNA i-inducible nucleic acid, a targeting vector, an antibody, and a dominant negative mutant. 4. The agent according to 4.

6. The agent according to claim 2, which is an agent for promoting differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells or an inhibitor of differentiation of hematopoietic stem cells into lymphocyte lineage cells.

7. The agent according to claim 2, which is an agent for promoting the differentiation of hematopoietic stem cells into erythroid lineage cells.

8. The agent according to claim 4, which is an agent for inhibiting the differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells, or an agent for promoting the differentiation of hematopoietic stem cells into lymphocyte lineage cells.

9. The agent according to claim 1, which is a prophylactic or therapeutic agent for hematopoietic disease, immune disease or allergic disease.

10. A method for regulating the differentiation of hematopoietic stem cells, comprising culturing hematopoietic stem cells in a medium in the presence of a substance that regulates the expression or function of F g f21.

1 1. The method according to claim 10, wherein the substance that regulates the expression or function of F g f 21 is F g f 21.

1 2. The method according to claim 10, wherein the method further comprises isolating an erythrocyte-myeloid lineage cell or a lymphocyte lineage cell that has been induced to differentiate from a hematopoietic stem cell. .

1 3. Further differentiation of erythrocyte-myeloid lineage cells induced to differentiate from hematopoietic stem cells I

41. The method of claim 10, further comprising:

14. Culturing hematopoietic stem cells in medium in the presence of F gf 21 and assessing the differentiation efficiency of hematopoietic stem cells into erythrocyte-myeloid or lymphocyte lineage cells. A method for determining the differentiation efficiency of stem cells.

1 5. A method for screening a substance capable of regulating the differentiation of hematopoietic stem cells, comprising evaluating whether a test substance can regulate the expression of F g f 21.

1 6. The method according to claim 15, comprising the following steps (a) to (c):

(a) contacting the test substance with a cell capable of measuring F g f 21 expression;

(c) A step of selecting a test substance that regulates the expression level of F g f 21 based on the comparison result of (b) above.

1 7. The method according to claim 15, comprising the following steps (a) to (c):

(a) administering a test substance to an animal;

(b) measuring the expression level of F g f 21 in an animal administered with the test substance, and comparing the expression level with the expression level of F g f 21 in a control animal not administered with the test substance;

18. A method for identifying an F g f 21 polymorphism that causes a change in the differentiation regulating ability of a hematopoietic stem cell, comprising analyzing the effect of a specific polymorphism of F g f 21 on the differentiation regulating ability of a hematopoietic stem cell.

1 9. Measure the expression level and Z or polymorphism of F gf 21 using biological samples collected from animals, and evaluate the animal's biological condition for hematopoietic stem cell differentiation based on the measurement results. For determining the biological state of an animal against

20. A diagnostic agent for the biological state of an animal for hematopoietic stem cell differentiation, comprising a reagent for measuring the expression level of F gf 21 and / or a polymorphism.

2 1. Kit including (a) and (b) below:

(a) a substance that modulates the expression or function of F g f 2 1; and

(b) a reagent for identifying hematopoietic stem cells or differentiated cells thereof, and a reagent for regulating differentiation of Z or hematopoietic stem cells.

22. A method for regulating differentiation of hematopoietic stem cells, comprising administering an effective amount of a substance that regulates the expression or function of F g f 21 to a subject.

23. Use of a substance that modulates the expression or function of F g f 21 in the production of a hematopoietic stem cell differentiation regulator.