WO2020184685A1 - Method for producing cytopenia model animal, cytopenia model animal, method for evaluating blood cell function, method for producing blood cells, method for screening cytopenia therapeutic drug material candidate, and method for producing cytopenia therapeutic drug material candidate - Google Patents

Method for producing cytopenia model animal, cytopenia model animal, method for evaluating blood cell function, method for producing blood cells, method for screening cytopenia therapeutic drug material candidate, and method for producing cytopenia therapeutic drug material candidate Download PDF

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WO2020184685A1
WO2020184685A1 PCT/JP2020/010961 JP2020010961W WO2020184685A1 WO 2020184685 A1 WO2020184685 A1 WO 2020184685A1 JP 2020010961 W JP2020010961 W JP 2020010961W WO 2020184685 A1 WO2020184685 A1 WO 2020184685A1
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blood
model animal
cytopenia
antibody
cells
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PCT/JP2020/010961
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French (fr)
Japanese (ja)
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善弘 熊谷
秀徳 広瀬
幸敬 伊東
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株式会社メガカリオン
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors

Definitions

  • the present invention relates to a method for producing a model animal for cytopenia, a model animal for cytopenia, a method for evaluating blood cell function, a method for producing blood cells, a method for screening a candidate substance for a therapeutic agent for cytopenia, and a therapeutic agent for cytopenia. Regarding the method for producing the candidate substance of.
  • Platelet preparations are administered to patients with decreased platelets during bleeding such as surgery or injury. Platelet preparations are currently produced from blood obtained from blood donations. However, due to changes in the population composition, there is concern that the amount of blood donated will decrease and that platelet preparations will be in short supply.
  • Non-Patent Document 1 a method for producing platelets in vitro has been developed.
  • platelets produced in vitro do not have a sufficient hemostatic effect, which is the function of platelets
  • a method capable of evaluating the function of the produced platelets is required. Therefore, as a method for evaluating the function of platelets, platelets are administered to a model animal in which the number of platelets is reduced as compared with a normal animal (hereinafter referred to as "thrombocytopenia model animal" or “model animal") to stop bleeding. We examined whether it could be evaluated using the recovery of function as an index.
  • thrombocytopenia model animal As a method of reducing platelets in animals, there is a method of systemically irradiating NOG mice (NOD / Shi-scid-IL2R ⁇ null mice) with X-rays to destroy the bone marrow of the animals, and a model mouse for thrombocytopenia, or It is used as a model animal with a decreased blood cell count (hereinafter, also referred to as "thrombocytopenia model animal"). Therefore, the present inventors attempted to evaluate the function of platelets using the X-ray irradiation-induced cytopenia model animal, and found that the X-ray irradiation-induced cytopenia model animal had a short bleeding time and was constant.
  • an object of the present invention is, for example, to provide a method for producing a model animal for blood cell hypoplasia in which the number of blood cells is further reduced as compared with the model animal for blood cell hypoplasia by X-ray irradiation.
  • the method for producing a model animal for blood cell hypoplasia of the present invention includes an irradiation step of irradiating a non-human animal with radiation.
  • the administration step of administering the anti-blood cell antibody to the non-human animal is included.
  • the first model animal for cytopenia of the present invention (hereinafter, also referred to as "first model animal") is obtained by the method for producing a model animal for cytopenia of the present invention.
  • the second model animal for cytopenia of the present invention (hereinafter, also referred to as “second model animal”) satisfies any one or more of the following conditions (1) to (3): (1) The number of platelets in the blood is 7.3 ⁇ 10 4 / ⁇ L or less. (2) The number of red blood cells in the blood is 500 ⁇ 10 4 cells / ⁇ L or less. (3) The number of white blood cells in the blood is 3 ⁇ 10 2 cells / ⁇ L or less.
  • the method for evaluating blood cell function of the present invention includes a step of administering blood cells to a model animal of blood cell hypoplasia and an evaluation step of evaluating the function of the blood cells.
  • the model animal for cytopenia is the model animal for cytopenia of the present invention.
  • the method for producing a blood cell of the present invention includes an evaluation step for evaluating the function of the blood cell of the test blood cell and an evaluation step.
  • the evaluation step includes a selection step of selecting blood cells satisfying the criteria as functional blood cells.
  • the evaluation step is carried out by the method for evaluating blood cell function of the present invention.
  • the method for screening a candidate substance for a therapeutic agent for cytopenia of the present invention includes a step of administering a test substance to a model animal for cytopenia and
  • the model animal includes a selection step of selecting a test substance that increases the number of blood cells in the blood as a candidate substance for a therapeutic agent for hypocytopenia.
  • the model animal is a model animal for cytopenia of the present invention.
  • the method for producing a candidate substance for a therapeutic agent for cytopenia of the present invention includes a selection step of selecting a candidate substance for a therapeutic agent for cytopenia from a test substance.
  • the selection step is carried out by the screening method of the present invention.
  • the present invention it is possible to produce a model animal of blood cell hypoplasia in which the number of blood cells is reduced as compared with a model animal of blood cell hypoplasia by X-ray irradiation.
  • FIG. 1 is a schematic diagram showing a concentration system in Example 1.
  • FIG. 2 is a graph showing a method for preparing mice of Examples, Comparative Examples, and Reference Examples in Example 1.
  • FIG. 3 is a graph showing the number of platelets in the blood of the mouse in Example 1.
  • FIG. 4 is a graph showing the bleeding time in Example 2.
  • the method for producing a model animal for hemocytopenia of the present invention includes an irradiation step of irradiating a non-human animal with radiation and an administration step of administering an anti-blood cell antibody to the non-human animal.
  • the method for producing a model animal of the present invention is characterized by carrying out the irradiation step and the administration step, and other steps and conditions are not particularly limited. According to the method for producing a model animal of the present invention, the number of blood cells in the blood of the obtained model animal can be reduced as compared with the method for producing a model animal by X-ray irradiation.
  • the number of blood cells in the blood of the obtained model animal can be reduced as compared with the blood cell hypoplasia model animal by X-ray irradiation.
  • the hemostasis time bleeding time
  • a model animal for cytopenia suitable for evaluation of blood cell function and acquisition of a candidate substance for treatment of cytopenia including thrombocytopenia can be produced.
  • the number of blood cells in the blood of the obtained model animal can be sufficiently reduced, so that the rate of decrease in blood cells is high and the variation among individuals.
  • the variability can be compared, for example, as a standard error.
  • cytopenia means that, for example, the number of blood cells in the blood is significantly reduced as compared with, for example, a normal animal (for example, an untreated animal) corresponding to the model animal.
  • the blood cell count in the target animal is, for example, 60% or less, 55% or less, 50% or less, 45 based on the blood cell count in the normal animal corresponding to the target animal. % Or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1%
  • the target animal can be said to have cytopenia.
  • the number of blood cells in the blood of the target mouse is, for example, 550 ⁇ 10 4 cells / ⁇ L or less, 500 ⁇ 10 4 cells / ⁇ L or less, 400 ⁇ 10 4 cells / ⁇ L or less, 300 ⁇ If 10 4 cells / ⁇ L or less, 200 ⁇ 10 4 cells / ⁇ L or less, 100 ⁇ 10 4 cells / ⁇ L or less, or 50 ⁇ 10 4 cells / ⁇ L or less, the target mouse has hypocytopenia. Can be done.
  • the type of blood cell is not particularly limited, and examples thereof include platelets, erythrocytes, and white blood cells.
  • white blood cells examples include monocytes; granulocytes such as neutrophils, eosinophils and basophils; and lymphocytes such as T cells, B cells, NK cells and NKT cells.
  • monocytes examples include monocytes; granulocytes such as neutrophils, eosinophils and basophils; and lymphocytes such as T cells, B cells, NK cells and NKT cells.
  • lymphocytes such as T cells, B cells, NK cells and NKT cells.
  • the type of blood cells to be reduced may be, for example, one type, two or more types, or all types.
  • the cytopenia can also be referred to as "thrombocytopenia".
  • the blood platelet count in the target animal is, for example, 40% or less, 35% or less, 30% or less, 25, based on the blood platelet count in the normal animal corresponding to the target animal. %, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, the target animal is said to have thrombocytopenia. be able to.
  • the number of platelets in the blood of the target animal is, for example, about 40% (for example, 35 to 45%) and about 20% (for example, 35 to 45%) based on the number of platelets in the blood of a normal animal corresponding to the target animal. For example, in the case of 15-25%), and about 10% (eg, 14% or less), the animal of interest can be said to have mild, moderate, and severe thrombocytopenia.
  • the blood platelet count in the target mouse is, for example, 50 ⁇ 10 4 cells / ⁇ L or less, 40 ⁇ 10 4 cells / ⁇ L or less, 30 ⁇ 10 4 cells / ⁇ L or less, 20 ⁇ If 10 4 / ⁇ L or less, 10 ⁇ 10 4 / ⁇ L or less, 5 ⁇ 10 4 / ⁇ L or less, or 4 ⁇ 10 4 / ⁇ L or less, the target mouse has thrombocytopenia. Can be done.
  • the blood cell depletion can also be referred to as "erythrocytopenia".
  • the number of red blood cells in the blood of the target animal is, for example, 60% or less, 55% or less, 50% or less, 45 based on the number of red blood cells in the blood of a normal animal corresponding to the target animal. % Or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1%
  • the target animal can be said to have erythrocytopenia.
  • the number of red blood cells in the blood of the target mouse is, for example, 500 ⁇ 10 4 / ⁇ L or less, 350 ⁇ 10 4 / ⁇ L or less, 100 ⁇ 10 4 / ⁇ L or less, or 50.
  • it is ⁇ 10 4 cells / ⁇ L or less, it can be said that the subject mouse has erythrocytopenia.
  • the hypocytopenia can also be referred to as "leukopenia".
  • the white blood cell count in the target animal is 35% or less, 30% or less, 25% or less, 20 based on, for example, the white blood cell count in the blood of a normal animal corresponding to the target animal.
  • % or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less it can be said that the target animal has leukopenia.
  • the number of white blood cells in the blood of the target mouse is, for example, 3 ⁇ 10 2 cells / ⁇ L or less, 2 ⁇ 10 2 cells / ⁇ L or less, or 1 ⁇ 10 2 cells / ⁇ L or less.
  • the subject mouse can be said to have leukopenia.
  • the blood cell count may be counted, for example, by using a blood cell counter, staining with a blood cell-specific antibody, and counting with a flow cytometer, or blood cell counting. It may be counted using an apparatus.
  • the "significance" when comparing a model animal with a normal animal, the "significance" can be determined based on, for example, a statistically significant difference.
  • a known statistical analysis method can be selected and used.
  • the statistical analysis method is not particularly limited, and examples thereof include one-way ANOVA, one-way iterative measurement ANOVA, Dunnett's test, Student's t-test, and Wilcoxon test, and these can be combined. For example, in the above test, it can be determined that a significant difference was observed when the P value was less than 0.05, while it could be determined that no significant difference was observed when the P value was 0.05 or more. ..
  • platelet is one of the cell components in blood and means a cell component in which CD41a and CD42b are positive.
  • the platelets for example, do not have a cell nucleus and are smaller in size than the megakaryocytes. Therefore, the platelets and the megakaryocytes can be distinguished by, for example, the presence and / or size of cell nuclei. It is known that the platelets play an important role in thrombus formation and hemostasis, and are also involved in the pathophysiology of tissue regeneration and inflammation after injury.
  • erythrocyte means a blood cell in blood, which is Ter119 (glycophorin-A) and / or CD235a positive.
  • white blood cell means a blood cell in blood, which is positive for CD2, CD3, CD4, CD8, CD13, CD19, CD21, and / or CD56.
  • the model animal is a non-human animal.
  • the type of the non-human animal is not particularly limited, and examples thereof include primates such as monkeys, gorillas, chimpanzees and marmosets; rodents such as mice, rats and guinea pigs; dogs, cats, rabbits, sheep and horses. Be done.
  • the model animal for example, an immunodeficient animal or an animal in which the reticle endothelial system is destroyed is preferable, and an immunodeficient mouse is more preferable because it is easy to maintain a state in which blood cells are reduced.
  • the immunodeficient animal include a model animal for severe combined immunodeficiency disease (SCID).
  • SCID severe combined immunodeficiency disease
  • Examples of the animal in which the reticuloendotheliatic system is destroyed include a cell-mediated immunodeficient animal.
  • Examples of the immunodeficient mouse include Scid mouse, NOD-Scid mouse, NOG mouse and the like, and NOG mouse is preferable.
  • the irradiation step is a step of irradiating the non-human animal with radiation.
  • the irradiation step can be carried out, for example, by irradiating the non-human animal with radiation generated by a radiation generator.
  • the non-human animal is preferably under anesthesia or in a fixed state because radiation can be uniformly irradiated to the entire non-human animal.
  • Examples of the types of radiation to be irradiated in the irradiation step include electromagnetic radiation such as X-rays and ⁇ -rays; particle radiation such as ⁇ -rays, ⁇ -rays, electron beams, proton rays, neutron rays, and heavy particle rays; Therefore, electromagnetic radiation such as X-rays and ⁇ -rays is preferable because it can effectively induce a decrease in the number of blood cells.
  • the radiation used in the irradiation step may be one type or two or more types.
  • the radiation irradiation amount (dose) in the irradiation step is not particularly limited, and can be appropriately set according to the non-human animal, for example.
  • the irradiation amount is, for example, 1.5 to 12 Gy, 1.5 to 10 Gy, 1.5 to 6 Gy, preferably 2.5 to 6 Gy, 4 to 6 Gy.
  • the irradiation amount is, for example, the amount of radiation per irradiation.
  • the irradiation of the radiation may be performed once or a plurality of times, for example.
  • the number of irradiations of the radiation is, for example, 2 to 5 times, preferably 2 to 3 times.
  • the total amount of the irradiation amount of the plurality of times is, for example, 3 to 12 Gy and 5 to 12 Gy, and the decrease in the number of blood cells can be effectively induced and It is preferably 8 to 12 Gy, 8 to 11 Gy, and 8 to 10 Gy because it can suppress radiation damage to non-human animals.
  • the execution interval of each time is not particularly limited, but for example, after one irradiation, the next irradiation may be carried out immediately, or the next irradiation may be carried out immediately. It may be carried out at intervals until irradiation.
  • the interval of each time is, for example, 12 to 48 hours, preferably about 24 hours (for example, 20 to 28 hours).
  • the irradiation step it is preferable to irradiate the non-human animal twice with 4 to 6 Gy of radiation because it can effectively induce a decrease in the number of blood cells and suppress radiation damage to the non-human animal.
  • the two irradiations are preferably carried out about one day (for example, 18 to 30 hours) apart.
  • the anti-blood cell antibody is administered to the non-human animal.
  • the administration of the anti-blood cell antibody can be carried out by, for example, a general antibody administration method, and as a specific example, an antibody solution containing the anti-blood cell antibody is intravenously, subcutaneously or intraperitoneally administered to the non-human animal. It can be carried out by doing.
  • the anti-blood cell antibody is an antibody against the blood cells, that is, an antibody capable of binding to the blood cells.
  • the anti-blood cell antibody is preferably an antibody against the cell membrane surface antigen of the blood cell.
  • the anti-blood cell antibody is not particularly limited, and can be appropriately determined, for example, depending on the type of the non-human animal, the type of the blood cell, and the cell surface antigen thereof.
  • the anti-blood cell antibody may be, for example, one type or two or more types. In the latter case, each anti-blood cell antibody may bind, for example, to the same antigen or to different antigens.
  • the anti-blood cell antibody is preferably a cell-removing functional antibody, a so-called Depletion antibody.
  • the cell removal function of an antibody is caused by, for example, antibody-dependent cellular cytotoxicity (ADCC) activity or complement-dependent cytotoxicity (CDC) activity of the antibody.
  • ADCC activity is controlled by, for example, the presence or absence of fucose in the N-glycosidic bond sugar chain in the Fc region of the antibody, particularly in the sugar chain.
  • the N-glycosidic bond sugar chain is bound to, for example, asparagine at position 297 of the IgG type antibody. Therefore, the cell-removing functionality of the anti-blood cell antibody can be controlled, for example, by the addition or removal of the N-glycoside-binding sugar chain of the anti-blood cell antibody and the addition or removal of fucose in the sugar chain.
  • the ADCC activity of the anti-blood cell antibody is reduced. ..
