JP2006246823A - Use of fgf21 as hematopoietic factor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide new medicines and reagents relating to Fgf21 and the useful means for developing them. <P>SOLUTION: This invention provides a differentiation controlling agent for hematopoietic stem cells including a substance controlling the expression or the function of Fgf21: a method for controlling the differentiation of hematopoietic stem cells by Fgf21 and a method for judging the efficiency of the differentiation: a screening method of judging whether the substance can control the differentiation of the hematopoietic stem cells or not, including that the test substance can control the expression or the functions of Fgf21 or not: the identification method for the polymorphic Fgf21 that causes the change in the differentiation control ability of hematopoietic stem cell: the judging method and the diagnostic agent for animal live body conditions to the differentiation of hematopoietic stem cells: a kit including a substance controlling the expression or the function of Fgf21. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention provides a hematopoietic stem cell differentiation regulator, a hematopoietic stem cell differentiation regulation method, a screening method for a substance capable of regulating hematopoietic stem cell differentiation, and the like.

  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 through hemangioblast, hematopoietic stem cells, and erythroid progenitor cells. The search for hematopoietic factors contributing to this developmental process and the analysis of their functions are thought to contribute to the elucidation, prevention and treatment of various hematopoietic diseases.

  Zebrafish are fertilized and developed outside the mother's body, their development is fast, and the embryo is transparent throughout the development period. In addition, experimental developmental analysis by introduction of foreign genes, cell transplantation, etc. can be easily performed. Moreover, 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 clarified that the blood cell differentiation process is well preserved from zebrafish to humans. Therefore, zebrafish is an excellent model animal for examining human blood cell differentiation processes (Non-patent Documents 1-2).

  It has been clarified that fibroblast growth factors (FGFs) currently exist in humans in 22 types from the homology of amino acid sequences. It is known that its actions are not only proliferative activity against fibroblasts but also deeply involved in various life phenomena such as morphogenesis, angiogenesis, tumor formation, wound healing, nerve survival maintenance, and metabolic regulation. (Nonpatent literature 3-4). The present inventors have previously identified human Fgf21 (Non-patent Document 5). However, its function is still unknown.

Thisse, C. and Zon, L.I. (2002) Science 295, 457-462. Davidson, A.J. and Zon, L.I. (2004) Oncogene 23, 7233-7246. Ornitz, D.M. and Itoh, N. (2001) Genome Biol. 21, REVIEW3005. Itoh, N. and Ornitz, D.M. (2004) Trends Genet. 20, 563-569. Nishimura, T. et al. (2000) Biochim. Biophys. Acta 1492, 203-206.

  Functional analysis of genes leads to the development of drugs or reagents having new action mechanisms for various diseases. The present invention provides a drug or a reagent having a new action mechanism for various diseases based on the knowledge obtained by functional analysis of the Fgf21 gene, and a useful means for developing a drug or a reagent. The purpose is to provide.

  The present inventors suppressed the function of Fgf21 in zebrafish and analyzed its trait. As a result, the erythrocytes disappeared or decreased in the zebrafish, and the disappearance or decrease of erythrocytes was caused by the erythrocyte-bone marrow of hematopoietic stem cells. It was found that the differentiation into sphere lineage cells 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 Fgf21 in zebrafish. Therefore, Fgf21 differentiates hematopoietic stem cells into erythrocyte-myeloid lineage cells (for example, erythrocytes, megakaryocytes, eosinophils, neutrophils, basophils, monocytes, dendritic cells) or lymphocytes of hematopoietic stem cells. It is considered that differentiation into sphere lineage cells (for example, T cells, B cells, NK cells) can be controlled. In addition, the substance that regulates the expression or function of Fgf21 has the ability to regulate the differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphocyte lineage cells and / or diseases caused by abnormalities in blood cells (for example, hematopoiesis) It is considered that it may be useful for development of medicines and research reagents for 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 Fgf21;
[2] The agent according to [1] above, wherein the substance that regulates the expression or function of Fgf21 is a substance that promotes the expression or function of Fgf21;
[3] The agent according to [2] above, wherein the substance that promotes the expression or function of Fgf21 is Fgf21 or an expression vector thereof;
[4] The agent according to [1] above, wherein the substance that regulates the expression or function of Fgf21 is a substance that suppresses the expression or function of Fgf21;
[5] The agent according to [4] above, wherein the substance that suppresses the expression or function of Fgf21 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;
[6] The agent according to [2] above, 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 [2] above, which is an agent for promoting differentiation of hematopoietic stem cells into erythroid lineage cells;
[8] The agent of [4] above, which is an agent for suppressing differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells, or an agent for promoting differentiation of hematopoietic stem cells into lymphocyte lineage cells;
[9] The agent according to [1] above, which is a prophylactic 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 Fgf21;
[11] The method of [10] above, wherein the substance that regulates the expression or function of Fgf21 is Fgf21;
[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 of [10] above, further comprising further differentiating erythrocyte-myeloid lineage cells that have been induced to differentiate from hematopoietic stem cells;
[14] The differentiation efficiency of hematopoietic stem cells by Fgf21, comprising culturing hematopoietic stem cells in a medium in the presence of Fgf21 and evaluating the differentiation efficiency of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphocyte lineage cells. Judgment method;
[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 Fgf21;
[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 Fgf21 expression;
(B) measuring the expression level of Fgf21 in a cell contacted with a test substance and comparing the expression level with the expression level of Fgf21 in a control cell not contacted with the test substance;
(C) a step of selecting a test substance that regulates the expression level of Fgf21 based on the comparison result of (b) above;
[17] The method according to [15] above, comprising the following steps (a) to (c):
(A) administering a test substance to an animal;
(B) measuring the expression level of Fgf21 in an animal administered with the test substance, and comparing the expression level with the expression level of Fgf21 in a control animal not administered with the test substance;
(C) a step of selecting a test substance that regulates the expression level of Fgf21 based on the comparison result of (b) above;
[18] A method for identifying an Fgf21 polymorphism that causes a change in the ability to regulate differentiation of hematopoietic stem cells, comprising analyzing the influence of a specific polymorphism of Fgf21 on the ability to regulate differentiation of hematopoietic stem cells;
[19] An animal for hematopoietic stem cell differentiation, comprising measuring the expression level and / or polymorphism of Fgf21 using a biological sample collected from the animal, and evaluating the biological state of the animal for hematopoietic stem cell differentiation based on the measurement result Method for determining the biological state of
[20] A diagnostic agent for the biological state of an animal for hematopoietic stem cell differentiation, comprising a reagent for measuring the expression level and / or polymorphism of Fgf21;
[21] Kit including the following (a) and (b):
(A) a substance that regulates the expression or function of Fgf21; and (b) a reagent for identifying hematopoietic stem cells or differentiated cells thereof and / or a reagent for regulating differentiation of differentiated cells of hematopoietic stem cells.

  The agent of the present invention may be useful for regulating differentiation of hematopoietic stem cells and for preventing or treating predetermined diseases such as hematopoietic diseases and immune diseases. The method for regulating differentiation of the present invention is useful for regulating differentiation of hematopoietic stem cells into CMP cells, CLP cells or their differentiated cells, in particular, regulating the differentiation of hematopoietic stem cells into CMP cells. The method for determining differentiation efficiency of the present invention is a method for predicting the therapeutic effect of a substance that regulates the expression or function of Fgf21 in the treatment of a patient with a disease (eg, hematopoietic disease, immune disease) caused by abnormal blood cells. It is useful for making possible. 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.

(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 Fgf21.