  • the anti-blood cell antibody has the N-glycosidic bond sugar chain removed, or when fucose is removed from the sugar chain of the anti-blood cell antibody, the ADCC activity of the anti-blood cell antibody is improved.
  • the antibody against the cell membrane surface antigen of the platelets is, for example, an anti-CD41 antibody, an anti-CD42a antibody, an anti-CD42b antibody, an anti-CD42c antibody, an anti-CD42d antibody, an anti-CD49b.
  • examples include antibodies, anti-CD61 antibodies, anti-CD109 antibodies, anti-GPVI antibodies and the like.
  • examples of the anti-CD41 antibody include an anti-CD41a antibody.
  • MWReg30 clone name
  • the anti-CD42d antibody for example, 1C2 (clone name) can be used.
  • the anti-blood cell antibody is an antibody against the cell membrane surface antigen of erythrocytes
  • examples of the antibody against the cell membrane surface antigen of erythrocytes include anti-Ter119 antibody and anti-CD235a antibody.
  • anti-TER-119 antibody for example, TER-119 (clone name) can be used.
  • anti-CD235a antibody for example, 11E4B-7-6 (clone name) can be used.
  • the antibody against the cell membrane surface antigen of the leukocytes is, for example, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD13 antibody, anti-CD19.
  • examples include antibodies, anti-CD21 antibodies, anti-CD56 antibodies and the like.
  • SFCI3Pt2H9 (clone name) can be used as the anti-CD2 antibody.
  • UCHT1 (clone name) can be used.
  • SFCI12T4D11 (clone name) can be used as the anti-CD4 antibody.
  • SFCI21Thy2D3 (clone name) can be used as the anti-CD8 antibody.
  • SJ1D1 (clone name) can be used.
  • HD237 (clone name) can be used as the anti-CD19 antibody.
  • BL13 (clone name) can be used as the anti-CD21 antibody.
  • N901 (clone name) can be used.
  • the dose of the anti-blood cell antibody to be administered to the non-human animal is not particularly limited, and is appropriately determined according to, for example, the type of the non-human animal, its body weight, the number of blood cells before administration, and the like. Can be set.
  • the dose is, for example, 1 to 1000 ⁇ g / kg body weight, 10 to 500 ⁇ g / kg body weight, or 20 to 100 ⁇ g / kg body weight.
  • the non-human animal is a mouse
  • the dose is, for example, 1 to 1000 ⁇ g / kg body weight, 10 to 100 ⁇ g / kg body weight, or 30 to 70 ⁇ g / kg body weight.
  • the dose may be set high, for example, when the number of platelets before the administration is relatively large, or may be set low when the number of platelets before the administration is relatively small.
  • the number of administrations of the anti-blood cell antibody to the non-human animal may be, for example, once or a plurality of times.
  • the number of administrations of the antibody is, for example, It is 2 to 5 times, preferably 2 to 3 times.
  • the total amount of the plurality of antibody doses is, for example, 2 to 2000 ⁇ g / kg body weight, 20 to 1000 ⁇ g / kg body weight, or 40 to 200 ⁇ g / kg body weight. It is preferably 40 to 200 ⁇ g / kg body weight or 60 to 100 ⁇ g / kg body weight because it can effectively induce a decrease in blood cell count.
  • the total amount of the multiple antibody doses is, for example, 2-2000 ⁇ g / kg body weight, 20-200 ⁇ g / kg body weight, or 40-200 ⁇ g / kg body weight, preferably 60. It has a body weight of ⁇ 140 ⁇ g / kg.
  • the execution interval of each administration is not particularly limited, but for example, the next administration may be carried out immediately after one administration. It may be carried out at intervals until the next administration.
  • the interval between each time is, for example, 12 to 48 hours, preferably about 24 hours (for example, 20 to 28 hours).
  • the order of the irradiation step and the administration step is not particularly limited.
  • the administration step may be carried out after the irradiation step is carried out, or the irradiation step may be carried out after the administration step is carried out. May be good.
  • the method for producing a model animal of the present invention may carry out the irradiation step and the administration step in parallel. In this case, in the method for producing a model animal of the present invention, the administration step may be carried out during the irradiation in the irradiation step, or the irradiation step may be carried out during the administration in the administration step.
  • the order of the irradiation and the administration is not particularly limited.
  • the method for producing a model animal of the present invention can induce a decrease in blood cell count more effectively, it is preferable to carry out the administration step after the irradiation step.
  • the reduction of blood cells by irradiation takes time, for example.
  • the decrease in blood cells due to the administration of anti-blood cell antibody is presumed to occur at an early stage, for example. Therefore, by carrying out the administration step after the irradiation step, in particular, by carrying out the administration step in accordance with the peak time of the decrease of blood cells after the irradiation step (about 7 days after the irradiation). The decrease in blood cell count can be induced more effectively.
  • the interval from the irradiation step to the administration step is not particularly limited.
  • the administration step may be carried out immediately after the irradiation step, or may be carried out at regular intervals.
  • the administration step is, for example, 4 to 14 days, 5 to 13 days after the irradiation step with reference to the time of the first irradiation, further reducing the blood cell count. It is preferably 6 to 10 days and 7 to 9 days because it can be effectively induced.
  • the execution time of the administration step may be determined, for example, based on the blood cell count of the non-human animal after the irradiation step.
  • the blood cell count in the non-human animal is, for example, 40% or less, 35% or less, based on the blood cell count in the normal animal corresponding to the target animal. 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, with respect to the non-human animal It is preferable to carry out the administration step.
  • the blood cells are, for example, any one or more of platelets, red blood cells, and white blood cells.
  • the time of carrying out the administration step is, for example, the platelet count, erythrocyte count, or leukocyte count in the non-human animal. It can be determined using the number as an index.
  • the method for producing the model animal of the present invention can produce the model animal of the present invention described later.
  • the first model animal for cytopenia of the present invention can be obtained by the method for producing a model animal of the present invention as described above.
  • the first model animal of the present invention is characterized by being obtained by the method for producing a model animal of the present invention, and other configurations and conditions are not particularly limited.
  • the first model animal of the present invention can be suitably used for the evaluation method of the present invention, the blood cell production method screening method, and the therapeutic drug candidate production method described later.
  • the description of the method for producing the model animal of the present invention can be incorporated.
  • the first model animal satisfies, for example, any one or more of the following conditions (1) to (3).
  • the first model animal is a thrombocytopenia model animal, it is preferable that the first model animal satisfies the following condition (1).
  • the first model animal is a erythrocytopenia model animal, it is preferable that the first model animal satisfies the following condition (2).
  • the first model animal is a leukopenia model animal, it is preferable that the first model animal satisfies the following condition (3).
  • (1) The number of platelets in the blood is 7.3 ⁇ 10 4 / ⁇ L or less.
  • the number of red blood cells in the blood is 500 ⁇ 10 4 cells / ⁇ L or less.
  • the number of white blood cells in the blood is 3 ⁇ 10 2 cells / ⁇ L or less.
  • the number of platelets in the blood is, for example, 1 to 13 days, preferably 1 to 13 days, based on the time of the final treatment in the irradiation step or the administration step in the method for producing a model animal of the present invention.
  • the number of platelets in the blood is preferably 5 ⁇ 10 4 / ⁇ L or less, and more preferably 4 ⁇ 10 4 / ⁇ L or less.
  • the number of platelets in the blood can be counted using, for example, a blood cell component, and for example, the number of isolated whole blood and its blood cell fraction can be counted by carrying out the above-mentioned blood cell count counting method. ..
  • the platelet count in blood means, for example, the average value of the platelet count in the blood of one first model animal or the blood platelet count of a plurality of first model animals.
  • the number of red blood cells in the blood is preferably 300 ⁇ 10 4 cells / ⁇ L or less, and more preferably 100 ⁇ 10 4 cells / ⁇ L or less.
  • the number of red blood cells in the blood can be counted using, for example, a blood cell component, and for example, the number of isolated whole blood and its blood cell fraction can be counted by carrying out the above-mentioned blood cell count counting method. ..
  • the number of red blood cells in blood means, for example, the average number of red blood cells in the blood of one first model animal or the number of red blood cells in the blood of a plurality of first model animals.
  • the white blood cell count in the blood is preferably 2 ⁇ 10 2 / ⁇ L or less, and more preferably 1 ⁇ 10 2 / ⁇ L or less.
  • the white blood cell count in the blood can be counted using, for example, a blood cell component, and can be counted, for example, by carrying out the above-mentioned blood cell count counting method for the isolated whole blood and its blood cell fraction. ..
  • the white blood cell count in blood means, for example, the average value of the white blood cell count in the blood of one first model animal or the white blood cell count in the blood of a plurality of first model animals.
  • the first model animal of the present invention may satisfy any one or more of the above conditions (1) to (3), may satisfy one, may satisfy a plurality, and satisfy all. You may.
  • the first model animal satisfies, for example, at least one of the following conditions (4) and (5).
  • the first model animal is a model animal for thrombocytopenia
  • the time from the start of bleeding to hemostasis exceeds 5 minutes (5)
  • the blood of the model animal contains an anti-blood cell antibody.
  • the method for measuring the hemostasis time is, for example, the time from the start of the bleeding to the stop of the bleeding when the blood vessel of the model animal is bleeding.
  • the blood vessels to be bleeding include, for example, arteries and veins, preferably peripheral arteries.
  • the bleeding artery is a tail artery, preferably a ventral tail artery.
  • the liquid is, for example, an isotonic liquid such as physiological saline.
  • the hemostasis time is, for example, more than 5 minutes, preferably 6 minutes or more, 7 minutes or more, 8 minutes or more, or 9 minutes or more, more preferably 10 minutes or more. is there.
  • the hemostasis time means, for example, the average value of the hemostasis time of one first model animal or the hemostasis time of a plurality of first model animals.
  • the anti-blood cell antibody is, for example, the same as the anti-blood cell antibody administered in the method for producing a first model animal of the present invention.
  • the anti-blood cell antibody can be carried out by, for example, a method for detecting an antibody against a specific antigen, for example, an ELISA method (Enzyme-Linked ImmunoSorbent Assay), a Western blotting method, a flow cytometry method or the like.
  • the blood may contain, for example, the humoral component of the blood, and specific examples thereof include whole blood, plasma, and serum.
  • the first model animal of the present invention may satisfy any one or more of the above conditions (4) to (5), one may be satisfied, or all may be satisfied, but all of them may be satisfied. It is preferable to meet.
  • the second model animal for cytopenia of the present invention satisfies any one or more of the following conditions (1) to (3): (1) The number of platelets in the blood is 7.3 ⁇ 10 4 / ⁇ L or less. (2) The number of red blood cells in the blood is 500 ⁇ 10 4 cells / ⁇ L or less. (3) The number of white blood cells in the blood is 3 ⁇ 10 2 cells / ⁇ L or less.
  • the second model animal of the present invention is characterized in that it satisfies any one or more of the above conditions (1) to (3), and other configurations and conditions are not particularly limited.
  • the second model animal of the present invention can be suitably used for the evaluation method of the present invention, the blood cell production method screening method, and the therapeutic drug candidate production method described later.
  • the method for producing the model animal of the present invention and the description of the first model animal can be incorporated.
  • the second model animal satisfies, for example, at least one of the following conditions (4) and (5).
  • the second model animal is a model animal for thrombocytopenia
  • the time from the start of bleeding to hemostasis exceeds 5 minutes (5)
  • the blood of the model animal contains an anti-blood cell antibody.
  • the method for evaluating blood cell function of the present invention includes a step of administering blood cells to a model animal of blood cell hypoplasia and an evaluation step of evaluating the function of the blood cells. It is a model animal of the blood cell hypoplasia of the present invention.
  • the evaluation method of the present invention is characterized in that the model animal for hemocytopenia used in the evaluation step is the first model animal and / or the second model animal of the present invention, and other steps and conditions. Is not particularly limited.
  • the evaluation method of the present invention uses the first model animal and / or the second model animal of the present invention in which the number of blood cells is reduced as compared with the blood cell hypoplasia model animal by X-ray irradiation, the function of blood cells. Can be evaluated more sensitively.
  • the description of the method for producing a model animal of the present invention, the first model animal, and the second model animal can be incorporated.
  • the "function of blood cells” means, for example, the function that the blood cells exert in a living body.
  • the function of the blood cell is, for example, a hemostatic function.
  • the function of the blood cell is the function of carrying oxygen and carbon dioxide.
  • the function of the blood cell is, for example, a biological defense function for recognizing and removing non-self substances, pathogens or abnormal cells.
  • the evaluation method of the present invention may evaluate, for example, one function or a plurality of functions.
  • the blood cell to be evaluated may be, for example, a blood cell isolated from an animal or a blood cell derived from a progenitor cell such as a pluripotent cell or a stem cell.
  • a progenitor cell such as a pluripotent cell or a stem cell.
  • the pluripotent cells include embryonic stem cell (ES) cells and induced pluripotent stem (iPS) cells.
  • the evaluation method of the present invention includes, for example, a step of isolating a test blood cell from an animal.
  • the blood cell may be, for example, isolated whole blood or a blood cell fraction thereof. Examples of the animal include humans and the non-human animals.
  • the method for producing blood cells of the present invention includes, for example, a step of inducing blood cells to be tested from progenitor cells.
  • the test cell is a platelet
  • for the induction of the platelet for example, International Publication No. 2011/034073 and International Publication No. 2012/157586 can be referred to.
  • the progenitor cell means, for example, a cell capable of inducing differentiation into a test blood cell.
  • the blood cells may be formulated blood cells, and examples thereof include blood products such as blood cell preparations; blood cell function mimics; and the like. Examples of the blood cell preparation include platelet preparations, red blood cell preparations, leukocyte preparations and the like.
  • the blood cell function mimetic agent is, for example, a preparation containing an alternative that mimics the function of blood cells, and specific examples thereof include a platelet function mimetic agent (Platelet functional mimetics), an erythrocyte function mimetic agent (Erythrocyte functional mimetics), and a leukocyte function mimicry. Agents (Leukocyte functional mimetics) and the like can be mentioned.
  • the evaluation method of the present invention may be carried out for the entire blood cell group, or the blood cells. It may be performed on a part of the group. In the latter case, the evaluation method of the present invention can be, for example, a sampling inspection.
  • the model animal for cytopenia is the first model animal and / or the second model animal of the present invention.
  • the first model animal and / or the second model animal of the present invention can be obtained by, for example, the method for producing the model animal of the present invention. Therefore, the evaluation method of the present invention may include a production step of producing a model animal for hemocytopenia by the method for producing a model animal of the present invention. The manufacturing step is carried out, for example, prior to the administration step.
  • blood cells blood cells to be evaluated
  • the model animal for cytopenia is, for example, a model animal in which the blood cells to be administered are decreased.
  • the number of blood cells to be administered to the model animal of cytopenia is not particularly limited, and can be appropriately set according to, for example, the type and body weight of the model animal.
  • the dose of platelets in the administration step may be, for example, 1 ⁇ 10 7 to 1 ⁇ 10 9 platelets / animal or 1 to 3 ⁇ 10 8 platelets / animal.
  • the dose of red blood cells in the administering step may be, for example, 300 ⁇ 10 7 ⁇ 500 ⁇ 10 9 erythrocytes / animal, or 300 ⁇ 500 ⁇ 10 8 red blood cells / mouse and the like.
  • the dose of leukocytes in the administration step is, for example, 1 ⁇ 10 1 to 1 ⁇ 10 7 leukocytes / animal, 2 ⁇ 10 1 to 5 ⁇ 10 3 leukocytes / animal, or 2 to 5 ⁇ . 10 2 white blood cells / animal can be given.
  • the type of the model animal and the type of animal from which the blood cells to be administered to the model animal are derived may be the same or different.
  • the blood cell to be evaluated may be a rodent blood cell or a blood cell other than a rodent (for example, a human blood cell).
  • the blood cell to be evaluated is, for example, human platelet.
  • the function of the blood cell is evaluated.
  • the method for evaluating the function of blood cells can be appropriately determined, for example, according to the type of blood cells.
  • the evaluation step can evaluate the function of the platelets, more specifically, the hemostatic function, based on, for example, bleeding the model animal or the hemostatic time of the model animal.
  • the bleeding and hemostasis times of the model animal can be incorporated into the above description.
  • the function of the platelets can be evaluated based on a predetermined standard.
  • the criteria may be set based on, for example, the hemostasis time obtained using platelets isolated from animals, or may be set by the evaluator at a desired time according to the purpose.
  • the standard hemostasis time is, for example, 5 minutes or less, preferably 2 minutes or less. In this case, in the evaluation step, if the hemostasis time of the blood cells is 5 minutes or less, preferably 2 minutes or less, it can be determined that the platelets satisfy the criteria.
  • the evaluation step evaluates the function of red blood cells, more specifically, the oxygen carrying function, based on anemia symptoms such as pallor of mucous membrane or skin, decreased activity, and tachypnea. it can.
  • the evaluation step administers a target such as, for example, an antigen, a bacterium or a virus to the model animal and an immune response to the target (eg, antibody production, production of inflammatory cytokines, specific).
  • a target such as, for example, an antigen, a bacterium or a virus
  • an immune response to the target eg, antibody production, production of inflammatory cytokines, specific.
  • the function of leukocytes more specifically, the biological defense function can be evaluated.
  • the method for producing blood cells of the present invention comprises an evaluation step of evaluating the function of blood cells for a test blood cell and a selection step of selecting blood cells satisfying the criteria as functional blood cells in the evaluation step. Including, the evaluation step is carried out by the method for evaluating blood cell function of the present invention.
  • the method for producing blood cells of the present invention is characterized in that the evaluation step is carried out by the method for evaluating blood cell function of the present invention, and other steps and conditions are not particularly limited. According to the method for producing a blood cell of the present invention, the function of the blood cell is evaluated by the evaluation method of the present invention, so that the function of the blood cell can be evaluated with high sensitivity.
  • the method for producing blood cells of the present invention for example, more functional blood cells can be produced.
  • the description of the method for producing a model animal of the present invention, the first model animal, the second model animal, and the evaluation method can be incorporated.
  • the blood cell to be tested may be, for example, a blood cell isolated from an animal, or a blood cell derived from a progenitor cell such as a pluripotent cell or a stem cell.
  • a progenitor cell such as a pluripotent cell or a stem cell.
  • the pluripotent cells include embryonic stem cell (ES) cells and induced pluripotent stem (iPS) cells.
  • the method for producing a blood cell of the present invention includes, for example, a step of isolating the blood cell to be tested from an animal.
  • the blood cell may be, for example, isolated whole blood or a blood cell fraction thereof. Examples of the animal include humans and the non-human animals.
  • the method for producing blood cells of the present invention includes, for example, a step of inducing blood cells to be tested from progenitor cells.
  • the test cell is a platelet
  • for the induction of the platelet for example, International Publication No. 2011/034073 and International Publication No. 2012/157586 can be referred to.
  • the blood cells may be formulated blood cells, and examples thereof include blood products such as blood cell preparations; blood cell function mimics; and the like.
  • the blood cell preparation include platelet preparations, red blood cell preparations, leukocyte preparations and the like.
  • the blood cell function mimetic agent is, for example, a preparation containing an alternative that mimics the function of blood cells, and specific examples thereof include a platelet function mimetic agent (Platelet functional mimetics), an erythrocyte function mimetic agent (Erythrocyte functional mimetics), and a leukocyte function mimicry. Agents (Leukocyte functional mimetics) and the like can be mentioned.
  • the evaluation step can be carried out, for example, in the same manner as the evaluation method of the present invention.
  • blood cells that meet the criteria in the evaluation step are selected as functional blood cells.
  • functional blood cells may be selected, for example, by collecting blood cells administered to the model animal for thrombocytopenia from the model animal for thrombocytopenia.
  • the blood cell to be tested is the blood cell group described above, in the selection step, for example, a blood cell group obtained by extracting blood cells satisfying the criteria may be selected as a functional blood cell.
  • the method for producing blood cells of the present invention may include a formulation step of producing a blood cell preparation from the selected functional blood cells.
  • the formulation step can be carried out in the same manner as, for example, a known method for producing a blood product.
  • the method for screening a candidate substance for a therapeutic agent for blood cell hypoplasia of the present invention includes a step of administering a test substance to a model animal of blood cell hypoplasia and a subject in which the number of blood cells in the blood increases in the model animal.
  • the model animal includes a selection step of selecting a test substance as a candidate substance for a therapeutic agent for blood cell hypoplasia, and the model animal is a model animal for blood cell hypoplasia of the present invention.
  • the screening method of the present invention is characterized by being the first model animal and / or the second model animal of the present invention as the model animal of the hemocytopenia, and other steps and conditions are particularly limited. Not done.
  • the screening method of the present invention uses the first model animal and / or the second model animal of the present invention in which the number of blood cells is reduced as compared with the blood cell hypoplasia model animal by X-ray irradiation, the function of blood cells. Can be evaluated more sensitively. Therefore, according to the screening method of the present invention, for example, a more effective candidate substance for a therapeutic agent for cytopenia can be screened.
  • the description of the method for producing a model animal of the present invention, the first model animal, the second model animal, and the evaluation method can be incorporated.
  • the model animal for cytopenia is the first model animal and / or the second model animal of the present invention.
  • the first model animal and / or the second model animal of the present invention can be obtained by, for example, the method for producing the model animal of the present invention. Therefore, the screening method of the present invention may include a production step of producing a model animal for hemocytopenia by the method for producing a model animal of the present invention. The manufacturing step is carried out, for example, prior to the administration step.
  • the test substance is administered to a model animal of cytopenia.
  • the test substance is not particularly limited, and is, for example, low molecular weight compound; nucleic acid molecule; protein; peptide; compound library; expression product of gene library; cell extract; cell culture supernatant; fermented microbial product; ocean. Biological extracts; plant extracts and the like.
  • the peptide may be a cyclic peptide such as a special cyclic peptide.
  • the method of administering the test substance to the model animal for cytopenia of the present invention is not particularly limited.
  • the administration method include oral administration and parenteral administration.
  • the parenteral administration include intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, nasal administration, pulmonary administration and transdermal administration.
  • the test substance is a protein and a peptide
  • a viral vector having a gene encoding the protein or the like is constructed, and the gene is used as a model animal for hemocytopenia of the present invention by utilizing its infectivity. It may be administered and expressed in the body.
  • a test substance that increases the number of blood cells in the blood of the model animal is selected as a candidate substance for a therapeutic agent for hypocytopenia.
  • the increase in the number of blood cells in the blood may be determined based on, for example, the number of blood cells, or may be determined in comparison with the comparison target.
  • the comparison target includes the blood cell count in the blood of the model animal before administration of the test substance, the blood cell count of the control model animal to which the test substance is not administered, and the like.
  • the comparison target is the blood cell count in the blood of the model animal before the administration of the test substance
  • the blood cell count is significantly increased as compared with the blood cell count in the blood of the model animal before the administration of the test substance. If so, it can be determined that the number of blood cells in the blood is increasing in the selection step.
  • the comparison target is the blood cell count of the control model animal to which the test substance is not administered
  • the blood cell count is significantly increased as compared with the control model animal to which the test substance is not administered. If so, it can be determined that the number of blood cells in the blood is increasing in the selection step.
  • the method for producing a candidate substance for a therapeutic agent for blood cell hypoplasia of the present invention includes a selection step of selecting a candidate substance for a therapeutic agent for blood cell hypoplasia from a test substance, and the selection step includes the present invention. It is carried out by the screening method of the present invention.
  • the method for producing a candidate substance for a therapeutic agent of the present invention is characterized in that the selection step is carried out by the screening method of the present invention, and other steps and conditions are not particularly limited.
  • the selection step is carried out by the screening method of the present invention, for example, a more effective candidate substance for a therapeutic agent for hemocytopenia can be produced.
  • the description of the method for producing a model animal of the present invention, the first model animal, the second model animal, the evaluation method, and the screening method can be incorporated.
  • Example 1 It was confirmed that the method for producing a model animal for thrombocytopenia of the present invention can produce a model animal for thrombocytopenia in which platelets are reduced.
  • TKDN SeV2 and NIH5 human fetal skin fibroblast-derived iPS cells established using Sendai virus
  • differentiation culture into blood cells was carried out. Specifically, human ES / iPS cell colonies were co-cultured with C3H10T1 / 2 feeder cells in the presence of 20 ng / mL VEGF (manufactured by R & D SYSTEMS) for 14 days to prepare hematopoietic progenitor cells (HPC). ..
  • the gene transfer system used the lentiviral vector system.
  • the lentiviral vector is a Tetracycline-controlled Tet-on® gene expression induction system vector. It was made by recombining the mOKS cassette of LV-TRE-mOKS-Ubc-tTA-I2G (Reference 2 below) with c-MYC, BMI1, or BCL-xL. Vectors with c-MYC, BMI1, or BCL-xL introduced into LV-TRE-c-Myc-Ubc-tTA-I2G, LVTRE-BMI1-Ubc-tTA-I2G, and LV-TRE-BCL-, respectively.
  • the c-MYC, BMI1, and BCL-xL viruses were prepared by gene transfer into 293T cells with the lentiviral vector. By infecting the cells of interest with the resulting virus, the c-MYC, BMI1, and BCL-xL genes are introduced into the genomic sequence of the cells of interest. These genes, which have been stably introduced into the genome sequence, can be forcibly expressed by adding doxycycline (clontech # 631311) to the medium.
  • the spin infection was performed twice every 12 hours.
  • the medium was basal medium (15% Fetal Bovine Serum (GIBCO), 1% Penicillin-Streptomycin-Glutamine (GIBCO), 1% Insulin, Transferrin, Selenium Solution (ITS-G) (GIBCO), 0.45 mmol / L 1-Thioglycerol).
  • IMDM Iscove's Modified Dulbecco's Medium
  • Sigma-Aldrich 50 ⁇ g / mL L-Ascorbic Acid
  • 50 ng / mL Human thrombopoietin TPO
  • Protamine was further added to the medium (hereinafter referred to as differentiation medium) to which 50 ng / mL Human Stem Cell Factor (SCF) (R & D SYSTEMS) and 2 ⁇ g / mL Doxycycline (Dox, clontech # 631311) were added so as to be the final concentration.
  • the medium was added so that the value was 10 ⁇ g / mL.
  • virus-infected blood cells obtained by the above method were collected by pipetting, and the supernatant was removed by centrifugation at 1200 rpm for 5 minutes. It was then suspended in a new differentiation medium and seeded on new C3H10T1 / 2 feeder cells (6 well plate). Subculture was performed by performing the same operation on the 9th day of infection. At the time of re-seeding, after counting the number of cells, the cells were seeded on C3H10T1 / 2 feeder cells so as to have 1 ⁇ 10 5 cells / 2 mL / well (6 well plate).
  • BCL-xL virus infection in megakaryocyte autoproliferation strain BCL-xL was introduced into the megakaryocyte autoproliferation strain on the 14th day of infection by the lentivirus method using BCL-xL virus.
  • Virus particles were added to the medium to achieve MOI 10 and infected by spin infection (32 ° C, 900 rpm, centrifuge for 60 minutes). Forced expression of the BCL-xL gene was carried out by adding Dox to the medium to a concentration of 1 ⁇ g / mL Dox.
  • the BCL-xL infection day 24 were collected megakaryocyte self-propagating strains transformed gene, cells 1.0 ⁇ 10 5 per anti-human CD41a-APC antibody (BioLegend), anti-human CD42b-PE antibody (eBioscience), and Anti-human CD235ab-Pacific Blue (Anti-CD235ab-PB; BioLegend) antibodies were immunostained with 2 ⁇ L, 1 ⁇ L, and 1 ⁇ L, respectively, and then analyzed with FACS Verse TM. Then, on the 24th day of infection, a strain having a CD41a positive rate of 50% or more was designated as an immortalized megakaryocyte cell line. These cells, which were able to proliferate for 24 days or more after infection, were designated as immortalized megakaryocyte cell lines SeV2-MKCL and NIH5-MKCL.
  • SeV2-MKCL and NIH5-MKCL were statically cultured in a 10 cm dish (10 mL / dish).
  • IMDM was used as the basic medium, and the following components were added (concentration is final concentration).
  • the culture conditions were 27 ° C. and 5% CO 2 .
  • the cells were cultured in the presence of the platelet production medium for 6 days to produce platelets, thereby producing a culture of megakaryocytes.
  • the cleaning storage liquid bags 1 and 2 include a cleaning storage liquid.
  • the wash and storage solution was prepared by adding 20% (v / v%) ACD and 2.5% (w / v%) human serum albumin to a bicanate infusion solution (bicarbon infusion solution, manufactured by Otsuka Pharmaceutical Co., Ltd.) and adjusting the pH to 7.2 with NaOH. I used the one that I did.
  • the culture of the megakaryocytes was concentrated using a hollow fiber membrane (Plasmaflow OP, manufactured by Asahi Kasei Medical Co., Ltd.), and the obtained concentrate of the culture of megakaryocytes was placed in a storage bag. Recovered.
  • the cell bag containing the culture to which the ACD-A solution was added was bonded to the ACP215 disposable set using a sterile bonding device. Then, I started ACP215 in service mode and set the rotation speed to 2500 rpm (350 x g). ACP215 was started and the culture in the cell bag was introduced into a separation bowl at about 100 mL / min. The liquid component flowing out of the separation bowl was collected in a collection bag. After introducing the entire amount of the culture in the cell bag into the separation bowl, an additional 500 mL of a wash storage solution was introduced into the separation bowl. After introducing the washing and preserving solution into the separation bowl, centrifugation was stopped and a collection bag containing the recovery solution (recovered liquid component containing platelets) was separated using a tube sealer.
  • a recovery bag containing a recovery solution (including platelets) was joined to the new ACP215 disposable set using the aseptic joining device.
  • the collection bag containing the collection liquid was installed on the stand.
  • centrifugal speed of ACP215 was changed to 5000 rpm (1398.8 x g), and centrifugation was started.
  • automatic injection was changed to manual injection.
  • the recovered solution was introduced into the separation bowl at an introduction rate of about 100 mL / min. After adding the entire amount of the recovered solution to the separation bowl, an additional 500 mL of a washing and preserving solution was added.
  • Model animal for thrombocytopenia A model animal for thrombocytopenia was prepared according to FIG. Specifically, as a model animal of the example, an 8-week-old male NOG mouse (NOD.Shi-scid, IL-2R ⁇ KO Jic (NOG), manufactured by Central Institute for Experimental Animals) was used as ketamine. A mixture of (100 mg / kg body weight) and xylazine (10 mg / kg body weight) was intraperitoneally administered under anesthesia. Next, the anesthetized mouse was placed in an X-ray irradiation device (MX-80Labo, Mediextech Co., Ltd.) and irradiated with a dose of 5 Gy (day 0). One day later, the similarly anesthetized mice were irradiated with a dose of 5 Gy again (day 1).
  • NOG 8-week-old male NOG mouse
  • IL-2R ⁇ KO Jic manufactured by Central Institute for Experimental Animals
  • test substance On the 9th day, 200 ⁇ L of the platelet preparation prepared in (3) above was added to half of each mouse prepared in (4) above as a test substance, 2 ⁇ such that 10 8 platelets, were administered into the tail vein using a needle and syringe (platelet product administration group, ACP161N5). In place of the platelet preparation, 200 ⁇ L of the vicanate infusion (Vehicle) was administered to the other half as a test substance (Vehicle administration group).
  • Rat IgG1, ⁇ Isotype Control antibody and APC Mouse IgG1, ⁇ Isotype Control antibody labeled with PE / Cy7 and APC Mouse IgG1, ⁇ Isotype Control antibody (for unstained sample, sample for staining calibration: Cat.No. 400416, 400120, Biolegend, Inc. ) Or PE-Cy7 labeled anti-mouse CD41 antibody and APC anti-human CD41 antibody (for stained samples: Cat. No. 133916, 303710, Biolegend, Inc.) were added in 5 ⁇ L increments and stirred at room temperature. The reaction was allowed to stand for 15 minutes under shading. PBS was added to each tube after the reaction so as to be 400 ⁇ L / tube, and used for the measurement. Each sample was stored at room temperature in the dark until measurement.
  • the platelet fraction in the unstained sample was gated and developed on a PE-Cy7 histogram. Upon development, the sensitivity of PE-Cy7 was adjusted so that the negative peak was displayed appropriately. Then, the sample for staining calibration was set on the flow cytometer and developed by the histogram of APC. Upon deployment, the sensitivity of the APC was adjusted so that the negative and positive peaks were in the appropriate positions.