  Hematopoietic stem cells differentiate into common myeloid progenitor cells (CMP cells) or lymphocyte progenitor cells (CLP cells). CMP cells differentiate into granulocyte / macrophage progenitor cells (GMP cells) or megakaryocyte / erythroid progenitor cells (MEP cells), and GMP cells are further differentiated into bone marrow. It differentiates into eosinophils, neutrophils, basophils, monocytes, etc. via myelocytic cells or monocytic cells, and MEP cells further differentiate into erythroblasts or megakaryocytes. On the other hand, CLP cells are T-cell progenitor cells (CTP cells), B-cell progenitor cells (CBP cells) or NK progenitor cells (NKP cells). ). The present inventors are able to promote / suppress a substance that promotes / suppresses the expression or function of Fgf21 into a hematopoietic stem cell into a CMP cell or a differentiated cell thereof (eg, Gata1-positive cell, mpx-positive cell), In addition, it has been found that the differentiation of hematopoietic stem cells into CLP cells or differentiated cells thereof (for example, Ikaros positive cells) can be suppressed / promoted.

As used herein, the following terminology “lineage cells” is used where appropriate.
The “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, “erythrocyte lineage cell” comprehensively means CMP cells, MEP cells, erythroblasts, and erythrocytes. The “megakaryocyte lineage cell” comprehensively means CMP cells, MEP cells, and megakaryocytes. “Myeloid lineage cell” generically means CMP cells, GMP cells, myeloid cells, monocytic cells, eosinophils, neutrophils, basophils, monocytes.

  “Lymphocyte lineage cell” generically means CLP cells and differentiated cells thereof, and includes T cell lineage cells, B cell lineage cells, and NK cell lineage cells. Here, “T cell lineage cells” comprehensively mean CLP cells, CTP cells, and T cells. “B cell lineage cell” comprehensively means CLP cells, CBP cells, and B cells. “NK cell lineage cells” comprehensively mean CLP cells, NKP cells, and NK cells.

  Fgf21 refers to human Fgf21 (see, for example, GenBank accession number: AB021975) or orthologs thereof, or mutants thereof (including SNPs and haplotypes). The orthologue of Fgf21 is not particularly limited, and is derived from any animal, for example, fish, birds, mammals (eg, cows, sheep, pigs, goats, monkeys, rabbits, rats, hamsters, guinea pigs, mice). obtain. Fgf21 is a secreted protein, and a signal sequence (for example, corresponding to the 1st to 28th amino acid residues in the amino acid sequence shown in SEQ ID NO: 1) can be removed by processing. In the method of the present invention, as Fgf21, any of those from which the signal sequence has been removed and those from which the signal sequence has not been removed can be used, but those from which the signal sequence has been removed are preferred.

  In one embodiment, the substance that modulates Fgf21 expression or function may be a substance that promotes Fgf21 expression.

  The expression of Fgf21 means that a translation product (ie, protein) from Fgf21 is produced and functionally localized at the site of action. Therefore, the substance that promotes the expression of Fgf21 may act at any stage of Fgf21 transcription, post-transcriptional regulation, translation, post-translational modification, localization, and protein folding. As used herein, promotion of Fgf21 expression includes supplementation with Fgf21 (protein).

  An example of a substance that promotes the expression of Fgf21 can be Fgf21 or an expression vector containing a nucleic acid encoding Fgf21.

  Fgf21 can be a natural protein or a recombinant protein. Fgf21 can be prepared by a method known per se, for example, a) Fgf21 may be recovered from a biological sample (eg, blood) containing Fgf21, and b) a host cell (eg, Escherichia, Bacillus, Yeast, insect cells, insects, animal cells) may be used to prepare a transformant by introducing an Fgf21 expression vector (described later), and Fgf21 produced by the transformant may be recovered. C) rabbit reticulocyte Fgf21 may be synthesized by a cell-free system using lysate, wheat germ lysate, E. coli lysate or the like. Fgf21 is a method that utilizes solubility such as salting out and solvent precipitation; a method that mainly uses a difference in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; A method utilizing charge difference such as exchange chromatography; a method utilizing specific affinity such as affinity chromatography and use of Fgf21 antibody; a method utilizing hydrophobic difference such as reverse phase high performance liquid chromatography; etc. A method using a difference in isoelectric point such as an electric point electrophoresis method;

  In another embodiment, the substance that modulates the expression or function of Fgf21 can be a substance that suppresses the expression of Fgf21. The substance that suppresses the expression of Fgf21 may act at any stage such as transcription of Fgf21, post-transcriptional regulation, translation, post-translational modification, localization, and protein folding.

  An example of a substance that suppresses the expression of Fgf21 is an antisense nucleic acid against a transcription product of Fgf21, specifically, mRNA or an initial transcription product. The antisense nucleic acid is composed of a base sequence capable of hybridizing with the target mRNA (initial transcript) under physiological conditions of a cell expressing the target mRNA (initial transcript), and the target mRNA in the hybridized state. A nucleic acid capable of inhibiting translation of a polypeptide encoded by (early transcript). The type of the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. In addition, since the phosphodiester bond of a natural type antisense nucleic acid is easily degraded by a nucleolytic enzyme present in cells, the antisense nucleic acid of the present invention has a thiophosphate type (phosphorus) that is stable to the degrading enzyme. It is also possible to synthesize using modified nucleotides such as P′O of acid bond and P═S) or 2′-O-methyl type. Other important factors for the design of antisense nucleic acid include enhancement of water solubility and cell membrane permeability. These can also be overcome by devising dosage forms such as the use of liposomes and microspheres. The length of the antisense nucleic acid is not particularly limited as long as it can specifically hybridize with the transcription product of Fgf21. The short one is about 15 bases, and the long one is complementary to the entire mRNA (initial transcription product) sequence. The sequence may contain 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. Furthermore, antisense nucleic acids not only hybridize with Fgf21 transcripts and inhibit translation, but also bind to double-stranded DNA to form triplex and inhibit transcription into mRNA. It may be a thing.

  Another example of a substance that suppresses the expression of Fgf21 is to specifically cleave the transcription product of Fgf21, specifically mRNA or the initial transcription product, within the coding region (including the intron portion in the case of the initial transcription product). To obtain ribozyme. Ribozyme refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that an oligo DNA having a base sequence of the enzyme active site also has a nucleic acid cleaving activity. As long as it has specific nucleic acid cleavage activity, it is used as a concept including DNA. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. Further, when the ribozyme is used in the form of an expression vector containing DNA encoding the ribozyme, it can be a hybrid ribozyme in which a sequence modified with tRNA is further linked in order to promote the transfer to the cytoplasm [ Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

  Yet another example of a substance that suppresses the expression of Fgf21 is an RNAi-inducible nucleic acid. An RNAi-inducible nucleic acid refers to a nucleic acid that can induce an RNAi effect when introduced into a cell, and is preferably RNA. The RNAi effect refers to 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 RNAi 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 chain provided by a single RNA stem-loop structure. Examples of the RNAi-inducible nucleic acid include siRNA, stRNA, miRNA and the like.

  Another example of a substance that suppresses the expression of Fgf21 is a targeting vector. The targeting vector used in the present invention includes a first polynucleotide and a second polynucleotide homologous to the Fgf21 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 Fgf21. When the genomic DNA containing the Fgf21 gene lacks a genomic DNA partial region existing between two regions homologous to the first and second polynucleotides, the first and second polynucleotides Selected to result in a functional deficiency. Selectable markers include positive selectable markers (eg, neomycin resistance gene, hygromycin B phosphotransferase (BPH) gene, blasticidin S deaminase gene, puromycin resistance gene), negative selectable markers (eg, herpes simplex virus (HSV)). Thymidine kinase (tk) gene, diphtheria toxin A fragment (DTA) gene) and the like. The targeting vector can include either a positive selection marker, a negative selection marker, or both. The targeting vector may also include two or more recombinase target sequences (eg, loxP sequence used in the Cre / loxP system derived from bacteriophage P1, FRT sequence used in the FLP / FRT system derived from yeast).

  In another embodiment, the substance that modulates the expression or function of Fgf21 may be a substance that suppresses the function of Fgf21. The substance that suppresses the function of Fgf21 is not particularly limited as long as it is a substance that can interfere with the action of Fgf21. Examples thereof include an antibody against Fgf21, a dominant negative mutant of Fgf21, and an expression vector containing a nucleic acid encoding them.