  • the staining calibration sample was developed with a two-dimensional histogram of APC / PE-Cy7, and fluorescence correction was performed as necessary. Based on the conditions set above, the number of platelets in the stained sample was measured. The measurement was completed when 6,000 Trucount Tubes beads were measured. The sensitivity was adjusted once for each measurement, and the conditions were not changed between the samples.
  • FIG. 3 is a graph showing the number of platelets in the blood of each mouse.
  • the horizontal axis represents the sample type and date
  • the vertical axis represents the platelet count.
  • the platelet count was approximately 250 ⁇ 10 4 platelets / ⁇ L in all groups.
  • platelet count in blood becomes less 7.3 ⁇ 10 4 platelets / [mu] L It was confirmed that thrombocytopenia had occurred.
  • the mice in comparative examples and reference examples the number of platelets compared to the blood to day 0 is reduced, in part, the number of platelets in the blood becomes less 7.3 ⁇ 10 4 platelets / [mu] L cage, although thrombocytopenia occurs, most greater than 7.3 ⁇ 10 4 platelets / [mu] L, no thrombocytopenia occurs, it was found that a large individual difference. Therefore, the model animal for platelet hypoplasia can be produced by the method for producing a model animal of the present invention, and the model animal obtained by the method for producing a model animal of the present invention is compared with the model animal obtained by X-ray irradiation.
  • Example 2 The bleeding time of a model animal for thrombocytopenia prepared by the production method of the present invention was confirmed.
  • the mice were anesthetized with isoflurane anesthesia.
  • the ventral tail artery of each individual was incised at a portion 2 cm from the tip of the tail using the edge portion of a 23 G injection needle. After confirming bleeding from the incision, 45 mL of physiological saline kept at 37 ° C.
  • mice used in Example 1 one individual was designated as a satellite from Examples, Comparative Examples, and Reference Examples, so it was not used in this test.
  • the bleeding time of each mouse is shown in FIG. 4 and Table 3 below.
  • FIG. 4 is a graph showing the bleeding time.
  • the horizontal axis represents the type of sample and the type of test substance administered
  • the vertical axis represents the bleeding time.
  • the average value of the bleeding time in the Vehicle-administered group was 177 ⁇ 23 seconds (Mean ⁇ SE (standard error), hereinafter the same), and the platelet preparation.
  • the average bleeding time in the administration group was 97 ⁇ 9 seconds, and there was a tendency for the bleeding time to be shortened by the administration of platelet preparations.
  • the average value of the bleeding time in the Vehicle-administered group was 111 ⁇ 10 seconds, and the average value of the bleeding time in the platelet preparation-administered group was 83 ⁇ 17 seconds, and bleeding due to the administration of the platelet preparation. No tendency to shorten the time was observed.
  • the bleeding time of all the individuals exceeded 600 seconds, and a remarkable prolongation of the bleeding time was observed.
  • the average value of the bleeding time in the platelet preparation group in the mice of the examples was 213 ⁇ 34 seconds, and a clear reduction in the bleeding time due to the administration of the platelet preparation was observed.
  • the model animal for thrombocytopenia prepared by the production method of the present invention has a decreased platelet count and a remarkable prolongation of bleeding time, and is therefore suitable for evaluation of platelet function. It turned out.
  • mice general condition, changes in body weight, changes in platelet count, and bleeding time due to different intensity X-ray irradiation were confirmed.
  • the time of the first X-ray irradiation is set as the 0th day.
  • untreated (X-ray non-irradiated group, Normal) mice were also added to the test. Then, on days 0, 5, and 9, the weight of each mouse was recorded.
  • the antibody to be added to the sample was labeled with PE / Cy7, Rat IgG1, ⁇ Isotype Control antibody (unstained sample and stained calibration sample: Cat. No. 400416, Biolegend, Inc.), Alternatively, the same as in Examples 1 (6-3) and (6-4) except that the anti-mouse CD41 antibody labeled with PE-Cy7 (stained sample: Cat. No. 133916, Biolegend, Inc.) was 5 ⁇ L. Then, the platelet count was measured, the flow cytometer was measured, and the data was analyzed. In addition, after the blood sampling on the 9th day, the bleeding time was measured in the same manner as in Example 2 except that the test substance was not administered to each mouse. Since one of the mice in the 6 Gy irradiation group rebleeded after confirming hemostasis, the measurement of the bleeding time was restarted, and the measurement was terminated when no bleeding was observed again.
  • the platelet count of the mouse tended to decrease significantly as the X-ray irradiation intensity increased.
  • the number of platelets is reduced to about 76 ⁇ 10 4 platelets / ⁇ L, the number of platelets is reduced to 7.3 ⁇ 10 4 platelets / ⁇ L or less by administration of the antiplatelet antibody. Therefore, it was suggested that a model animal for thrombocytopenia can be produced by administering an antiplatelet antibody after irradiation of 4 to 6 Gy each time when X-rays are irradiated twice.
  • the mice irradiated with X-rays tended to prolong the bleeding time depending on the amount of X-ray irradiation as compared with the mice in the X-ray non-irradiated group, but the variation among individuals was very large. It was big.
  • the dose at the time of X-ray irradiation on the 0th day was 1.5 Gy, 2.0 Gy or 2.5 Gy (1.5 Gy).
  • the time of the first X-ray irradiation is set as the 0th day.
  • a model animal for thrombocytopenia can be produced by administering an antiplatelet antibody after X-ray irradiation of 1.5 Gy or more each time when X-ray is irradiated once.
  • the platelet count can be reduced as the irradiation dose is increased.
  • a model animal for thrombocytopenia can be produced by administering an antiplatelet antibody after each irradiation with 1.5 Gy or more of X-rays.
  • Appendix 1 Irradiation process to irradiate non-human animals and A method for producing a model animal for cytopenia, which comprises an administration step of administering an anti-blood cell antibody to the non-human animal.
  • Appendix 2 The production method according to Appendix 1, wherein the anti-blood cell antibody is an antibody against a cell membrane surface antigen of blood cells.
  • Appendix 3 The production method according to Appendix 1 or 2, wherein the anti-blood cell antibody is an antibody against a cell membrane surface antigen of platelets.
  • the antibody against the cell membrane surface antigen of the platelets comprises a group consisting of an anti-CD41 antibody, an anti-CD42a antibody, an anti-CD42b antibody, an anti-CD42c antibody, an anti-CD42d antibody, an anti-CD49b antibody, an anti-CD61 antibody, an anti-CD109 antibody, and an anti-GPVI antibody.
  • (Appendix 5) The production method according to any one of Supplementary note 1 to 4, wherein the anti-blood cell antibody is a cell-removing functional antibody.
  • (Appendix 10) The production method according to any one of Supplementary note 1 to 9, wherein the administration step is carried out on 5 to 13 days after the irradiation step with reference to the time of the first irradiation.
  • (Appendix 11) The production method according to any one of Appendix 1 to 10, wherein the non-human animal is a rodent.
  • (Appendix 12) The production method according to any one of Appendix 1 to 11, wherein the non-human animal is a mouse.
  • (Appendix 13) The production method according to Appendix 12, wherein the mouse is an immunodeficient mouse or a mouse in which the reticuloendothelial system is destroyed.
  • (Appendix 14) The production method according to Appendix 13, wherein the immunodeficient mouse is a NOG mouse.
  • Appendix 15 The production method according to any one of Supplementary note 1 to 14, wherein the blood cell is at least one selected from the group consisting of platelets, erythrocytes, and white blood cells.
  • Appendix 16 A model animal for cytopenia obtained by the method for producing a model animal for cytopenia according to any one of Appendix 1 to 15.
  • (Appendix 17) The model animal for cytopenia according to Appendix 16, which satisfies any one or more of the following conditions (1) to (3): (1) The number of platelets in the blood is 7.3 ⁇ 10 4 / ⁇ L or less.
  • the number of red blood cells in the blood is 500 ⁇ 10 4 cells / ⁇ L or less.
  • the number of white blood cells in the blood is 3 ⁇ 10 2 cells / ⁇ L or less.
  • a model animal for cytopenia that satisfies any one or more of the following conditions (1) to (3): (1) The number of platelets in the blood of the model animal is 7.3 ⁇ 10 4 cells / ⁇ L or less.
  • the number of red blood cells in the blood is 500 ⁇ 10 4 cells / ⁇ L or less.
  • the number of white blood cells in the blood is 3 ⁇ 10 2 cells / ⁇ L or less.
  • (Appendix 19) The model animal according to any one of Appendix 16 to 18, which satisfies at least one of the following conditions (4) and (5): (4) The time from the start of bleeding to hemostasis exceeds 5 minutes; (5) The blood of the model animal contains an anti-blood cell antibody. (Appendix 20) It includes a step of administering blood cells to a model animal of cytopenia and an evaluation step of evaluating the function of the blood cells. The method for evaluating blood cell function, wherein the model animal for cytopenia is the model animal for cytopenia according to any one of Appendix 16 to 19.
  • the blood cells are platelets and The evaluation method according to Appendix 20, wherein in the evaluation step, the model animal is bleeding to evaluate the function of the platelets.
  • the blood cells are platelets and The evaluation method according to Appendix 20 or 21, wherein in the evaluation step, the function of the platelets is evaluated based on the hemostasis time.
  • the blood cells are platelets and The evaluation method according to any one of Appendix 20 to 22, wherein the function of the platelet is a hemostatic function.
  • the blood cells are platelets and The evaluation method according to any one of Appendix 20 to 23, wherein the platelet is a platelet preparation or a platelet functional mimetics.
  • the blood cells are platelets and The evaluation method according to any one of Appendix 20 to 24, wherein the platelet is a human platelet.
  • An evaluation process for evaluating the function of blood cells to be tested and The evaluation step includes a selection step of selecting blood cells satisfying the criteria as functional blood cells.
  • the method for producing blood cells, wherein the evaluation step is carried out by the method for evaluating blood cell function according to any one of Appendix 20 to 26.
  • Appendix 28 The production method according to Appendix 27, wherein the blood cells are platelets.
  • Appendix 29 The process of administering the test substance to a model animal for cytopenia, The model animal includes a selection step of selecting a test substance that increases the number of blood cells in the blood as a candidate substance for a therapeutic agent for hypocytopenia.
  • Appendix 30 The screening according to Appendix 29, wherein in the selection step, the test substance having an increased blood cell count is selected as the therapeutic candidate substance as compared with the control model animal to which the test substance is not administered.
  • Appendix 31 The screening method according to Appendix 29 or 30, wherein the test substance is at least one selected from the group consisting of low molecular weight compounds, peptides, proteins and nucleic acids.
  • Appendix 32 The screening method according to any of Supplementary note 29 to 31, wherein the blood cells are platelets.
  • Appendix 33 The screening method according to any one of Annex 29 to 32, which comprises a manufacturing step of producing a model animal for cytopenia by the method for producing a model animal for cytopenia according to any one of Supplements 1 to 15.
  • Appendix 34 Includes a selection process that selects candidate substances for the treatment of cytopenia from the test substances.
  • the selection step is a method for producing a candidate substance for a therapeutic drug for cytopenia, which is carried out by the screening method for a candidate substance for a therapeutic drug for cytopenia according to any one of Supplementary notes 29 to 33.
  • the number of blood cells in the blood of the obtained model animal can be reduced as compared with the method for producing a model animal by X-ray irradiation. Further, according to the present invention, for example, the number of blood cells in the blood of the obtained model animal can be reduced as compared with the blood cell hypoplasia model animal by X-ray irradiation. Therefore, when the blood cells are platelets, Hemostasis time (bleeding time) at the time of bleeding can be extended. Therefore, according to the present invention, it is possible to produce a model animal for cytopenia suitable for, for example, evaluation of blood cell function and acquisition of a candidate substance for treatment of cytopenia including thrombocytopenia. Therefore, the present invention is extremely useful in, for example, the field of cell medicine using blood cells, the field of blood products, the field of medical treatment, and the like.

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Abstract

Provided is a method for producing a cytopenia model animal having a lower blood cell count than an X-ray irradiation-induced cytopenia model animal. This method for producing a cytopenia model animal comprises: an irradiation step for irradiating a non-human animal with radiation; and an administration step for administering an anti-blood cell antibody to the non-human animal.

Description

血球減少症のモデル動物の製造方法、血球減少症モデル動物、血球機能の評価方法、血球の製造方法、血球減少症の治療薬候補物質のスクリーニング方法、および血球減少症の治療薬の候補物質の製造方法Method for producing a model animal for blood cell hypoplasia, model animal for blood cell hypoplasia, method for evaluating blood cell function, method for producing blood cells, method for screening candidate substances for therapeutic drug for blood cell hypoplasia, and candidate substances for therapeutic drug for blood cell hypoplasia Production method
　本発明は、血球減少症のモデル動物の製造方法、血球減少症モデル動物、血球機能の評価方法、血球の製造方法、血球減少症の治療薬候補物質のスクリーニング方法、および血球減少症の治療薬の候補物質の製造方法に関する。 The present invention relates to a method for producing a model animal for cytopenia, a model animal for cytopenia, a method for evaluating blood cell function, a method for producing blood cells, a method for screening a candidate substance for a therapeutic agent for cytopenia, and a therapeutic agent for cytopenia. Regarding the method for producing the candidate substance of.
　血小板製剤は、手術、傷害等の出血時、その他血小板の減少を伴う患者に対して投与される。血小板製剤は、献血で得られた血液から現在製造されている。しかしながら、人口構成の変化から、献血量が低減し、血小板製剤が不足することが懸念されている。 Platelet preparations are administered to patients with decreased platelets during bleeding such as surgery or injury. Platelet preparations are currently produced from blood obtained from blood donations. However, due to changes in the population composition, there is concern that the amount of blood donated will decrease and that platelet preparations will be in short supply.
　また、献血の提供者が細菌等の感染症に罹患している場合、血液が細菌汚染されている可能性があるため、細菌汚染された血小板製剤の投与による感染症のリスクがある。このため、in vitroで血小板を製造する方法が開発されている（非特許文献１）。 Also, if the blood donor is suffering from an infectious disease such as bacteria, there is a risk of infectious disease due to administration of a bacterially contaminated platelet preparation because the blood may be contaminated with bacteria. Therefore, a method for producing platelets in vitro has been developed (Non-Patent Document 1).
　in vitroで製造された血小板の中には、血小板の機能である止血作用を十分に有さない血小板も存在するため、製造された血小板の機能を評価できる方法が求められている。そこで、血小板の機能の評価方法として、正常な動物と比較して、血小板の数が減少したモデル動物（以下、「血小板減少症モデル動物」または「モデル動物」という）に血小板を投与し、止血機能が回復するかを指標に評価可能かを検討した。 Since some platelets produced in vitro do not have a sufficient hemostatic effect, which is the function of platelets, a method capable of evaluating the function of the produced platelets is required. Therefore, as a method for evaluating the function of platelets, platelets are administered to a model animal in which the number of platelets is reduced as compared with a normal animal (hereinafter referred to as "thrombocytopenia model animal" or "model animal") to stop bleeding. We examined whether it could be evaluated using the recovery of function as an index.
　動物において血小板を減少させる方法としては、ＮＯＧマウス（NOD/Shi-scid-IL2Rγnullマウス）にＸ線を全身照射し、前記動物の骨髄を破壊する方法があり、血小板減少症のモデルマウス、または血球数が減少したモデル動物（以下、「血球減少症モデル動物」ともいう）として使用されている。そこで、本発明者らは、前記Ｘ線照射による血球減少症モデル動物を用いて、血小板の機能を評価しようとしたところ、前記Ｘ線照射による血球減少症モデル動物では、出血時間が短く、一定しないため、血小板の機能評価が困難という問題が生じることを見出した。これは、前記Ｘ線照射による血球減少症モデル動物では、血小板数は減少しており、血小板減少症の病態を分析するモデル動物としては使用できるが、血小板の止血機能を評価するには、血小板の減少の程度または個体間のばらつきの抑制が不十分であるためと推定された。また、血小板以外の血球についても、同様の問題を見出した。 As a method of reducing platelets in animals, there is a method of systemically irradiating NOG mice (NOD / Shi-scid-IL2Rγ null mice) with X-rays to destroy the bone marrow of the animals, and a model mouse for thrombocytopenia, or It is used as a model animal with a decreased blood cell count (hereinafter, also referred to as "thrombocytopenia model animal"). Therefore, the present inventors attempted to evaluate the function of platelets using the X-ray irradiation-induced cytopenia model animal, and found that the X-ray irradiation-induced cytopenia model animal had a short bleeding time and was constant. Therefore, it was found that the problem of difficulty in evaluating the function of platelets arises. This can be used as a model animal for analyzing the pathophysiology of thrombocytopenia because the platelet count is decreased in the model animal for cytopenia caused by X-ray irradiation. However, in order to evaluate the hemostatic function of platelets, platelets are used. It was presumed that the degree of decrease or the suppression of variation among individuals was insufficient. We also found similar problems with blood cells other than platelets.