The antibody against Fgf21 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 Fgf21.

  For example, a polyclonal antibody can be obtained by using a commercially available adjuvant (for example, Fgf21 or a fragment thereof (if necessary, a complex cross-linked to a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin)) as an antigen. And complete or incomplete Freund's adjuvant) administered subcutaneously or intraperitoneally 2-4 times every 2 to 3 weeks (the antibody titer of the partially collected serum is measured by a known antigen-antibody reaction, and its rise It can be obtained by collecting whole blood about 3 to about 10 days after the final immunization 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 be obtained by cell fusion methods (for example, Takeshi Watanabe, Principles of Cell Fusion Methods and Production of Monoclonal Antibodies, Akira Taniuchi, Toshitada Takahashi, “Monoclonal Antibodies and Cancer: Basic and Clinical”, 2-14). Page, Science Forum Publishing, 1985). For example, the factor is administered to a mouse subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and about 3 days after the final administration, the spleen or lymph node is collected and white blood cells are collected. This leukocyte and myeloma cells (for example, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the factor. The cell fusion may be PEG method [J. Immunol. Methods, 81 (2): 223-228 (1985)] or voltage pulse method [Hybridoma, 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 hybridoma producing the monoclonal antibody can be cultured 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 antibody of the present invention may be a chimeric antibody, a humanized or humanized antibody. Chimeric antibodies include, for example, “Experimental Medicine (Special Issue), Vol. 6, No. 10, 1988”, Japanese Patent Publication No. 3-73280, and humanized antibodies include, for example, Japanese Patent Publication No. 4-506458, JP-A-62-296890 and the like, human antibodies include, for example, `` Nature Genetics, Vol.15, p.146-156, 1997 '', `` Nature Genetics, Vol.7, p.13-21, 1994 '', JP 4-504365 Publication, International Application Publication No. WO 94/25585 Publication, “Nikkei Science, June, 40 to 50, 1995”, “Nature, Vol.368, p.856-859, 1994 ", Japanese Patent Publication No. 6-500233, and the like.

  The dominant negative mutant of Fgf21 refers to a substance whose activity is reduced by introducing a mutation into Fgf21. The dominant negative mutant can indirectly inhibit its activity by competing with natural Fgf21. The dominant negative mutant can be prepared by introducing a mutation into a nucleic acid encoding Fgf21. 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 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 acid, ribozyme, RNAi-inducible nucleic acid, targeting vector, antibody, and dominant negative mutant may be abbreviated as “inhibitory substance”.

  When the substance that regulates the expression or function of Fgf21 is a nucleic acid molecule or a protein molecule, the agent of the present invention can also contain an expression vector containing a nucleic acid molecule encoding the nucleic acid molecule or protein molecule as an active ingredient. In the expression vector, the oligonucleotide or polynucleotide encoding the nucleic acid molecule must be operably linked to a promoter capable of exhibiting promoter activity in a mammalian cell to be administered. The promoter to be used is not particularly limited as long as it can function in the mammal to be administered. For example, SV40-derived early promoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived LTR, adenovirus-derived early promoter Examples include viral promoters such as promoters, and mammalian constituent protein gene promoters such as β-actin gene promoters, PGK gene promoters, transferrin gene promoters, and the like.

  The expression vector preferably contains a transcription termination signal, ie a terminator region, downstream of the oligo (poly) nucleotide encoding the nucleic acid molecule. In addition, selectable marker genes for selection of transformed cells (such as genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, genes that complement auxotrophic mutations, etc.) You can also

  The vector may be a basic skeleton vector, a plasmid or a viral vector used as an expression vector, but suitable vectors for administration to mammals such as humans include adenovirus, retrovirus, adeno-associated virus, herpes virus, vaccinia virus. And viral vectors such as poxvirus, poliovirus, Sindbis virus, Sendai virus, Epstein-Barr virus, and the like.

  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 Fgf21. Examples of pharmaceutically acceptable carriers include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate and other excipients, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone. , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate , Aerosil, Talc, Sodium lauryl sulfate lubricant, Citric acid, Menthol, Glycyllysine / Ammonium salt, Glycine, Orange powder, Fragrance, Sodium benzoate Preservatives such as lithium, sodium hydrogen sulfite, methyl paraben, propyl paraben, stabilizers such as citric acid, sodium citrate, acetic acid, suspensions such as methyl cellulose, polyvinyl pyrrolidone, aluminum stearate, dispersants such as surfactants, Examples include, but are not limited to, water, physiological saline, diluents such as orange juice, base waxes such as cacao butter, polyethylene glycol, and white kerosene.

  Preparations suitable for oral administration include solutions in which an effective amount of a substance is dissolved in a diluent such as water or physiological saline, capsules, sachets or tablets containing an effective amount of the substance as a solid or granule. Examples thereof include a suspension in which an effective amount of a substance is suspended in a suitable dispersion medium, and an emulsion 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 infusion, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. Aqueous and non-aqueous sterile suspensions are also included, 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 dosage 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. The amount of active ingredient per day for an adult is about 0.001 to about 500 mg / 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 Fgf21, it is useful as an agent for promoting differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells. The present inventors have found that Fgf21 is a factor responsible for promoting differentiation of hematopoietic stem cells (eg, gata2 positive) into CMP cells (eg, gata1 positive). Therefore, it is considered that a substance that promotes the expression or function of Fgf21 can promote the differentiation of hematopoietic stem cells into CMP cells, thereby increasing the number of red blood cells, megakaryocytes, myelocytes, and the like. In order to increase the number of cells such as erythrocytes more efficiently, the agent of the present invention further promotes differentiation into erythrocytes (for example, erythropoietin), a substance that promotes differentiation into megakaryocytes (for example, thrombopoietin), bone marrow A substance that promotes differentiation into spheres (for example, granulocyte-macrophage colony-stimulating factor) may further be contained. In this case, the agent of the present invention can be used for diseases in which an increase in the number of cells such as red blood cells, platelets, and myeloid cells is desired. Examples of the disease for 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 the disease 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, after cancer chemotherapy and radiation therapy). Moreover, the agent of this invention can also be used for the purpose of the improvement of the biological function (for example, motor function) regarding oxygen conveyance.

  Further, when the agent of the present invention contains a substance that promotes the expression or function of Fgf21, it is useful as an agent for suppressing differentiation of hematopoietic stem cells into lymphocyte lineage cells. A substance that promotes the expression or function of Fgf21 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, thereby causing lymphocyte lineage It is thought that the number of 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 the disease for which a decrease in 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 Fgf21, it is useful as an agent for inhibiting the differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells. The present inventors have found that suppression of the function of Fgf21 suppresses differentiation of hematopoietic stem cells into CMP cells. Therefore, it is considered that a substance that suppresses the expression or function of Fgf21 can suppress the differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells, thereby reducing the number of erythrocytes, megakaryocytes, myelocytes, and the like. .

  Moreover, when the agent of the present invention contains a substance that suppresses the expression or function of Fgf21, it is useful as an agent for promoting differentiation of hematopoietic stem cells into lymphocyte lineage cells. A substance that suppresses the expression or function of Fgf21 indirectly promotes differentiation of hematopoietic stem cells into CLP cells as a result of inhibiting differentiation of hematopoietic stem cells into CMP cells, thereby increasing the number of lymphocyte lineage cells. It is thought to get. The agent of the present invention may further contain a substance that promotes differentiation into lymphocyte lineage cells in order to increase the number of lymphocyte lineage cells more efficiently. Examples of other substances that promote differentiation into lymphocyte lineage cells include interleukin-7. 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.

  When the modulator of the present invention contains other substances in addition to substances that promote the expression or function of Fgf21, they are combined together (for example, stored as a mixture in the same container) or separated from each other It can be provided in a form (eg, stored in a different container). Note that the modulator of the present invention can be used in vivo or in vitro.