　そこで、本発明は、例えば、Ｘ線照射による血球減少症モデル動物と比較して、より血球数が減少した血球減少症のモデル動物の製造方法の提供を目的とする。 Therefore, an object of the present invention is, for example, to provide a method for producing a model animal for blood cell hypoplasia in which the number of blood cells is further reduced as compared with the model animal for blood cell hypoplasia by X-ray irradiation.
　前記目的を達成するために、本発明の血球板減少症のモデル動物の製造方法（以下、「モデル動物の製造方法」ともいう）は、非ヒト動物に放射線を照射する照射工程と、
前記非ヒト動物に抗血球抗体を投与する投与工程とを含む。 In order to achieve the above object, the method for producing a model animal for blood cell hypoplasia of the present invention (hereinafter, also referred to as "method for producing a model animal") includes an irradiation step of irradiating a non-human animal with radiation.
The administration step of administering the anti-blood cell antibody to the non-human animal is included.
　本発明の第１の血球減少症のモデル動物（以下、「第１のモデル動物」ともいう）は、前記本発明の血球減少症のモデル動物の製造方法により得られる。 The first model animal for cytopenia of the present invention (hereinafter, also referred to as "first model animal") is obtained by the method for producing a model animal for cytopenia of the present invention.
　本発明の第２の血球減少症のモデル動物（以下、「第２のモデル動物」ともいう）は、下記条件（１）～（３）のいずれか一つ以上を満たす：
（１）血液中の血小板数が、７．３×１０４個／μＬ以下である。
（２）血液中の赤血球数が、５００×１０４個／μＬ以下である。
（３）血液中の白血球数が、３×１０２個／μＬ以下である。 The second model animal for cytopenia of the present invention (hereinafter, also referred to as “second model animal”) satisfies any one or more of the following conditions (1) to (3):
(1) The number of platelets in the blood is 7.3 × 10 4 / μL or less.
(2) The number of red blood cells in the blood is 500 × 10 4 cells / μL or less.
(3) The number of white blood cells in the blood is 3 × 10 2 cells / μL or less.
　本発明の血球機能の評価方法（以下、「評価方法」ともいう）は、血球減少症のモデル動物に血球を投与する工程と
前記血球の機能を評価する評価工程とを含み、
前記血球減少症のモデル動物は、前記本発明の血球減少症のモデル動物である。 The method for evaluating blood cell function of the present invention (hereinafter, also referred to as “evaluation method”) includes a step of administering blood cells to a model animal of blood cell hypoplasia and an evaluation step of evaluating the function of the blood cells.
The model animal for cytopenia is the model animal for cytopenia of the present invention.
　本発明の血球の製造方法は、被検血球について、血球の機能を評価する評価工程と、
前記評価工程において、基準を満たす血球を機能性の血球として選抜する選抜工程とを含み、
前記評価工程は、前記本発明の血球機能の評価方法により実施される。 The method for producing a blood cell of the present invention includes an evaluation step for evaluating the function of the blood cell of the test blood cell and an evaluation step.
The evaluation step includes a selection step of selecting blood cells satisfying the criteria as functional blood cells.
The evaluation step is carried out by the method for evaluating blood cell function of the present invention.
　本発明の血球減少症の治療薬候補物質のスクリーニング方法（以下、「スクリーニング方法」ともいう）は、被検物質を血球減少症のモデル動物に投与する工程と、
前記モデル動物に血液中の血球数が増加する被検物質を、血球減少症の治療薬の候補物質として選択する選択工程とを含み、
前記モデル動物は、本発明の血球減少症のモデル動物である。 The method for screening a candidate substance for a therapeutic agent for cytopenia of the present invention (hereinafter, also referred to as “screening method”) includes a step of administering a test substance to a model animal for cytopenia and
The model animal includes a selection step of selecting a test substance that increases the number of blood cells in the blood as a candidate substance for a therapeutic agent for hypocytopenia.
The model animal is a model animal for cytopenia of the present invention.
　本発明の血球減少症の治療薬の候補物質の製造方法は、被検物質から血球減少症の治療薬の候補物質を選抜する選抜工程を含み、
前記選抜工程は、前記本発明のスクリーニング方法で実施される。 The method for producing a candidate substance for a therapeutic agent for cytopenia of the present invention includes a selection step of selecting a candidate substance for a therapeutic agent for cytopenia from a test substance.
The selection step is carried out by the screening method of the present invention.
　本発明によれば、Ｘ線照射による血球減少症モデル動物と比較して、血球数が減少した血球減少症のモデル動物を製造できる。 According to the present invention, it is possible to produce a model animal of blood cell hypoplasia in which the number of blood cells is reduced as compared with a model animal of blood cell hypoplasia by X-ray irradiation.
図１は、実施例１における濃縮システムを示す模式図である。FIG. 1 is a schematic diagram showing a concentration system in Example 1. 図２は、実施例１における実施例、比較例および参考例のマウスの調製方法を示すグラフである。FIG. 2 is a graph showing a method for preparing mice of Examples, Comparative Examples, and Reference Examples in Example 1. 図３は、実施例１におけるマウスの血液中の血小板数を示すグラフである。FIG. 3 is a graph showing the number of platelets in the blood of the mouse in Example 1. 図４は、実施例２における出血時間を示すグラフである。FIG. 4 is a graph showing the bleeding time in Example 2.
＜血球減少症のモデル動物の製造方法＞
　本発明の血球減少症のモデル動物の製造方法は、前述のように、非ヒト動物に放射線を照射する照射工程と、前記非ヒト動物に抗血球抗体を投与する投与工程とを含む。本発明のモデル動物の製造方法は、前記照射工程と前記投与工程とを実施することが特徴であり、その他の工程および条件は、特に制限されない。本発明のモデル動物の製造方法によれば、前記Ｘ線照射によるモデル動物の製造方法と比較して、得られたモデル動物の血中の血球数を減少させることができる。また、本発明のモデル動物の製造方法によれば、例えば、Ｘ線照射による血球減少症モデル動物と比較して、得られたモデル動物の血中の血球数を減少させることができるため、前記血球が血小板の場合、出血時の止血時間（出血時間）が延長できる。このため、本発明のモデル動物の製造方法によれば、例えば、血球機能の評価、血小板減少症を含む血球減少症の治療用候補物質の取得に適した血球減少症のモデル動物を製造できる。また、本発明のモデル動物の製造方法によれば、例えば、得られたモデル動物の血中の血球数を十分に減少させることができるため、血球の減少率の高い再現性および個体間のばらつきが抑制されたモデル動物を製造できる。前記ばらつきは、例えば、標準誤差として、比較できる。 <Manufacturing method of model animals for cytopenia>
As described above, the method for producing a model animal for hemocytopenia of the present invention includes an irradiation step of irradiating a non-human animal with radiation and an administration step of administering an anti-blood cell antibody to the non-human animal. The method for producing a model animal of the present invention is characterized by carrying out the irradiation step and the administration step, and other steps and conditions are not particularly limited. According to the method for producing a model animal of the present invention, the number of blood cells in the blood of the obtained model animal can be reduced as compared with the method for producing a model animal by X-ray irradiation. Further, according to the method for producing a model animal of the present invention, for example, the number of blood cells in the blood of the obtained model animal can be reduced as compared with the blood cell hypoplasia model animal by X-ray irradiation. When the blood cells are platelets, the hemostasis time (bleeding time) at the time of bleeding can be extended. Therefore, according to the method for producing a model animal of the present invention, for example, a model animal for cytopenia suitable for evaluation of blood cell function and acquisition of a candidate substance for treatment of cytopenia including thrombocytopenia can be produced. Further, according to the method for producing a model animal of the present invention, for example, the number of blood cells in the blood of the obtained model animal can be sufficiently reduced, so that the rate of decrease in blood cells is high and the variation among individuals. Can be produced as a model animal in which is suppressed. The variability can be compared, for example, as a standard error.
　本発明において、「血球減少症」は、例えば、前記モデル動物と対応する正常な動物（例えば、未処理の動物）と比較して、血中の血球数が有意に減少していることを意味する。具体例として、対象の動物における血中の血球数が、例えば、前記対象の動物と対応する正常な動物における血中の血球数を基準として、６０％以下、５５％以下、５０％以下、４５％以下、４０％以下、３５％以下、３０％以下、２５％以下、２０％以下、１５％以下、１０％以下、５％以下、４％以下、３％以下、２％以下、または１％以下の場合、前記対象の動物は、血球減少症であるということができる。前記動物がＮＯＧマウスの場合、対象のマウスにおける血中の血球数が、例えば、５５０×１０４個／μＬ以下、５００×１０４個／μＬ以下、４００×１０４個／μＬ以下、３００×１０４個／μＬ以下、２００×１０４個／μＬ以下、１００×１０４個／μＬ以下、または５０×１０４個／μＬ以下の場合、前記対象のマウスは、血球減少症であるということができる。前記血球の種類は、特に制限されず、例えば、血小板、赤血球、白血球等があげられる。前記白血球は、例えば、単球；好中球、好酸球、好塩基球等の顆粒球；Ｔ細胞、Ｂ細胞、ＮＫ細胞、ＮＫＴ細胞等のリンパ球；等があげられる。前記「血球減少症」において、減少する血球の種類は、例えば、１種類でもよいし、２種類以上でもよいし、全種類でもよい。 In the present invention, "cytopenia" means that, for example, the number of blood cells in the blood is significantly reduced as compared with, for example, a normal animal (for example, an untreated animal) corresponding to the model animal. To do. As a specific example, the blood cell count in the target animal is, for example, 60% or less, 55% or less, 50% or less, 45 based on the blood cell count in the normal animal corresponding to the target animal. % Or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% In the following cases, the target animal can be said to have cytopenia. When the animal is a NOG mouse, the number of blood cells in the blood of the target mouse is, for example, 550 × 10 4 cells / μL or less, 500 × 10 4 cells / μL or less, 400 × 10 4 cells / μL or less, 300 × If 10 4 cells / μL or less, 200 × 10 4 cells / μL or less, 100 × 10 4 cells / μL or less, or 50 × 10 4 cells / μL or less, the target mouse has hypocytopenia. Can be done. The type of blood cell is not particularly limited, and examples thereof include platelets, erythrocytes, and white blood cells. Examples of the white blood cells include monocytes; granulocytes such as neutrophils, eosinophils and basophils; and lymphocytes such as T cells, B cells, NK cells and NKT cells. In the above-mentioned "cytopenia", the type of blood cells to be reduced may be, for example, one type, two or more types, or all types.
　前記血球として血小板が減少している場合、前記血球減少症は、「血小板減少症」ということもできる。具体例として、対象の動物における血中の血小板数が、例えば、前記対象の動物と対応する正常な動物における血中の血小板数を基準として、４０％以下、３５％以下、３０％以下、２５％以下、２０％以下、１５％以下、１０％以下、５％以下、４％以下、３％以下、２％以下、または１％以下の場合、前記対象の動物は、血小板減少症であるということができる。前記対象の動物における血中の血小板数が、例えば、前記対象の動物と対応する正常な動物における血中の血小板数を基準として、約４０％（例えば、３５～４５％）、約２０％（例えば、１５～２５％）、および約１０％（例えば、１４％以下）の場合、前記対象の動物は、軽度、中度、および重度の血小板減少症であるということができる。前記動物がＮＯＧマウスの場合、対象のマウスにおける血中の血小板数が、例えば、５０×１０４個／μＬ以下、４０×１０４個／μＬ以下、３０×１０４個／μＬ以下、２０×１０４個／μＬ以下、１０×１０４個／μＬ以下、５×１０４個／μＬ以下、または４×１０４個／μＬ以下の場合、前記対象のマウスは、血小板減少症であるということができる。 When the number of platelets is decreased as the blood cells, the cytopenia can also be referred to as "thrombocytopenia". As a specific example, the blood platelet count in the target animal is, for example, 40% or less, 35% or less, 30% or less, 25, based on the blood platelet count in the normal animal corresponding to the target animal. %, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, the target animal is said to have thrombocytopenia. be able to. The number of platelets in the blood of the target animal is, for example, about 40% (for example, 35 to 45%) and about 20% (for example, 35 to 45%) based on the number of platelets in the blood of a normal animal corresponding to the target animal. For example, in the case of 15-25%), and about 10% (eg, 14% or less), the animal of interest can be said to have mild, moderate, and severe thrombocytopenia. When the animal is a NOG mouse, the blood platelet count in the target mouse is, for example, 50 × 10 4 cells / μL or less, 40 × 10 4 cells / μL or less, 30 × 10 4 cells / μL or less, 20 × If 10 4 / μL or less, 10 × 10 4 / μL or less, 5 × 10 4 / μL or less, or 4 × 10 4 / μL or less, the target mouse has thrombocytopenia. Can be done.
　前記血球として赤血球が減少している場合、前記血球減少症は、「赤血球減少症」ということもできる。具体例として、対象の動物における血中の赤血球数が、例えば、前記対象の動物と対応する正常な動物における血中の赤血球数を基準として、６０％以下、５５％以下、５０％以下、４５％以下、４０％以下、３５％以下、３０％以下、２５％以下、２０％以下、１５％以下、１０％以下、５％以下、４％以下、３％以下、２％以下、または１％以下の場合、前記対象の動物は、赤血球減少症であるということができる。前記動物がＮＯＧマウスの場合、対象のマウスにおける血中の赤血球数が、例えば、５００×１０４個／μＬ以下、３５０×１０４個／μＬ以下、１００×１０４個／μＬ以下、または５０×１０４個／μＬ以下の場合、前記対象のマウスは、赤血球減少症であるということができる。 When the number of red blood cells is reduced as the blood cells, the blood cell depletion can also be referred to as "erythrocytopenia". As a specific example, the number of red blood cells in the blood of the target animal is, for example, 60% or less, 55% or less, 50% or less, 45 based on the number of red blood cells in the blood of a normal animal corresponding to the target animal. % Or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% In the following cases, the target animal can be said to have erythrocytopenia. When the animal is a NOG mouse, the number of red blood cells in the blood of the target mouse is, for example, 500 × 10 4 / μL or less, 350 × 10 4 / μL or less, 100 × 10 4 / μL or less, or 50. When it is × 10 4 cells / μL or less, it can be said that the subject mouse has erythrocytopenia.
　前記血球として白血球が減少している場合、前記血球減少症は、「白血球減少症」ということもできる。具体例として、対象の動物における血中の白血球数が、例えば、前記対象の動物と対応する正常な動物における血中の白血球数を基準として、３５％以下、３０％以下、２５％以下、２０％以下、１５％以下、１０％以下、５％以下、４％以下、３％以下、２％以下、または１％以下の場合、前記対象の動物は、白血球減少症であるということができる。前記動物がＮＯＧマウスの場合、対象のマウスにおける血中の白血球数が、例えば、３×１０２個／μＬ以下、２×１０２個／μＬ以下、または１×１０２個／μＬ以下の場合、前記対象のマウスは、白血球減少症であるということができる。 When the number of leukocytes is reduced as the blood cells, the hypocytopenia can also be referred to as "leukopenia". As a specific example, the white blood cell count in the target animal is 35% or less, 30% or less, 25% or less, 20 based on, for example, the white blood cell count in the blood of a normal animal corresponding to the target animal. When% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, it can be said that the target animal has leukopenia. When the animal is a NOG mouse, the number of white blood cells in the blood of the target mouse is, for example, 3 × 10 2 cells / μL or less, 2 × 10 2 cells / μL or less, or 1 × 10 2 cells / μL or less. , The subject mouse can be said to have leukopenia.
　本発明において、前記血球数は、例えば、血球計算盤を用いて計数してもよいし、血球特異的な抗体により染色し、フローサイトメーターでカウントすることにより計数してもよいし、血球計数装置を用いて計数してもよい。 In the present invention, the blood cell count may be counted, for example, by using a blood cell counter, staining with a blood cell-specific antibody, and counting with a flow cytometer, or blood cell counting. It may be counted using an apparatus.
　本発明において、モデル動物と正常な動物と比較する場合、前記「有意」は、例えば、統計学的有意差に基づき判断できる。前記統計解析手法は、公知の統計解析手法を選択して使用できる。前記統計解析手法は、特に制限されず、例えば、一元配置ＡＮＯＶＡ、一元配置反復測定ＡＮＯＶＡ、ダネット検定、スチューデントのｔ検定およびウィルコクソン検定があげられ、これらを組み合わせることもできる。例えば、前記検定において、Ｐ値が０．０５未満であった場合に有意差が認められたと判断し、一方、Ｐ値が０．０５以上であった場合に有意差が認められなかったと判断できる。 In the present invention, when comparing a model animal with a normal animal, the "significance" can be determined based on, for example, a statistically significant difference. As the statistical analysis method, a known statistical analysis method can be selected and used. The statistical analysis method is not particularly limited, and examples thereof include one-way ANOVA, one-way iterative measurement ANOVA, Dunnett's test, Student's t-test, and Wilcoxon test, and these can be combined. For example, in the above test, it can be determined that a significant difference was observed when the P value was less than 0.05, while it could be determined that no significant difference was observed when the P value was 0.05 or more. ..
　本発明において、「血小板」は、血液中の細胞成分の一つであり、CD41aおよびCD42bが陽性である細胞成分を意味する。前記血小板は、例えば、細胞核を有さず、また、前記巨核球と比較して、大きさが小さい。このため、前記血小板と前記巨核球とは、例えば、細胞核の有無および／または大きさにより区別できる。前記血小板は、血栓形成と止血において重要な役割を果たすとともに、損傷後の組織再生や炎症の病態生理にも関与することが知られている。また、前記血小板は、出血等により血小板が活性化されると、その膜上にIntegrin αIIBβ3（glycoprotein IIb/IIIa; CD41aとCD61の複合体）等の細胞接着因子の受容体が発現することが知られているまた、前記血小板が活性化されると、血小板同士が凝集し、血小板から放出される各種の血液凝固因子によってフィブリンが凝固することにより、血栓が形成され、止血が進む。 In the present invention, "platelet" is one of the cell components in blood and means a cell component in which CD41a and CD42b are positive. The platelets, for example, do not have a cell nucleus and are smaller in size than the megakaryocytes. Therefore, the platelets and the megakaryocytes can be distinguished by, for example, the presence and / or size of cell nuclei. It is known that the platelets play an important role in thrombus formation and hemostasis, and are also involved in the pathophysiology of tissue regeneration and inflammation after injury. It is also known that when platelets are activated by bleeding or the like, receptors for cell adhesion factors such as Integrin αIIBβ3 (glycoprotein IIb / IIIa; a complex of CD41a and CD61) are expressed on the platelets. When the platelets are activated, the platelets aggregate with each other, and fibrin is coagulated by various blood coagulation factors released from the platelets, thereby forming a thrombus and promoting hemostasis.
　本発明において、「赤血球」は、血液中の血液細胞であり、Ter119（glycophorin-A）および／またはＣＤ２３５ａ陽性である細胞を意味する。 In the present invention, "erythrocyte" means a blood cell in blood, which is Ter119 (glycophorin-A) and / or CD235a positive.
　本発明において、「白血球」は、血液中の血液細胞であり、ＣＤ２、ＣＤ３、ＣＤ４、ＣＤ８、ＣＤ１３、ＣＤ１９、ＣＤ２１、および／またはＣＤ５６が陽性である細胞を意味する。 In the present invention, "white blood cell" means a blood cell in blood, which is positive for CD2, CD3, CD4, CD8, CD13, CD19, CD21, and / or CD56.
　本発明において、モデル動物は、非ヒト動物である。前記非ヒト動物の種類は、特に制限されず、例えば、サル、ゴリラ、チンパンジー、マーモセット等の霊長類；マウス、ラット、モルモット等の齧歯類；イヌ、ネコ、ウサギ、ヒツジ、ウマ等があげられる。前記モデル動物は、例えば、血球が減少した状態を維持し易いことから、免疫不全動物または細網内皮系が破壊された動物が好ましく、免疫不全マウスがより好ましい。前記免疫不全動物は、例えば、重症複合免疫不全症（ＳＣＩＤ）のモデル動物があげられる。前記細網内皮系が破壊された動物は、例えば、細胞性免疫不全動物があげられる。前記免疫不全マウスは、例えば、Ｓｃｉｄマウス、ＮＯＤ－Ｓｃｉｄマウス、ＮＯＧマウス等があげられ、好ましくは、ＮＯＧマウスである。 In the present invention, the model animal is a non-human animal. The type of the non-human animal is not particularly limited, and examples thereof include primates such as monkeys, gorillas, chimpanzees and marmosets; rodents such as mice, rats and guinea pigs; dogs, cats, rabbits, sheep and horses. Be done. As the model animal, for example, an immunodeficient animal or an animal in which the reticle endothelial system is destroyed is preferable, and an immunodeficient mouse is more preferable because it is easy to maintain a state in which blood cells are reduced. Examples of the immunodeficient animal include a model animal for severe combined immunodeficiency disease (SCID). Examples of the animal in which the reticuloendotheliatic system is destroyed include a cell-mediated immunodeficient animal. Examples of the immunodeficient mouse include Scid mouse, NOD-Scid mouse, NOG mouse and the like, and NOG mouse is preferable.
　前記照射工程は、前記非ヒト動物に放射線を照射する工程である。前記照射工程は、例えば、放射線発生装置で発生した放射線を、前記非ヒト動物に照射することにより実施できる。前記照射工程において、例えば、前記非ヒト動物の全体に均一に放射線を照射できることから、前記非ヒト動物は、麻酔下または固定状態であることが好ましい。 The irradiation step is a step of irradiating the non-human animal with radiation. The irradiation step can be carried out, for example, by irradiating the non-human animal with radiation generated by a radiation generator. In the irradiation step, for example, the non-human animal is preferably under anesthesia or in a fixed state because radiation can be uniformly irradiated to the entire non-human animal.
　前記照射工程において、照射する放射線の種類は、例えば、Ｘ線、γ線等の電磁放射線；α線、β線、電子線、陽子線、中性子線、重粒子線等の粒子放射線；等があげられ、血球数の減少を効果的に誘導できることから、好ましくは、Ｘ線、γ線等の電磁放射線である。前記照射工程において用いる放射線は、１種類でもよいし、２種類以上でもよい。 Examples of the types of radiation to be irradiated in the irradiation step include electromagnetic radiation such as X-rays and γ-rays; particle radiation such as α-rays, β-rays, electron beams, proton rays, neutron rays, and heavy particle rays; Therefore, electromagnetic radiation such as X-rays and γ-rays is preferable because it can effectively induce a decrease in the number of blood cells. The radiation used in the irradiation step may be one type or two or more types.
　前記照射工程における放射線照射量（線量）は、特に制限されず、例えば、前記非ヒト動物に応じて、適宜設定できる。具体例として、前記放射線照射量は、例えば、１．５～１２Ｇｙ、１．５～１０Ｇｙ、１．５～６Ｇｙであり、好ましくは、２．５～６Ｇｙ、４～６Ｇｙである。前記放射線照射量は、例えば、１回の照射あたりの放射線照射量である。 The radiation irradiation amount (dose) in the irradiation step is not particularly limited, and can be appropriately set according to the non-human animal, for example. As a specific example, the irradiation amount is, for example, 1.5 to 12 Gy, 1.5 to 10 Gy, 1.5 to 6 Gy, preferably 2.5 to 6 Gy, 4 to 6 Gy. The irradiation amount is, for example, the amount of radiation per irradiation.
　前記照射工程において、前記放射線の照射は、例えば、１回行なってもよいし、複数回実施してもよい。前記放射線の照射を複数回行なう場合、前記放射線の照射回数は、例えば、２～５回であり、好ましくは、２～３回である。前記照射工程において、前記放射線の照射を複数回行なう場合、複数回の放射線照射量の合計量は、例えば、３～１２Ｇｙ、５～１２Ｇｙであり、血球数の減少を効果的に誘導でき、かつ非ヒト動物の放射線障害を抑制できることから、好ましくは、８～１２Ｇｙ、８～１１Ｇｙ、８～１０Ｇｙである。 In the irradiation step, the irradiation of the radiation may be performed once or a plurality of times, for example. When the irradiation of the radiation is performed a plurality of times, the number of irradiations of the radiation is, for example, 2 to 5 times, preferably 2 to 3 times. When the irradiation of the radiation is performed a plurality of times in the irradiation step, the total amount of the irradiation amount of the plurality of times is, for example, 3 to 12 Gy and 5 to 12 Gy, and the decrease in the number of blood cells can be effectively induced and It is preferably 8 to 12 Gy, 8 to 11 Gy, and 8 to 10 Gy because it can suppress radiation damage to non-human animals.
　前記照射工程において、前記放射線の照射を複数回実施する場合、各回の実施間隔は、特に制限されないが、例えば、１回の照射後、つぎの照射をすぐに実施してもよいし、次の照射まで間隔をあけて実施してもよい。前記放射線の照射を、間隔を開けて複数回実施する場合、各回の間隔は、例えば、１２～４８時間、好ましくは、約２４時間（例えば、２０～２８時間）である。 When the irradiation of the radiation is carried out a plurality of times in the irradiation step, the execution interval of each time is not particularly limited, but for example, after one irradiation, the next irradiation may be carried out immediately, or the next irradiation may be carried out immediately. It may be carried out at intervals until irradiation. When the irradiation of the radiation is carried out a plurality of times with an interval, the interval of each time is, for example, 12 to 48 hours, preferably about 24 hours (for example, 20 to 28 hours).
　前記照射工程では、血球数の減少を効果的に誘導でき、かつ非ヒト動物の放射線障害を抑制できることから、前記非ヒト動物に１回あたり４～６Ｇｙの放射線を２回照射することが好ましい。前記２回の照射は、約１日（例えば、１８～３０時間）あけて実施されることが好ましい。 In the irradiation step, it is preferable to irradiate the non-human animal twice with 4 to 6 Gy of radiation because it can effectively induce a decrease in the number of blood cells and suppress radiation damage to the non-human animal. The two irradiations are preferably carried out about one day (for example, 18 to 30 hours) apart.
　前記投与工程では、前記非ヒト動物に抗血球抗体を投与する。前記抗血球抗体の投与は、例えば、一般的な抗体の投与方法で実施でき、具体例として、前記抗血球抗体を含む抗体液を、前記非ヒト動物に静脈投与、皮下投与、または腹腔内投与することにより実施できる。 In the administration step, the anti-blood cell antibody is administered to the non-human animal. The administration of the anti-blood cell antibody can be carried out by, for example, a general antibody administration method, and as a specific example, an antibody solution containing the anti-blood cell antibody is intravenously, subcutaneously or intraperitoneally administered to the non-human animal. It can be carried out by doing.
　前記抗血球抗体は、前記血球に対する抗体、すなわち、前記血球に結合可能な抗体である。前記抗血球抗体は、好ましくは、前記血球の細胞膜表面抗原に対する抗体である。前記抗血球抗体は、特に制限されず、例えば、前記非ヒト動物の種類と、前記血球の種類およびその細胞表面抗原とに応じて、適宜決定できる。前記抗血球抗体は、例えば、１種類でもよいし、２種類以上でもよい。後者の場合、各抗血球抗体は、例えば、同じ抗原に結合してもよいし、異なる抗原に結合してもよい。前記抗血球抗体は、細胞除去機能性抗体、いわゆるデプリーション（Depletion）抗体であることが好ましい。 The anti-blood cell antibody is an antibody against the blood cells, that is, an antibody capable of binding to the blood cells. The anti-blood cell antibody is preferably an antibody against the cell membrane surface antigen of the blood cell. The anti-blood cell antibody is not particularly limited, and can be appropriately determined, for example, depending on the type of the non-human animal, the type of the blood cell, and the cell surface antigen thereof. The anti-blood cell antibody may be, for example, one type or two or more types. In the latter case, each anti-blood cell antibody may bind, for example, to the same antigen or to different antigens. The anti-blood cell antibody is preferably a cell-removing functional antibody, a so-called Depletion antibody.
　抗体の細胞除去機能は、例えば、抗体の抗体依存性細胞傷害（Antibody Dependent Cellular Cytotoxicity：ＡＤＣＣ）活性または補体依存性細胞傷害（Complement Dependent Cytotoxicity：ＣＤＣ）活性により生じる。また、ＡＤＣＣ活性は、例えば、抗体のＦｃ領域のＮ－グリコシド結合糖鎖、特に、前記糖鎖におけるフコースの有無より制御されている。前記Ｎ－グリコシド結合糖鎖は、例えば、ＩｇＧ型抗体の２９７番目のアスパラギンと結合している。このため、前記抗血球抗体の細胞除去機能性は、例えば、前記抗血球抗体のＮ－グリコシド結合糖鎖の付加または除去および前記糖鎖におけるフコースの付加または除去により制御できる。具体的には、前記抗血球抗体が、Ｎ－グリコシド結合糖鎖を付加された場合、または前記抗血球抗体の糖鎖にフコースが付加された場合、前記抗血球抗体は、ＡＤＣＣ活性が低下する。他方、前記抗血球抗体が、Ｎ－グリコシド結合糖鎖を除去された場合、または前記抗血球抗体の糖鎖からフコースが除去された場合、前記抗血球抗体は、ＡＤＣＣ活性が向上する。 The cell removal function of an antibody is caused by, for example, antibody-dependent cellular cytotoxicity (ADCC) activity or complement-dependent cytotoxicity (CDC) activity of the antibody. In addition, ADCC activity is controlled by, for example, the presence or absence of fucose in the N-glycosidic bond sugar chain in the Fc region of the antibody, particularly in the sugar chain. The N-glycosidic bond sugar chain is bound to, for example, asparagine at position 297 of the IgG type antibody. Therefore, the cell-removing functionality of the anti-blood cell antibody can be controlled, for example, by the addition or removal of the N-glycoside-binding sugar chain of the anti-blood cell antibody and the addition or removal of fucose in the sugar chain. Specifically, when the anti-blood cell antibody is added with an N-glycosidic bond sugar chain, or when fucose is added to the sugar chain of the anti-blood cell antibody, the ADCC activity of the anti-blood cell antibody is reduced. .. On the other hand, when the anti-blood cell antibody has the N-glycosidic bond sugar chain removed, or when fucose is removed from the sugar chain of the anti-blood cell antibody, the ADCC activity of the anti-blood cell antibody is improved.
　前記抗血球抗体が血小板の細胞膜表面抗原に対する抗体である場合、前記血小板の細胞膜表面抗原に対する抗体は、例えば、抗ＣＤ４１抗体、抗ＣＤ４２ａ抗体、抗ＣＤ４２ｂ抗体、抗ＣＤ４２ｃ抗体、抗ＣＤ４２ｄ抗体、抗ＣＤ４９ｂ抗体、抗ＣＤ６１抗体、抗ＣＤ１０９抗体、および抗ＧＰＶＩ抗体等があげられる。前記抗ＣＤ４１抗体は、例えば、抗ＣＤ４１ａ抗体があげられる。抗ＣＤ４１抗体としては、例えば、MWReg30（クローン名）が使用できる。抗ＣＤ４２ｄ抗体としては、例えば、1C2（クローン名）が使用できる。 When the anti-blood cell antibody is an antibody against the cell membrane surface antigen of platelets, the antibody against the cell membrane surface antigen of the platelets is, for example, an anti-CD41 antibody, an anti-CD42a antibody, an anti-CD42b antibody, an anti-CD42c antibody, an anti-CD42d antibody, an anti-CD49b. Examples include antibodies, anti-CD61 antibodies, anti-CD109 antibodies, anti-GPVI antibodies and the like. Examples of the anti-CD41 antibody include an anti-CD41a antibody. As the anti-CD41 antibody, for example, MWReg30 (clone name) can be used. As the anti-CD42d antibody, for example, 1C2 (clone name) can be used.