(2. Hematopoietic stem cell differentiation regulation method)
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 Fgf21 expression or function.

  Hematopoietic stem cells can be cells derived from any animal, such as fish, birds, mammals (eg, cows, sheep, pigs, goats, monkeys, humans, rabbits, rats, hamsters, guinea pigs, mice). 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. The hematopoietic stem cell may further be a cell induced to differentiate from a pluripotent stem cell such as an embryonic stem cell or a somatic stem cell. 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 cells derived from the intended individual, cells suitable for allogeneic transplantation can be obtained. Hematopoietic stem cells can be obtained by a method known per se (for example, FACS) using a cell surface marker. Examples of cell surface markers for hematopoietic stem cells include Sca-1 and ckit.

  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, a minimal essential medium (MEM), Dulbecco's modified minimal essential medium (DMEM), F12 medium or An RPMI1640 medium or a mixed medium thereof as a basic medium. Examples of the additive 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 method for regulating differentiation of the present invention comprises culturing hematopoietic stem cells in a medium in the presence of Fgf21. The culture of hematopoietic stem cells in the presence of Fgf21 is not particularly limited as long as it results in culturing hematopoietic stem cells in a medium containing Fgf21. Therefore, Fgf21 may not be present in the medium at the start of culture. Examples of culturing hematopoietic stem cells in the presence of Fgf21 include culturing hematopoietic stem cells in a medium supplemented with Fgf21, culturing hematopoietic stem cells introduced with an Fgf21 expression vector, and co-culturing hematopoietic stem cells with Fgf21-expressing cells. However, culture of hematopoietic stem cells in a medium supplemented with Fgf21 is preferred.

  When culturing hematopoietic stem cells in a medium supplemented with Fgf21, Fgf21 added to the medium can be a natural protein or a recombinant protein. Fgf21 can be prepared by a method known per se.

  When culturing hematopoietic stem cells into which an Fgf21 expression vector has been introduced, the expression vector introduced into the hematopoietic stem cells can be the same as the above-described Fgf21 expression vector. 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 method using a lipid such as a liposome or a cationic lipid. Moreover, 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, a method known per se, for example, a method using a retrovirus, a method using a targeting vector capable of homologous recombination, or the like can be used.

  When co-culturing Fgf21-expressing cells and hematopoietic stem cells, the cells that 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 Fgf21 expression vector into a host cell, or a natural cell that expresses Fgf21. As natural cells that express Fgf21, cells derived from Fgf21-expressing tissues (eg, bone marrow, liver, thymus) can be used. The Fgf21-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 Fgf21-expressing cell can also be a cell derived from the same animal or the same individual as the hematopoietic stem cell. The co-culture may be performed when hematopoietic stem cells and Fgf21-expressing cells are in physical contact, or when these cells are present in the same culture system but separated by a partition wall capable of transferring substances, This includes cases where contact is not possible. The case where hematopoietic stem cells and Fgf21-expressing cells are present in the same culture system but separated by a partition wall that allows the passage of substances and cannot physically contact the cells themselves is, for example, a filter (for example, a culture cell culture) The case where both cells are cultured separately using an insert) is mentioned.

  In another embodiment, the method for regulating differentiation of the present invention comprises culturing hematopoietic stem cells in a medium so as to suppress the expression or function of Fgf21. Examples of such hematopoietic stem cell culture include culture of hematopoietic stem cells in a medium supplemented with an Fgf21 inhibitory substance (described above).

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

  The differentiation regulation method of the present invention can also include further differentiation of cells induced to differentiate from hematopoietic stem cells. The method for further differentiating the cells induced to differentiate from hematopoietic stem cells is not particularly limited. For example, hematopoietic stem cells are cultured in the presence of a substance that regulates the expression or function of Fgf21 and another differentiation regulator. And a method of using other differentiation regulators after culturing hematopoietic stem cells in the presence of a substance that regulates the expression or function of Fgf21. 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, the substances and materials used in the culture are preferably unified with substances and materials derived from the same species as the subject undergoing cell therapy. The cells used in the culture are preferably cells derived from the same species as the subject undergoing cell therapy from the viewpoint of allogeneic transplantation, but from the viewpoint of allogeneic transplantation, it is more preferable to use cells derived from the subject. preferable.

(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 Fgf21. The determination method of the present invention includes, for example, culturing hematopoietic stem cells in a medium in the presence of Fgf21, and evaluating the differentiation efficiency 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 desired to determine the differentiation efficiency of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphocyte lineage cells. Hematopoietic stem cells collected from a subject (eg, a subject under consideration for treatment with a substance that modulates Fgf21 expression or function) can be used.

  Although the culture of hematopoietic stem cells in the medium in the presence of Fgf21 can be performed in the same manner as the differentiation regulation method described above, the culture of hematopoietic stem cells in a medium supplemented with Fgf21 is preferred.

  Evaluation of differentiation efficiency of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphocyte lineage cells can be performed by a method known per se. For example, whether or not the differentiation efficiency of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphocyte lineage cells is sufficient to affirm the usefulness of treatment using a substance that modulates the expression or function of Fgf21 Can be evaluated.

  This determination method can be useful, for example, in predicting the effect of treatment using a substance that modulates the expression or function of Fgf21 in a given patient and determining whether or not to adopt the treatment in the patient. .

(4. Screening method for substances capable of regulating the differentiation of hematopoietic stem cells)
The present invention also includes a method for screening a substance capable of regulating differentiation of hematopoietic stem cells, comprising evaluating whether a test substance can regulate the expression or function of Fgf21, and a substance obtained by the screening method, and Provided is a hematopoietic stem cell differentiation regulator comprising the substance.

  The test substance to be subjected to the screening method may be any known compound or novel compound, for example, a nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, compound prepared using combinatorial chemistry technology Examples include libraries, random peptide libraries prepared by solid phase synthesis and 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 Fgf21 expression;
(B) measuring the expression level of Fgf21 in a cell contacted with a test substance and comparing the expression level with the expression level of Fgf21 in a control cell not contacted with the test substance;
(C) A step of selecting a test substance that regulates the expression level of Fgf21 based on the comparison result of (b).

  In step (a) of the above method, the test substance is placed in contact with cells capable of measuring Fgf21 expression. Contact of the test substance with cells capable of measuring Fgf21 expression can be performed in a culture medium.

  The cell capable of measuring the expression of Fgf21 refers to a cell capable of directly or indirectly evaluating the expression level of a product of Fgf21, for example, a transcription product or a translation product. A cell capable of directly evaluating the expression level of the Fgf21 product may be a cell capable of naturally expressing Fgf21, whereas a cell capable of indirectly evaluating the expression level of the Fgf21 product may be an Fgf21 transcriptional regulatory region. Cells that allow reporter assays for. The cell capable of measuring the expression of Fgf21 can be an animal cell, for example, a mammalian cell such as a mouse, rat, hamster, guinea pig, rabbit, dog, monkey or human.

  Cells that can naturally express Fgf21 are not particularly limited as long as they 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 a reporter assay for the Fgf21 transcriptional regulatory region is a cell that contains an Fgf21 transcriptional regulatory region and a reporter gene operably linked to the region. The Fgf21 transcription regulatory region and reporter gene can be inserted into an expression vector. The Fgf21 transcriptional regulatory region is not particularly limited as long as it is a region that can control the expression of Fgf21. For example, a region from the transcription start point to about 2 kbp upstream, or one or more bases in the nucleotide sequence of this region is deleted. Examples thereof include a region consisting of a substituted or added base sequence and having the ability to control the transcription of Fgf21. The reporter gene may be any gene that encodes a detectable protein or an enzyme that produces a detectable substance. For example, the GFP (green fluorescent protein) gene, GUS (β-glucuronidase) gene, LUC (luciferase) gene, CAT (Chloramphenicol acetyltransferase) gene and the like.

  A cell into which a Fgf21 transcription regulatory region and a reporter gene operably linked to the region are introduced can be used as long as the Fgf21 transcription regulatory function can be evaluated, that is, as long as the expression level of the reporter gene can be quantitatively analyzed. It is not limited. However, since a physiological transcriptional regulatory factor for Fgf21 is expressed and considered to be more appropriate for the evaluation of the expression regulation of Fgf21, a cell capable of naturally expressing Fgf21 is preferable as the introduced cell.

  The medium in which the test substance is brought into contact with the cells capable of measuring the expression of Fgf21 is appropriately selected according to the type of cells used. For example, a minimal essential medium containing about 5 to 20% fetal calf serum (MEM), Dulbecco's modified minimal essential medium (DMEM), RPMI 1640 medium, 199 medium, and the like. 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, the culture temperature is usually about 30 to about 40 ° C., and the culture time is About 12 to about 72 hours.

  In step (b) of the above method, first, the expression level of Fgf21 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 a cell capable of naturally expressing Fgf21 is used as a cell capable of measuring Fgf21 expression, the expression level can be measured by a method known per se for a product of Fgf21, for example, a transcription product or a translation product. . 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. The expression level of the translation product can be measured by preparing an extract from the cells and using an immunological technique. As an immunological method, a radioisotope immunoassay (RIA method), an ELISA method (Methods in Enzymol. 70: 419-439 (1980)), a fluorescent antibody method, or the like can be used. On the other hand, when a cell capable of performing a reporter assay for the Fgf21 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 Fgf21 in the cells contacted with the test substance is compared with the expression level of Fgf21 in the control cells not contacted with the test substance. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference. The expression level of Fgf21 in the control cells not contacted with the test substance is the expression level measured at the same time, even if the expression level was measured in advance with respect to the measurement of the expression level of Fgf21 in the cells contacted with the test substance. However, the expression level is preferably measured simultaneously 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 Fgf21 is selected. The regulation of the expression level of Fgf21 can be an increase or decrease in the expression level. For example, a test substance that increases the expression level of Fgf21 (promotes the expression) has an action of promoting differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells or an action of suppressing differentiation of hematopoietic stem cells into lymphocyte lineage cells. And can be a preventive / 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 Fgf21 (suppresses the expression) has an action of suppressing differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells or an action of promoting differentiation of hematopoietic stem cells into lymphocyte lineage cells. And can be used as a preventive / therapeutic agent for diseases such as immunodeficiency diseases. Therefore, it becomes possible to select a candidate substance for a medicine such as a preventive / therapeutic agent for a predetermined disease or a research reagent, using the expression level of Fgf21 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 Fgf21 in an animal administered with the test substance, and comparing the expression level with the expression level of Fgf21 in a control animal not administered with the test substance;
(C) A step of selecting a test substance that regulates the expression level of Fgf21 based on the comparison result of (b).

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

  In step (b) of the above method, the expression level of Fgf21 can be measured by a method known per se. For example, the blood concentration of Fgf21 is measured as the expression level of Fgf21. The comparison of the expression level in this step (b) and the step (c) of the above method can be performed in the same manner as in the screening method using cells capable of measuring the expression of Fgf21.

  The screening method of the present invention makes it possible to screen for substances that can regulate the differentiation of hematopoietic stem cells, or preventive / therapeutic agents for certain diseases. Therefore, the screening method of the present invention is useful for the development of the above-mentioned pharmaceuticals or research reagents.

(5. Method for identifying Fgf21 polymorphism causing change in the ability to regulate differentiation of hematopoietic stem cells)
The present invention also includes a method for identifying an Fgf21 polymorphism that causes a change in the ability to regulate differentiation of hematopoietic stem cells, comprising analyzing the influence of a specific polymorphism of Fgf21 on the ability to regulate differentiation of hematopoietic stem cells. Provided is a protein / nucleic acid molecule containing the Fgf21 polymorphism that changes the ability to regulate differentiation of hematopoietic stem cells.

  The Fgf21 polymorphism means a nucleotide sequence mutation found at a certain frequency in genomic DNA containing the Fgf21 gene in a certain population, and substitution, deletion or addition of one or more DNAs in the genomic DNA containing the Fgf21 gene (Eg, SNP, haplotype), as well as repeats, inversions, translocations, etc. of partial regions in the genomic DNA. The polymorphism type of Fgf21 identified by the method of the present invention is a nucleotide that changes the differentiation efficiency of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphocyte lineage cells among all types of polymorphisms in Fgf21. It may be a sequence variation, or a nucleotide sequence variation that differs in frequency between animals affected and unaffected by a given disease (eg, hematopoietic disease, immune disease). The animal to be analyzed for the Fgf21 polymorphism is not particularly limited as described above, but is preferably a human.

  The analysis can be performed by a method known per se. For example, if there is a significant difference in the frequency of possessing a specific polymorphism depending on the frequency of occurrence and severity of a given disease (eg, hematopoietic disease, immune disease) as a result of an analysis method such as linkage analysis, the type This polymorphism can be determined to be a polymorphism that causes a change in the ability to regulate the differentiation of hematopoietic stem cells. Analysis can also be performed in vitro. For example, by culturing hematopoietic stem cells in the presence of Fgf21 containing a specific polymorphism, and comparing the differentiation efficiency of hematopoietic stem cells into red blood cell-myeloid lineage cells or lymphocyte lineage cells with that of control Fgf21 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 Fgf21 polymorphism. As the biological sample, not only a sample (eg, blood) containing Fgf21-expressing tissue or cells (eg, B cells) but also any tissue containing genomic DNA such as hair, nails, skin, and mucous membranes can be used. In view of availability, burden on the human body, and the like, the biological sample is preferably hair, nails, skin, mucous membrane, blood, plasma, serum, saliva and the like. Polymorphism can be determined by analyzing a large number of nucleotide sequences of genomes or transcripts contained in biological samples having different origins and identifying mutations found at a certain frequency in the determined nucleotide sequence.

  The identification method of the present invention and the Fgf21 polymorphism determined by the method are useful for determining the risk of developing a predetermined disease.

(6. Determination method and diagnostic agent of biological state of animal for hematopoietic stem cell differentiation)
(6.1. Determination method and diagnostic agent based on measurement of expression level)
The present invention provides a method for determining the biological state of an animal with respect to hematopoietic stem cell differentiation based on the expression level of Fgf21. 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) a step of measuring the expression level of Fgf21 in a biological sample collected from an animal;
(B) A step of evaluating the biological state of the animal against hematopoietic stem cell differentiation based on the expression level of Fgf21.

  In step (a) of the above method, the expression level of Fgf21 is measured in a biological sample collected from an animal. The animal is not particularly limited as described above, but a human is preferable. The biological sample is not particularly limited as long as it is a sample capable of measuring the expression level of Fgf21. Examples thereof include blood, bone marrow fluid, liver, and thymus, but blood that is less invasive is preferable. The expression level of Fgf21 can be measured 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 against hematopoietic stem cell differentiation can be evaluated based on the expression level of Fgf21. Specifically, first, the measured expression level of Fgf21 is compared with the expression level of Fgf21 in an animal without normal 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 Fgf21 in an animal without abnormal hematopoietic stem cell differentiation or in a normal animal can be determined by a method known per se.

  Next, based on the comparison result of the expression level of Fgf21, whether or not the animal may have an abnormality in hematopoietic stem cell differentiation, or may have a predetermined disease, or in the future It is possible to determine whether there is a high or low possibility of being affected. From the results of this example, when the expression level of Fgf21 is high, differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells is further promoted, and differentiation of hematopoietic stem cells into lymphocyte lineage cells is further suppressed, When the expression level of Fgf21 is low, it is considered that differentiation of hematopoietic stem cells into lymphocyte lineage cells is further promoted, and differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells can be further suppressed. 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 a specific gene are often observed before the onset of a specific disease. Therefore, it is considered possible to determine the abnormality of hematopoietic stem cell differentiation, the onset of a predetermined disease, or the risk of onset by analyzing the expression level of Fgf21.