　前記抗血球抗体が赤血球の細胞膜表面抗原に対する抗体である場合、前記赤血球の細胞膜表面抗原に対する抗体は、例えば、抗Ter119抗体、抗ＣＤ２３５ａ抗体等があげられる。抗TER-119抗体としては、例えば、TER-119（クローン名）が使用できる。抗ＣＤ２３５ａ抗体としては、例えば、11E4B-7-6（クローン名）が使用できる。 When the anti-blood cell antibody is an antibody against the cell membrane surface antigen of erythrocytes, examples of the antibody against the cell membrane surface antigen of erythrocytes include anti-Ter119 antibody and anti-CD235a antibody. As the anti-TER-119 antibody, for example, TER-119 (clone name) can be used. As the anti-CD235a antibody, for example, 11E4B-7-6 (clone name) can be used.
　前記抗血球抗体が白血球の細胞膜表面抗原に対する抗体である場合、前記白血球の細胞膜表面抗原に対する抗体は、例えば、抗ＣＤ２抗体、抗ＣＤ３抗体、抗ＣＤ４抗体、抗ＣＤ８抗体、抗ＣＤ１３抗体、抗ＣＤ１９抗体、抗ＣＤ２１抗体、抗ＣＤ５６抗体等があげられる。抗ＣＤ２抗体としては、SFCI3Pt2H9（クローン名）が使用できる。抗ＣＤ３抗体としては、UCHT1（クローン名）が使用できる。抗ＣＤ４抗体としては、SFCI12T4D11（クローン名）が使用できる。抗ＣＤ８抗体としては、SFCI21Thy2D3（クローン名）が使用できる。抗ＣＤ１３抗体としては、SJ1D1（クローン名）が使用できる。抗ＣＤ１９抗体としては、HD237（クローン名）が使用できる。抗ＣＤ２１抗体としては、BL13（クローン名）が使用できる。抗ＣＤ５６抗体としては、N901（クローン名）が使用できる。 When the anti-blood cell antibody is an antibody against the cell membrane surface antigen of leukocytes, the antibody against the cell membrane surface antigen of the leukocytes is, for example, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD13 antibody, anti-CD19. Examples include antibodies, anti-CD21 antibodies, anti-CD56 antibodies and the like. SFCI3Pt2H9 (clone name) can be used as the anti-CD2 antibody. As the anti-CD3 antibody, UCHT1 (clone name) can be used. SFCI12T4D11 (clone name) can be used as the anti-CD4 antibody. SFCI21Thy2D3 (clone name) can be used as the anti-CD8 antibody. As the anti-CD13 antibody, SJ1D1 (clone name) can be used. HD237 (clone name) can be used as the anti-CD19 antibody. BL13 (clone name) can be used as the anti-CD21 antibody. As the anti-CD56 antibody, N901 (clone name) can be used.
　前記投与工程において、前記非ヒト動物に投与される抗血球抗体の投与量は、特に制限されず、例えば、前記非ヒト動物の種類、その体重、および投与前における血球数等に応じて、適宜設定できる。具体例として、前記投与量は、例えば、１～１０００μｇ／ｋｇ体重、１０～５００μｇ／ｋｇ体重、または２０～１００μｇ／ｋｇ体重である。前記非ヒト動物がマウスの場合、前記投与量は、例えば、１～１０００μｇ／ｋｇ体重、１０～１００μｇ／ｋｇ体重、または３０～７０μｇ／ｋｇ体重である。前記投与量は、例えば、前記投与前における血小板の数が相対的に多い場合、多く設定してもよいし、前記投与前における血小板の数が相対的に少ない場合、少なく設定してもよい。 In the administration step, the dose of the anti-blood cell antibody to be administered to the non-human animal is not particularly limited, and is appropriately determined according to, for example, the type of the non-human animal, its body weight, the number of blood cells before administration, and the like. Can be set. As a specific example, the dose is, for example, 1 to 1000 μg / kg body weight, 10 to 500 μg / kg body weight, or 20 to 100 μg / kg body weight. When the non-human animal is a mouse, the dose is, for example, 1 to 1000 μg / kg body weight, 10 to 100 μg / kg body weight, or 30 to 70 μg / kg body weight. The dose may be set high, for example, when the number of platelets before the administration is relatively large, or may be set low when the number of platelets before the administration is relatively small.
　前記投与工程において、前記非ヒト動物への抗血球抗体の投与回数は、例えば、１回でもよいし、複数回でもよい、前記抗体の投与を複数回行なう場合、前記抗体の投与回数は、例えば、２～５回であり、好ましくは、２～３回である。前記投与工程において、前記抗体の投与を複数回行なう場合、複数回の抗体投与量の合計量は、例えば、２～２０００μｇ／ｋｇ体重、２０～１０００μｇ／ｋｇ体重、または４０～２００μｇ／ｋｇ体重であり、血球数の減少を効果的に誘導できることから、好ましくは、４０～２００μｇ／ｋｇ体重または６０～１００μｇ／ｋｇ体重である。前記非ヒト動物がマウスの場合、複数回の抗体投与量の合計量は、例えば、２～２０００μｇ／ｋｇ体重、２０～２００μｇ／ｋｇ体重、または４０～２００μｇ／ｋｇ体重であり、好ましくは、６０～１４０μｇ／ｋｇ体重である。 In the administration step, the number of administrations of the anti-blood cell antibody to the non-human animal may be, for example, once or a plurality of times. When the antibody is administered a plurality of times, the number of administrations of the antibody is, for example, It is 2 to 5 times, preferably 2 to 3 times. When the antibody is administered a plurality of times in the administration step, the total amount of the plurality of antibody doses is, for example, 2 to 2000 μg / kg body weight, 20 to 1000 μg / kg body weight, or 40 to 200 μg / kg body weight. It is preferably 40 to 200 μg / kg body weight or 60 to 100 μg / kg body weight because it can effectively induce a decrease in blood cell count. When the non-human animal is a mouse, the total amount of the multiple antibody doses is, for example, 2-2000 μg / kg body weight, 20-200 μg / kg body weight, or 40-200 μg / kg body weight, preferably 60. It has a body weight of ~ 140 μg / kg.
　前記投与工程において、前記抗血球抗体の投与を複数回実施する場合、各回の実施間隔は、特に制限されないが、例えば、１回の投与後、つぎの投与をすぐに実施してもよいし、次の投与まで間隔をあけて実施してもよい。前記抗血球抗体の投与を、間隔を開けて複数回実施する場合、各回の間隔は、例えば、１２～４８時間、好ましくは、約２４時間（例えば、２０～２８時間）である。 When the administration of the anti-blood cell antibody is carried out a plurality of times in the administration step, the execution interval of each administration is not particularly limited, but for example, the next administration may be carried out immediately after one administration. It may be carried out at intervals until the next administration. When the administration of the anti-blood cell antibody is carried out a plurality of times at intervals, the interval between each time is, for example, 12 to 48 hours, preferably about 24 hours (for example, 20 to 28 hours).
　前記照射工程および前記投与工程の順序は、特に制限されず、例えば、前記照射工程の実施後に、前記投与工程を実施してもよいし、前記投与工程の実施後に、前記照射工程を実施してもよい。また、前記照射工程における照射または前記投与工程における投与を複数回実施する場合、本発明のモデル動物の製造方法は、前記照射工程と前記投与工程とを並行して実施してもよい。この場合、本発明のモデル動物の製造方法は、前記照射工程における照射間に、前記投与工程を実施してもよいし、前記投与工程における投与間に、前記照射工程を実施してもよい。また、本発明のモデル動物の製造方法は、前記照射工程における照射および前記投与工程における投与を複数回実施する場合、前記照射および前記投与の順序は、特に制限されない。 The order of the irradiation step and the administration step is not particularly limited. For example, the administration step may be carried out after the irradiation step is carried out, or the irradiation step may be carried out after the administration step is carried out. May be good. Further, when the irradiation in the irradiation step or the administration in the administration step is carried out a plurality of times, the method for producing a model animal of the present invention may carry out the irradiation step and the administration step in parallel. In this case, in the method for producing a model animal of the present invention, the administration step may be carried out during the irradiation in the irradiation step, or the irradiation step may be carried out during the administration in the administration step. Further, in the method for producing a model animal of the present invention, when the irradiation in the irradiation step and the administration in the administration step are carried out a plurality of times, the order of the irradiation and the administration is not particularly limited.
　本発明のモデル動物の製造方法は、血球数の減少をより効果的に誘導できることから、前記照射工程後に前記投与工程を実施することが好ましい。一般的に、放射線照射による血球細胞の減少は、例えば、時間を要する。他方、抗血球抗体の投与による血球細胞の減少は、例えば、早期に生じると推定される。このため、前記照射工程後に前記投与工程を実施することで、特に、前記照射工程後の血球細胞の減少のピーク時期（照射後約７日）に合わせて、前記投与工程を実施することにより、血球数の減少をさらに効果的に誘導できる。 Since the method for producing a model animal of the present invention can induce a decrease in blood cell count more effectively, it is preferable to carry out the administration step after the irradiation step. In general, the reduction of blood cells by irradiation takes time, for example. On the other hand, the decrease in blood cells due to the administration of anti-blood cell antibody is presumed to occur at an early stage, for example. Therefore, by carrying out the administration step after the irradiation step, in particular, by carrying out the administration step in accordance with the peak time of the decrease of blood cells after the irradiation step (about 7 days after the irradiation). The decrease in blood cell count can be induced more effectively.
　前記照射工程後、前記投与工程までの間隔は、特に制限されず、例えば、前記照射工程後、すぐに前記投与工程を実施してもよいし、一定の間隔を開けて実施してもよい。前記一定の間隔を開けて実施する場合、前記投与工程は、例えば、前記照射工程後、最初の放射線照射時を基準として、４～１４日、５～１３日であり、血球数の減少をさらに効果的に誘導できることから、好ましくは、６～１０日、７～９日である。 The interval from the irradiation step to the administration step is not particularly limited. For example, the administration step may be carried out immediately after the irradiation step, or may be carried out at regular intervals. When carried out at regular intervals, the administration step is, for example, 4 to 14 days, 5 to 13 days after the irradiation step with reference to the time of the first irradiation, further reducing the blood cell count. It is preferably 6 to 10 days and 7 to 9 days because it can be effectively induced.
　前記照射工程後に前記投与工程を実施する場合、前記投与工程の実施時期は、例えば、前記照射工程後の非ヒト動物の血球数に基づき、決定してもよい。この場合、前記照射工程後、前記非ヒト動物における血中の血球数が、例えば、前記対象の動物と対応する正常な動物における血中の血球数を基準として、４０％以下、３５％以下、３０％以下、２５％以下、２０％以下、１５％以下、１０％以下、５％以下、４％以下、３％以下、２％以下、または１％以下の場合に、前記非ヒト動物に対して、前記投与工程を実施することが好ましい。前記血球は、例えば、血小板、赤血球、および白血球のうちいずれか一つ以上である。具体例として、前記血球減少症モデル動物として血小板、赤血球、または白血球の減少症モデル動物を製造する場合、前記投与工程の実施時期は、例えば、前記非ヒト動物における血小板数、赤血球数、または白血球数を指標として、決定できる。 When the administration step is carried out after the irradiation step, the execution time of the administration step may be determined, for example, based on the blood cell count of the non-human animal after the irradiation step. In this case, after the irradiation step, the blood cell count in the non-human animal is, for example, 40% or less, 35% or less, based on the blood cell count in the normal animal corresponding to the target animal. 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, with respect to the non-human animal It is preferable to carry out the administration step. The blood cells are, for example, any one or more of platelets, red blood cells, and white blood cells. As a specific example, when a platelet, erythrocyte, or leukocyte hypoplasia model animal is produced as the cytopenia model animal, the time of carrying out the administration step is, for example, the platelet count, erythrocyte count, or leukocyte count in the non-human animal. It can be determined using the number as an index.
　このようにして、本発明のモデル動物の製造方法は、後述の本発明のモデル動物を製造できる。 In this way, the method for producing the model animal of the present invention can produce the model animal of the present invention described later.
＜第１の血球減少症のモデル動物＞
　本発明の第１の血球減少症のモデル動物は、前述のように、前記本発明のモデル動物の製造方法により得られる。本発明の第１のモデル動物は、前記本発明のモデル動物の製造方法により得られることが特徴であり、その他の構成および条件は、特に制限されない。本発明の第１のモデル動物は、後述の本発明の評価方法、血球の製造方法スクリーニング方法および治療薬候補物質の製造方法に好適に使用できる。本発明の第１のモデル動物は、前記本発明のモデル動物の製造方法の説明を援用できる。 <Model animal of the first cytopenia>
The first model animal for cytopenia of the present invention can be obtained by the method for producing a model animal of the present invention as described above. The first model animal of the present invention is characterized by being obtained by the method for producing a model animal of the present invention, and other configurations and conditions are not particularly limited. The first model animal of the present invention can be suitably used for the evaluation method of the present invention, the blood cell production method screening method, and the therapeutic drug candidate production method described later. As the first model animal of the present invention, the description of the method for producing the model animal of the present invention can be incorporated.
　前記第１のモデル動物は、例えば、下記条件（１）～（３）のいずれか一つ以上を満たす。前記第１のモデル動物が血小板減少症モデル動物の場合、前記第１のモデル動物は、下記条件（１）を満たすことが好ましい。前記第１のモデル動物が赤血球減少症モデル動物の場合、前記第１のモデル動物は、下記条件（２）を満たすことが好ましい。前記第１のモデル動物が白血球減少症モデル動物の場合、前記第１のモデル動物は、下記条件（３）を満たすことが好ましい。
（１）血液中の血小板数が、７．３×１０４個／μＬ以下である。
（２）血液中の赤血球数が、５００×１０４個／μＬ以下である。
（３）血液中の白血球数が、３×１０２個／μＬ以下である。 The first model animal satisfies, for example, any one or more of the following conditions (1) to (3). When the first model animal is a thrombocytopenia model animal, it is preferable that the first model animal satisfies the following condition (1). When the first model animal is a erythrocytopenia model animal, it is preferable that the first model animal satisfies the following condition (2). When the first model animal is a leukopenia model animal, it is preferable that the first model animal satisfies the following condition (3).
(1) The number of platelets in the blood is 7.3 × 10 4 / μL or less.
(2) The number of red blood cells in the blood is 500 × 10 4 cells / μL or less.
(3) The number of white blood cells in the blood is 3 × 10 2 cells / μL or less.
　前記条件（１）において、前記血液中の血小板数は、例えば、前記本発明のモデル動物の製造方法における前記照射工程または前記投与工程における最終処理時を基準として、１～１３日、好ましくは、１～１１日における血小板数である。 In the condition (1), the number of platelets in the blood is, for example, 1 to 13 days, preferably 1 to 13 days, based on the time of the final treatment in the irradiation step or the administration step in the method for producing a model animal of the present invention. The number of platelets in 1 to 11 days.
　前記条件（１）において、前記血液中の血小板数は、好ましくは、５×１０４個／μＬ以下であり、より好ましくは、４×１０４個／μＬ以下である。前記血液中の血小板数は、例えば、血球成分を用いて計数でき、例えば、単離した全血およびその血球画分等に対して、前述の血球数の計数方法を実施することにより、計数できる。前記（１）において、血液中の血小板数は、例えば、１匹の第１のモデル動物の血液中の血小板数または複数の第１のモデル動物の血液中の血小板数の平均値を意味する。 Under the condition (1), the number of platelets in the blood is preferably 5 × 10 4 / μL or less, and more preferably 4 × 10 4 / μL or less. The number of platelets in the blood can be counted using, for example, a blood cell component, and for example, the number of isolated whole blood and its blood cell fraction can be counted by carrying out the above-mentioned blood cell count counting method. .. In (1) above, the platelet count in blood means, for example, the average value of the platelet count in the blood of one first model animal or the blood platelet count of a plurality of first model animals.
　前記条件（２）において、前記血液中の赤血球数は、好ましくは、３００×１０４個／μＬ以下であり、より好ましくは、１００×１０４個／μＬ以下である。前記血液中の赤血球数は、例えば、血球成分を用いて計数でき、例えば、単離した全血およびその血球画分等に対して、前述の血球数の計数方法を実施することにより、計数できる。前記（２）において、血液中の赤血球数は、例えば、１匹の第１のモデル動物の血液中の赤血球数または複数の第１のモデル動物の血液中の赤血球数の平均値を意味する。 Under the condition (2), the number of red blood cells in the blood is preferably 300 × 10 4 cells / μL or less, and more preferably 100 × 10 4 cells / μL or less. The number of red blood cells in the blood can be counted using, for example, a blood cell component, and for example, the number of isolated whole blood and its blood cell fraction can be counted by carrying out the above-mentioned blood cell count counting method. .. In (2) above, the number of red blood cells in blood means, for example, the average number of red blood cells in the blood of one first model animal or the number of red blood cells in the blood of a plurality of first model animals.