  The present invention also provides a diagnostic agent for the biological state of an animal against hematopoietic stem cell differentiation, comprising a reagent for measuring the expression level of Fgf21. The diagnostic agent can be useful, for example, as a diagnostic agent for the onset or risk of developing a predetermined disease in an animal.

The reagent for measuring the expression level of Fgf21 is not particularly limited as long as Fgf21 expression can be quantified. For example, an antibody against Fgf21 (described above), a nucleic acid probe against Fgf21 transcript, or a plurality of Fgf21 transcripts that can amplify Fgf21 transcript It may contain a primer. 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 the labeling substance include fluorescent substances such as FITC and FAM, luminescent substances such as luminol, luciferin, and lucigenin, radioisotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S, and ¹²³ I, biotin, and streptavidin. And the like, and the like.

  The nucleic acid probe for the Fgf21 transcript may be either DNA or RNA, but DNA is preferred 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 the transcript of Fgf21 can be detected, but is preferably about 15 to 1000 bp, more preferably about 50 to 500 bp. The probe may be provided in a form fixed on a substrate like a microarray.

  A plurality of primers (eg, primer pairs) capable of amplifying Fgf21 are selected such that a detectable size nucleotide fragment is amplified. A detectable size nucleotide fragment 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 Fgf21 can be amplified, but may preferably be about 15 to 100 bp, more preferably about 18 to 50 bp, and still more preferably about 20 to 30 bp. When the means capable of quantifying the Fgf21 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 onset risk.

(7.2. Determination method and diagnostic agent based on measurement of polymorphism)
The present invention provides a method for determining the biological state of an animal against hematopoietic cell differentiation based on the polymorphism of Fgf21. This method can be useful, for example, as a method for determining the onset risk of 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) a step of measuring Fgf21 polymorphism in a biological sample collected from an animal;
(B) A step of evaluating the biological state of the animal against hematopoietic stem cell differentiation based on the polymorphic type.

  In step (a) of the above method, the polymorphic type of Fgf21 is measured in a biological sample collected from an animal. The animals are as described above. The biological sample can be the same as described above in the identification method of the present invention.

  The measurement of the polymorphic type can be performed by a method known per se. For example, RFLP (restriction fragment length polymorphism) method, PCR-SSCP (single-stranded DNA higher-order structure polymorphism analysis) method, ASO (Allele Specific Oligonucleotide) hybridization method, direct sequencing method, ARMS (Amplification Refracting Mutation) System) method, denaturing gradient gel electrophoresis (Denaturing Gradient Gel Electrophoresis) 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 against hematopoietic stem cell differentiation can be evaluated based on the type of polymorphism. Specifically, it is determined whether or not an animal may have abnormal hematopoietic stem cell differentiation (excessive promotion or suppression), or whether the animal is likely to suffer from a given disease in the future. obtain. From the results of this Example, when Fgf21 contains a polymorphism that enhances its function, the differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells is further promoted, and the differentiation of hematopoietic stem cells into lymphocyte lineage cells. On the other hand, when Fgf21 contains a polymorphism that reduces its function, differentiation of hematopoietic stem cells into lymphocyte lineage cells is further promoted, and differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells It is thought that can be suppressed more. In addition, it is known that an animal that is likely to develop a specific disease often has a specific type of polymorphism in a gene associated with the disease. Therefore, it is considered that an animal containing a polymorphism that reduces the function of Fgf21 has a relatively high possibility of developing a disease such as anemia, an autoimmune disease, or an allergic disease. Similarly, an animal containing a polymorphism that enhances the function of Fgf21 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 development of a predetermined disease from the analysis of polymorphism. In addition, the type of polymorphism to be measured in this step can be obtained by, for example, the identification method of the present invention.

  The present invention also provides a diagnostic agent for the biological state of an animal against hematopoietic stem cell differentiation, comprising a reagent for measuring a polymorphism of Fgf21. This method can be useful, for example, as a diagnostic agent for the risk of developing a given disease in an animal.

  The Fgf21 polymorphism measuring reagent is not particularly limited as long as the Fgf21 polymorphism can be determined. The reagent may be labeled with a labeling substance. In addition, 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 Fgf21 polymorphism measuring reagent can specifically amplify Fgf21 having a specific type of polymorphism, or a nucleic acid probe capable of specifically measuring Fgf21 having a specific type of polymorphism. It may include a plurality of primers. Nucleic acid probes, primers can be for genomic DNA containing Fgf21 or Fgf21 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 Fgf21 having a specific type of polymorphism is not particularly limited as long as Fgf21 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 Fgf21 having a specific type of polymorphism can be selected, and may be, for example, a size of 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.

  A plurality of primers (eg, primer pairs) capable of specifically amplifying Fgf21 having a particular type of polymorphism are selected such that a measurable size nucleotide fragment is amplified. Such a plurality of primers is designed to include a polymorphic site at the 3 'end of any one of the primers, for example. Nucleotide fragments of measurable size 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 Fgf21 can be amplified, but may preferably be about 15 to 100 bp, more preferably about 18 to 50 bp, and still more preferably about 20 to 30 bp. When the means capable of measuring the polymorphism of Fgf21 is a primer pair for the Fgf21 transcript, the determination kit can further contain a reverse transcriptase.

  Examples of the reagent for measuring Fgf21 polymorphism include those containing a restriction enzyme that recognizes a specific type of polymorphic site. According to such a reagent, polymorphism analysis by RFLP becomes possible.

  The determination method and diagnostic agent of the present invention are useful for enabling determination of abnormal hematopoietic stem cell differentiation, risk of developing a predetermined disease, and providing an opportunity for improving lifestyle habits for the purpose of preventing a predetermined disease. It is.

(8. Kit)
The present invention provides a kit comprising the following (a) and (b):
(A) a substance that regulates the expression or function of Fgf21; and (b) a reagent for identifying hematopoietic stem cells or differentiated cells thereof and / or a reagent for regulating differentiation of differentiated cells of hematopoietic stem cells.

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

  As the reagent for identifying hematopoietic stem cells in (b) above, hematopoietic stem cell-specific cell surface markers (eg, Sca-1, kit), hematopoietic stem cell-specific cell non-surface markers (eg, Gata-1) And substances containing specific affinity (for example, antibodies).

  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 (for example, an antibody) having specific affinity for a cell non-surface marker specific for erythrocyte-myeloid lineage cells (for example, Globin, mpx) is used. Including. Examples of reagents for identifying lymphocyte lineage cells include cell surface markers specific to lymphocyte lineage cells (eg, B cell receptor, T cell receptor), cell non-surface markers specific to lymphocyte lineage cells (eg, Ikaros). Examples include substances containing a substance having a specific affinity (for example, an antibody).

  Examples of the differentiation regulating reagent for differentiated cells of the hematopoietic stem cell (b) include those for regulating differentiation of erythrocyte-myeloid lineage cells and lymphocyte lineage cells. Examples of the erythrocyte-myeloid lineage cell differentiation regulator include those containing thrombopoietin as a erythrocyte-myeloid lineage cell differentiation regulator. Examples of the differentiation regulator for lymphocyte lineage cells include those containing interleukin-7 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 performing the method of the present invention.

(9. Novel Fgf21 gene, protein, and related invention)
The present invention also provides novel Fgf21 polypeptides and polynucleotides.

  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. As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3, (a) 1 or 2 or more (for example, 1 to 50, preferably 1 to 30) in the amino acid sequence represented by SEQ ID NO: 3, More preferably 1-20, still more preferably 1-10, most preferably 1-5 amino acid sequences substituted, deleted, inserted or added, (b) shown in SEQ ID NO: 3 A significant amino acid sequence identity to the amino acid sequence (eg, about 70% or more, preferably about 80% or more, more preferably about 90% or more, even more preferably about 95% or more, most preferably about 97%, 98 % Or 99% or more amino acid sequence identity). 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 (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 percent identity is, for example, BLOSUM 62 matrix or PAM250 matrix, and gap weight: 16, It can be determined by using either 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 that is part of the GCG sequence alignment software package. When the ALIGN program is used to compare 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 first 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. As a polynucleotide having a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 2, (a) a high stringency condition for a polynucleotide comprising a complementary sequence of the nucleotide sequence represented by SEQ ID NO: 2 (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 preferred is a nucleotide sequence having about 95% or more, most preferably about 97%, 98% or 99% or more nucleotide sequence identity). As hybridization conditions under highly stringent conditions, after hybridization at 6 × SSC (sodium chloride / sodium citrate) / 45 ° C., 1 at 0.2 × SSC / 0.1% SDS / 50 to 65 ° C. Or the washing | cleaning of 2 times or more is 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 production cells (ie, a hybridoma), a polynucleotide encoding the polypeptide, an expression vector containing the polynucleotide, and an expression vector. Introduced transformant, novel Fgf21 inhibitor (eg, antisense nucleic acid, ribozyme, RNAi-inducible nucleic acid, dominant negative mutant), novel Fgf21 measurement reagent (eg, nucleic acid probe, primer pair or 3 or more) Primer sets), transgenic animals and cells of novel Fgf21 (eg, animals and cells overexpressing novel Fgf21, and animals and cells in which expression or function of novel Fgf21 is suppressed, ie, knockout animals and cells) Etc. The. These series of inventions can be made by the methods disclosed in this specification and other methods known in the art.

  The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1: Isolation and structural analysis of zebrafish Fgf21 cDNA Using the amino acid sequence of human Fgf21 as an index, zebrafish DNA database (genomic DNA, EST DNA) (GenBank: http: //www.ncbi.nlm.nih. A homology search was performed using gov /, Emsenble: http://www.ensemble.org/), and a zebrafish gene fragment expected to encode Fgf21 was found. Furthermore, from these sequences, primers (5′-cttagcagtcatgctttttgcc-3 ′ (SEQ ID NO: 4), 5′-tgctaatgtataagcatgtctttt-3 ′ (SEQ ID NO: 5)) capable of amplifying the entire translated sequence of Fgf21 were prepared by PCR. Fgf21 cDNA was amplified using zebrafish cDNA as a template. The amplified Fgf21 cDNA was then cloned. Furthermore, the base sequence (SEQ ID NO: 2) of the entire translation region of the Fgf21 cDNA was determined.
As a result, it was revealed from the nucleotide sequence (SEQ ID NO: 2) of the isolated zebrafish Fgf21 cDNA that Fgf21 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 (33.3% amino acid sequence identity) with human Fgf21 among known human proteins (FIG. 1A). The human Fgf21 gene is close to carbonic anhydrase XI (CA11) and dehydrogenase / reductase member 10 (DHRS10) on the chromosome (LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/). Similarly, the zebrafish Fgf21 gene was actually close to Ca11 and Dhrs10 on the zebrafish chromosome (Enzemble Zebrafish Genome Brower: http://www.ensemble.org/) (FIG. 1B).
From the above, this gene was confirmed to be the zebrafish Fgf21 gene.

Example 2 Fgf21 Function Inhibition Experiment by Introducing Fgf21 MO It has been clarified that introduction of a target gene MO into a fertilized egg of zebrafish effectively and specifically suppresses the function of the target gene (Nasevicius , A. and Ekker, SC (2000) Nat. Genet. 26, 153-60). Therefore, Fgf21 MO was introduced into a fertilized egg to suppress the function of Fgf21, and its phenotype was examined. Specifically, a morpholino-modified antisense oligonucleotide (MO) having a sequence (5′-GGCAAAAAAGCATGACTGAACTAAGCT-3 ′ (SEQ ID NO: 6), 25 bases) complementary to the 5 ′ untranslated sequence and part of the translated sequence of Fgf21 mRNA. ) Was introduced into a fertilized egg (10 ng / fertilized egg), and the phenotype of the embryo in which the function of Fgf21 was suppressed was analyzed. In addition, as a control experiment, control MO (5′-GGAAATAAGCATCACTGAGTAACCT-3 ′ (SEQ ID NO: 7), 25 bases) in which 5 bases in the 25 base sequence of Fgf21 MO were replaced with other bases was introduced (10 ng / fertilized egg) The phenotype was also analyzed in the embryos. Furthermore, as a function recovery experiment, Fgf21 MO and Fgf21 mRNA were simultaneously introduced into a fertilized egg (Fgf21 MO: 10 ng / fertilized egg, Fgf21 mRNA: 10 pg / fertilized egg), and the phenotype was analyzed. Fgf21 mRNA was prepared by incorporating the translation region of Fgf21 cDNA into a pCS2 + vector and using it as a template.
Red blood cells were observed as an analysis of embryo phenotype. Observation of erythrocytes was performed by removing the egg membranes of zebrafish embryos 24 hours after fertilization, immersing them in an o-dianisidine solution for 15 minutes in the dark and adding H ₂ O ₂ for color development.
As a result, it was confirmed that the Fgf21-introduced fetus had a curved body axis (FIG. 2A) and disappearance or reduction of red blood cells (FIG. 2B, Table 1). As a control experiment, almost no abnormality was observed in embryos into which control MO was introduced (Table 1). Furthermore, in the embryos in which Fgf21 MO and Fgf21 mRNA were simultaneously introduced as a function recovery experiment, the rate of disappearance or reduction of erythrocytes observed when Fgf21 MO alone was introduced was reduced (Table 1).

  From the above, it was considered that the disappearance of red blood cells confirmed by the introduction of Fgf21 MO was specific to the function suppression of Fgf21.

Example 3: Examination of the role of Fgf21 in angiogenesis As a cause of disappearance or reduction of red blood cells in Fgf21 function-suppressed embryos, 1) blood does not flow due to abnormal angiogenesis, or 2) red blood cells are not generated 2 One can be considered. Therefore, in order to clarify the cause of the disappearance or reduction of red blood cells, first, the blood vessel formation of Fgf21 function-suppressed embryos was examined.
Dextran labeled with a 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, flk1, 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 hybridized with a digoxigenin-labeled flk1 cRNA probe.
As a result, the blood vessel formation of the Fgf21 function-suppressed embryo was normal (FIG. 3A). The expression of the vascular endothelial cell marker, flk1 (Thisse, C. and Zon, LI (2002) Science 295, 457-462) was also normal (FIG. 3B).
From the above, it was considered that the disappearance or decrease of erythrocytes in Fgf21 function-suppressed embryos was not due to abnormal angiogenesis but due to abnormal production of erythrocytes.

Example 4: Examination of the role of Fgf21 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 preserved among vertebrates (Thisse and Zon , 2002; Davidson and Zon, 2004). That is, pattern formation of the mesoderm occurs in the gastrulation stage of early development, and hemangioblast, a progenitor cell common to blood cells and vascular endothelial cells, is generated. Furthermore, the blood cell lineage 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 proceed in a tissue called an intermediate cell mass (ICM) in a zebrafish fetus. Therefore, it was examined in which stage of blood cell development Fgf21 is involved.
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 digoxigenin labeled marker cRNA probe. Various hematopoietic cell markers include scl (hemangioblast), gata2 (hematopoietic stem cell), gata1 (erythrocyte-myeloid progenitor cell), mpx (myeloid), ikaros (lymphocyte progenitor cell) (Thisse, C. and Zon, LI (2002) Science 295, 457-462) was used.
As a result, the expression of the hemangioblast marker gene, scl, was normal in the Fgf21 function-suppressed embryo in the lateral plate mesoderm that will form ICM in the future (FIG. 5A). Moreover, the expression of the hematopoietic stem cell marker gene, gata2, was also normal (FIG. 5B). However, the expression of the erythrocyte-myeloid progenitor cell marker gene, gata1, and the myeloid marker gene, mpx, disappeared (FIGS. 5C and D). Thus, it was revealed that Fgf21 plays an essential role in the differentiation from hematopoietic stem cells to erythrocyte-myeloid progenitor cells. On the other hand, the expression of the lymphocyte progenitor cell marker gene, ikaros, was increased (FIG. 5E).
From the above, it was shown that suppression of the function of Fgf21 inhibits the differentiation of erythrocyte-myeloid progenitor cells from hematopoietic stem cells and increases lymphocyte progenitor cells.

(Conclusion)
This study revealed that Fgf21 has an important function in erythropoiesis. It was revealed that Fgf21 is a hematopoietic factor that acts on hematopoietic stem cells and plays an important role in the generation of erythrocyte-myeloid progenitor cells (FIG. 6). Erythropoietin, well known as a hematopoietic factor, acts on erythroid progenitor cells to promote their proliferation and differentiation into erythrocytes (FIG. 6). Therefore, Fgf21 is expected to be clinically applied as a new hematopoietic factor having a mechanism of action different from that of erythropoietin.

  The agent of the present invention enables the regulation of differentiation of hematopoietic stem cells and the prevention or treatment of predetermined diseases such as hematopoietic diseases and immune diseases. The differentiation regulation method of the present invention enables the regulation of 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 of the present invention is a method for predicting the therapeutic effect of a substance that regulates the expression or function of Fgf21 in the treatment of a patient with a disease (eg, hematopoietic disease, immune disease) caused by abnormal blood cells. Is possible. The screening method of the present invention makes it possible to develop pharmaceuticals for diseases caused by abnormal blood cells. The determination method and diagnostic agent of the present invention make it possible to evaluate the onset or risk of developing diseases caused by abnormal blood cells.

The amino acid sequence of zebrafish Fgf21 and the position of the gene on the chromosome are shown. (A) Comparison of amino acid sequences of zebrafish Fgf21 and human Fgf21. The amino acid sequence homology is 33.3%. (B) The position of the zebrafish Fgf21 gene and the human Fgf21 gene on the chromosome. Both are adjacent to Dhrs10 and Ca11. Mb, megabase Fig. 5 shows erythropoiesis in a zebrafish fetus with suppressed Fgf21 function. (A) Fgf21 MO (10 ng) was introduced into a fertilized egg, and fetus observation 24 hours after fertilization. Wild-type (left) and Fgf21 function-suppressed fetus (right). (B) In order to observe erythropoiesis, the area surrounded by the square in FIG. A was enlarged. Wild-type (left) and Fgf21 function-suppressed fetus (right). Arrows indicate red blood cells. Fig. 5 shows angiogenesis in an Fgf21 function-suppressed zebrafish fetus. (A) Fgf21 MO (10 ng) 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 Fgf21 function-suppressed fetus (right). Arrows indicate blood vessels. (B) Fgf21 MO (10 ng) was introduced into a fertilized egg, and the expression of the vascular endothelial cell gene marker, flk1, 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. The model figure of the differentiation process of a hematopoietic system cell is shown. Bold letters indicate marker genes of hematopoietic cells. The expression of the marker gene of the hematopoietic cell of a zebrafish fetus which suppresses Fgf21 function is shown. Fgf21 MO (10 ng) was introduced into a fertilized egg, and the expression of hematopoietic cell gene markers in the fetus was observed by whole mount in situ hybridization. Arrows indicate the disappearance or decrease of expression in Fgf21 function-suppressed fetuses. (A) Expression of scl, a marker gene of hemangioblast, in the lateral plate mesoderm of fetuses of 5 somites. (B) Expression of the hematopoietic stem cell marker gene, gata2, in fetal ICM 18 hours after fertilization. (C) Expression of erythroid-myeloid progenitor marker gene, gata1, in fetal ICM 24 hours after fertilization. (D) Expression of myeloid cell marker gene, mpx, in fetal ICM 24 hours after fertilization. (E) Expression of the lymphocyte precursor cell marker gene, ikaros, in fetal ICM 24 hours after fertilization. The action model figure of Fgf21 is shown. Fgf21 acts on the differentiation process from hematopoietic stem cells to erythrocyte-myeloid progenitor cells. On the other hand, erythropoietin acts on the differentiation process from erythrocyte-myeloid progenitor cells to erythrocytes.

Claims (21)

A hematopoietic stem cell differentiation regulator comprising a substance that regulates the expression or function of Fgf21. The agent according to claim 1, wherein the substance that regulates the expression or function of Fgf21 is a substance that promotes the expression or function of Fgf21. The agent according to claim 2, wherein the substance that promotes the expression or function of Fgf21 is Fgf21 or an expression vector thereof. The agent according to claim 1, wherein the substance that regulates the expression or function of Fgf21 is a substance that suppresses the expression or function of Fgf21. The agent according to claim 4, wherein the substance that suppresses the expression or function of Fgf21 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. The agent according to claim 2, which is an agent for promoting differentiation of hematopoietic stem cells into erythrocyte-myeloid lineage cells, or an agent for inhibiting differentiation of hematopoietic stem cells into lymphocyte lineage cells. The agent according to claim 2, which is an agent for promoting differentiation of hematopoietic stem cells into erythroid lineage cells. 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 differentiation of hematopoietic stem cells into lymphocyte lineage cells. The agent according to claim 1, which is a prophylactic or therapeutic agent for hematopoietic disease, immune disease or allergic disease. 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 Fgf21. 11. The method according to claim 10, wherein the substance that regulates the expression or function of Fgf21 is Fgf21. The method according to claim 10, wherein the method further comprises isolating erythrocyte-myeloid lineage cells or lymphocyte lineage cells induced to differentiate from hematopoietic stem cells. 11. The method according to claim 10, further comprising further differentiating erythrocyte-myeloid lineage cells that have been induced to differentiate from hematopoietic stem cells. A method for determining the differentiation efficiency of hematopoietic stem cells by Fgf21, comprising culturing hematopoietic stem cells in a medium in the presence of Fgf21, and evaluating the differentiation efficiency of hematopoietic stem cells into erythrocyte-myeloid lineage cells or lymphocyte lineage cells. A screening method for a substance capable of regulating the differentiation of hematopoietic stem cells, comprising evaluating whether or not a test substance can regulate the expression of Fgf21. 16. The method of claim 15, comprising the following steps (a) to (c):
(A) contacting the test substance with a cell capable of measuring Fgf21 expression;
(B) measuring the expression level of Fgf21 in a cell contacted with a test substance and comparing the expression level with the expression level of Fgf21 in a control cell not contacted with the test substance;
(C) A step of selecting a test substance that regulates the expression level of Fgf21 based on the comparison result of (b). 16. The method of claim 15, comprising the following steps (a) to (c):
(A) administering a test substance to an animal;
(B) measuring the expression level of Fgf21 in an animal administered with the test substance, and comparing the expression level with the expression level of Fgf21 in a control animal not administered with the test substance;
(C) A step of selecting a test substance that regulates the expression level of Fgf21 based on the comparison result of (b). A method for identifying an Fgf21 polymorphism that causes a change in the differentiation regulating ability of a hematopoietic stem cell, comprising analyzing the influence of a specific polymorphism of Fgf21 on the differentiation regulating ability of a hematopoietic stem cell. The biological state of the animal with respect to hematopoietic stem cell differentiation, comprising measuring the expression level and / or polymorphism of Fgf21 using a biological sample collected from the animal, and evaluating the biological state of the animal with respect to hematopoietic stem cell differentiation based on the measurement result Judgment method. A diagnostic agent for the biological state of an animal against hematopoietic stem cell differentiation, comprising a reagent for measuring the expression level of Fgf21 and / or polymorphism. A kit comprising the following (a) and (b):
(A) a substance that regulates the expression or function of Fgf21; and (b) a reagent for identifying hematopoietic stem cells or differentiated cells thereof and / or a reagent for regulating differentiation of differentiated cells of hematopoietic stem cells.