　前記条件（３）において、前記血液中の白血球数は、好ましくは、２×１０２個／μＬ以下であり、より好ましくは、１×１０２個／μＬ以下である。前記血液中の白血球数は、例えば、血球成分を用いて計数でき、例えば、単離した全血およびその血球画分等に対して、前述の血球数の計数方法を実施することにより、計数できる。前記（３）において、血液中の白血球数は、例えば、１匹の第１のモデル動物の血液中の白血球数または複数の第１のモデル動物の血液中の白血球数の平均値を意味する。 Under the condition (3), the white blood cell count in the blood is preferably 2 × 10 2 / μL or less, and more preferably 1 × 10 2 / μL or less. The white blood cell count in the blood can be counted using, for example, a blood cell component, and can be counted, for example, by carrying out the above-mentioned blood cell count counting method for the isolated whole blood and its blood cell fraction. .. In (3) above, the white blood cell count in blood means, for example, the average value of the white blood cell count in the blood of one first model animal or the white blood cell count in the blood of a plurality of first model animals.
　本発明の第１のモデル動物は、前記条件（１）～（３）のうちいずれか一つ以上を満たせばよく、一つを満たしてもよいし、複数を満たしてもよく、全部を満たしてもよい。 The first model animal of the present invention may satisfy any one or more of the above conditions (1) to (3), may satisfy one, may satisfy a plurality, and satisfy all. You may.
　前記第１のモデル動物は、例えば、下記条件（４）および（５）の少なくとも一方を満たす。前記第１のモデル動物が血小板減少症のモデル動物である場合、前記第１のモデル動物は、例えば、下記条件（４）を満たすことが好ましい。
（４）出血開始から止血までの時間（出血時間または止血時間）が５分を超える
（５）前記モデル動物の血液は、抗血球抗体を含む The first model animal satisfies, for example, at least one of the following conditions (4) and (5). When the first model animal is a model animal for thrombocytopenia, it is preferable that the first model animal satisfies, for example, the following condition (4).
(4) The time from the start of bleeding to hemostasis (bleeding time or hemostasis time) exceeds 5 minutes (5) The blood of the model animal contains an anti-blood cell antibody.
　前記条件（４）において、前記止血時間の測定方法は、例えば、前記モデル動物の血管から出血させた際に、前記出血開始から前記出血が停止するまでの時間である。出血させる血管は、例えば、動脈、静脈があげられ、好ましくは、末梢の動脈である。具体例として、前記モデル動物がマウス等の齧歯類の場合、前記出血させる動脈は、尾動脈があげられ、好ましくは、腹側の尾動脈である。前記止血時間の測定では、前記出血させた後、出血箇所を液体に浸漬することが好ましい。前記液体は、例えば、生理的食塩水等の等張液である。 In the condition (4), the method for measuring the hemostasis time is, for example, the time from the start of the bleeding to the stop of the bleeding when the blood vessel of the model animal is bleeding. The blood vessels to be bleeding include, for example, arteries and veins, preferably peripheral arteries. As a specific example, when the model animal is a rodent such as a mouse, the bleeding artery is a tail artery, preferably a ventral tail artery. In the measurement of the hemostasis time, it is preferable to immerse the bleeding site in a liquid after the bleeding. The liquid is, for example, an isotonic liquid such as physiological saline.
　前記条件（４）おいて、前記止血時間は、例えば、５分を超え、好ましくは、６分以上、７分以上、８分以上、または９分以上であり、より好ましくは、１０分以上である。前記（４）において、止血時間は、例えば、１匹の第１のモデル動物の止血時間または複数の第１のモデル動物の止血時間の平均値を意味する。 Under the condition (4), the hemostasis time is, for example, more than 5 minutes, preferably 6 minutes or more, 7 minutes or more, 8 minutes or more, or 9 minutes or more, more preferably 10 minutes or more. is there. In (4) above, the hemostasis time means, for example, the average value of the hemostasis time of one first model animal or the hemostasis time of a plurality of first model animals.
　前記条件（５）において、前記抗血球抗体は、例えば、本発明の第１のモデル動物の製造方法において投与された抗血球抗体と同じである。前記抗血球抗体は、例えば、特定の抗原に対する抗体の検出方法により実施でき、例えば、ＥＬＩＳＡ法（Enzyme-Linked Immuno Sorbent Assay）、ウエスタンブロッティング法、フローサイトメトリー法等により実施できる。前記血液は、例えば、前記血液の液性成分を含めばよく、具体例として、全血、血漿、血清等があげられる。 Under the condition (5), the anti-blood cell antibody is, for example, the same as the anti-blood cell antibody administered in the method for producing a first model animal of the present invention. The anti-blood cell antibody can be carried out by, for example, a method for detecting an antibody against a specific antigen, for example, an ELISA method (Enzyme-Linked ImmunoSorbent Assay), a Western blotting method, a flow cytometry method or the like. The blood may contain, for example, the humoral component of the blood, and specific examples thereof include whole blood, plasma, and serum.
　本発明の第１のモデル動物は、前記条件（４）～（５）のうちいずれか一つ以上を満たせばよく、一つを満たしてもよいし、全部を満たしてもよいが、全部を満たすことが好ましい。 The first model animal of the present invention may satisfy any one or more of the above conditions (4) to (5), one may be satisfied, or all may be satisfied, but all of them may be satisfied. It is preferable to meet.
＜第２の血球減少症のモデル動物＞
　本発明の第２の血球減少症のモデル動物は、前述のように、下記条件（１）～（３）のいずれか一つ以上を満たす：
（１）血液中の血小板数が、７．３×１０４個／μＬ以下である。
（２）血液中の赤血球数が、５００×１０４個／μＬ以下である。
（３）血液中の白血球数が、３×１０２個／μＬ以下である。 <Model animal for second cytopenia>
As described above, the second model animal for cytopenia of the present invention satisfies any one or more of the following conditions (1) to (3):
(1) The number of platelets in the blood is 7.3 × 10 4 / μL or less.
(2) The number of red blood cells in the blood is 500 × 10 4 cells / μL or less.
(3) The number of white blood cells in the blood is 3 × 10 2 cells / μL or less.
　本発明の第２のモデル動物は、前記条件（１）～（３）のいずれか一つ以上を満たすことが特徴であり、その他の構成および条件は、特に制限されない。本発明の第２のモデル動物は、後述の本発明の評価方法、血球の製造方法スクリーニング方法および治療薬候補物質の製造方法に好適に使用できる。本発明の第２のモデル動物は、前記本発明のモデル動物の製造方法および第１のモデル動物の説明を援用できる。 The second model animal of the present invention is characterized in that it satisfies any one or more of the above conditions (1) to (3), and other configurations and conditions are not particularly limited. The second model animal of the present invention can be suitably used for the evaluation method of the present invention, the blood cell production method screening method, and the therapeutic drug candidate production method described later. As the second model animal of the present invention, the method for producing the model animal of the present invention and the description of the first model animal can be incorporated.
　前記第２のモデル動物は、例えば、下記条件（４）および（５）の少なくとも一方を満たす。前記第２のモデル動物が血小板減少症のモデル動物である場合、前記第２のモデル動物は、例えば、下記条件（４）を満たすことが好ましい。
（４）出血開始から止血までの時間（止血時間）が５分を超える
（５）前記モデル動物の血液は、抗血球抗体を含む The second model animal satisfies, for example, at least one of the following conditions (4) and (5). When the second model animal is a model animal for thrombocytopenia, it is preferable that the second model animal satisfies, for example, the following condition (4).
(4) The time from the start of bleeding to hemostasis (hemostatic time) exceeds 5 minutes (5) The blood of the model animal contains an anti-blood cell antibody.
＜血球機能の評価方法＞
　本発明の血球機能の評価方法は、前述のように、血球減少症のモデル動物に血球を投与する工程と前記血球の機能を評価する評価工程とを含み、前記血球減少症のモデル動物は、前記本発明の血球減少症のモデル動物である。本発明の評価方法は、前記評価工程で用いる血球減少症のモデル動物が、前記本発明の第１のモデル動物および／または第２のモデル動物であることが特徴であり、その他の工程および条件は、特に制限されない。本発明の評価方法では、Ｘ線照射による血球減少症モデル動物と比較して、血球数が減少した前記本発明の第１のモデル動物および／または第２のモデル動物を用いるため、血球の機能をより感度よく評価できる。本発明の評価方法は、前記本発明のモデル動物の製造方法、第１のモデル動物、および第２のモデル動物の説明を援用できる。 <Evaluation method of blood cell function>
As described above, the method for evaluating blood cell function of the present invention includes a step of administering blood cells to a model animal of blood cell hypoplasia and an evaluation step of evaluating the function of the blood cells. It is a model animal of the blood cell hypoplasia of the present invention. The evaluation method of the present invention is characterized in that the model animal for hemocytopenia used in the evaluation step is the first model animal and / or the second model animal of the present invention, and other steps and conditions. Is not particularly limited. Since the evaluation method of the present invention uses the first model animal and / or the second model animal of the present invention in which the number of blood cells is reduced as compared with the blood cell hypoplasia model animal by X-ray irradiation, the function of blood cells. Can be evaluated more sensitively. As the evaluation method of the present invention, the description of the method for producing a model animal of the present invention, the first model animal, and the second model animal can be incorporated.
　本発明において、「血球の機能」は、例えば、前記血球が生体において発揮している機能を意味する。前記血球が血小板の場合、前記血球の機能は、例えば、止血機能である。前記血球が赤血球の場合、前記血球の機能は、酸素および二酸化炭素の運搬機能である。前記血球が白血球の場合、前記血球の機能は、例えば、非自己物質、病原体または異常な細胞を認識して除去するための生物的防御機能である。前記血球が複数の機能を発揮する場合、本発明の評価方法は、例えば、一つの機能を評価してもよいし、複数の機能を評価してもよい。 In the present invention, the "function of blood cells" means, for example, the function that the blood cells exert in a living body. When the blood cell is a platelet, the function of the blood cell is, for example, a hemostatic function. When the blood cell is a red blood cell, the function of the blood cell is the function of carrying oxygen and carbon dioxide. When the blood cell is a white blood cell, the function of the blood cell is, for example, a biological defense function for recognizing and removing non-self substances, pathogens or abnormal cells. When the blood cells exert a plurality of functions, the evaluation method of the present invention may evaluate, for example, one function or a plurality of functions.
　本発明の評価方法において、評価対象の血球は、例えば、動物から単離した血球でもよいし、多能性細胞、幹細胞等の前駆細胞から誘導した血球でもよい。前記多能性細胞は、例えば、胚性幹細胞（ＥＳ）細胞、人工多能性幹（ｉＰＳ）細胞等があげられる。前記評価対象の血球が単離した血球の場合、本発明の評価方法は、例えば、動物から被検血球を単離する工程を含む。前記血球は、例えば、単離した全血でもよいし、その血球画分でもよい。前記動物は、例えば、ヒトおよび前記非ヒト動物があげられる。前記被検血球が誘導した血球の場合、本発明の血球の製造方法は、例えば、前駆細胞から被検血球を誘導する工程を含む。前記被検細胞が血小板の場合、前記血小板の誘導は、例えば、国際公開第２０１１／０３４０７３号公報および国際公開第２０１２／１５７５８６号公報を参照できる。前記前駆細胞は、例えば、誘導する被検血球への分化能を有する細胞を意味する。また、前記血球は、製剤化された血球でもよく、例えば、血球製剤等の血液製剤；血球機能模倣剤；等があげられる。前記血球製剤は、例えば、血小板製剤、赤血球製剤、白血球製剤等があげられる。前記血球機能模倣剤は、例えば、血球の機能を模倣する代替物を含む製剤であり、具体例として、血小板機能模倣剤（Platelet functional mimetics）、赤血球機能模倣剤（Erythrocyte functional mimetics）、白血球機能模倣剤（Leukocyte functional mimetics）等があげられる。 In the evaluation method of the present invention, the blood cell to be evaluated may be, for example, a blood cell isolated from an animal or a blood cell derived from a progenitor cell such as a pluripotent cell or a stem cell. Examples of the pluripotent cells include embryonic stem cell (ES) cells and induced pluripotent stem (iPS) cells. When the blood cell to be evaluated is an isolated blood cell, the evaluation method of the present invention includes, for example, a step of isolating a test blood cell from an animal. The blood cell may be, for example, isolated whole blood or a blood cell fraction thereof. Examples of the animal include humans and the non-human animals. In the case of blood cells induced by the blood cells to be tested, the method for producing blood cells of the present invention includes, for example, a step of inducing blood cells to be tested from progenitor cells. When the test cell is a platelet, for the induction of the platelet, for example, International Publication No. 2011/034073 and International Publication No. 2012/157586 can be referred to. The progenitor cell means, for example, a cell capable of inducing differentiation into a test blood cell. Further, the blood cells may be formulated blood cells, and examples thereof include blood products such as blood cell preparations; blood cell function mimics; and the like. Examples of the blood cell preparation include platelet preparations, red blood cell preparations, leukocyte preparations and the like. The blood cell function mimetic agent is, for example, a preparation containing an alternative that mimics the function of blood cells, and specific examples thereof include a platelet function mimetic agent (Platelet functional mimetics), an erythrocyte function mimetic agent (Erythrocyte functional mimetics), and a leukocyte function mimicry. Agents (Leukocyte functional mimetics) and the like can be mentioned.
　前記血球が、単離された血球、誘導された血球等の多数の血球を含む血球群である場合、本発明の評価方法は、前記血球群全体に対して実施してもよいし、前記血球群の一部に対して実施してもよい。後者の場合、本発明の評価方法は、例えば、抜き取り検査ということもできる。 When the blood cell is a blood cell group containing a large number of blood cells such as isolated blood cells and induced blood cells, the evaluation method of the present invention may be carried out for the entire blood cell group, or the blood cells. It may be performed on a part of the group. In the latter case, the evaluation method of the present invention can be, for example, a sampling inspection.
　前記血球減少症のモデル動物は、前述のように、本発明の第１のモデル動物および／または第２のモデル動物である。また、本発明の第１のモデル動物および／または第２のモデル動物は、例えば、前記本発明のモデル動物の製造方法により得ることができる。このため、本発明の評価方法は、前記本発明のモデル動物の製造方法により、血球減少症のモデル動物を製造する製造工程を含んでもよい。前記製造工程は、例えば、前記投与工程に先立ち実施する。 As described above, the model animal for cytopenia is the first model animal and / or the second model animal of the present invention. Further, the first model animal and / or the second model animal of the present invention can be obtained by, for example, the method for producing the model animal of the present invention. Therefore, the evaluation method of the present invention may include a production step of producing a model animal for hemocytopenia by the method for producing a model animal of the present invention. The manufacturing step is carried out, for example, prior to the administration step.
　前記投与工程では、前記血球減少症のモデル動物に血球（評価対象の血球）を投与する。前記血球減少のモデル動物は、例えば、前記投与対象の血球が減少しているモデル動物である。前記投与工程において、前記血球減少症のモデル動物に投与する血球数は、特に制限されず、例えば、前記モデル動物の種類および体重に応じて、適宜設定できる。具体例として、前記血球が血小板の場合、前記投与工程における血小板の投与量は、例えば、１×１０７～１×１０９血小板／匹、または１～３×１０８血小板／匹があげられる。前記血球が赤血球の場合、前記投与工程における赤血球の投与量は、例えば、３００×１０７～５００×１０９赤血球／匹、または３００～５００×１０８赤血球／匹があげられる。前記血球が白血球の場合、前記投与工程における白血球の投与量は、例えば、１×１０１～１×１０７白血球／匹、２×１０１～５×１０３白血球／匹、または２～５×１０２白血球／匹があげられる。前記投与工程において、前記モデル動物の種類と、前記モデル動物に投与する血球が由来する動物の種類とは、同じもよいし、異なってもよい。具体例として、前記モデル動物がマウス等の齧歯類の場合、前記評価対象の血球は、齧歯類の血球でもよいし、齧歯類以外の血球（例えば、ヒトの血球）でもよい。前記評価対象の血球は、例えば、ヒト血小板である。 In the administration step, blood cells (blood cells to be evaluated) are administered to the model animal of the cytopenia. The model animal for cytopenia is, for example, a model animal in which the blood cells to be administered are decreased. In the administration step, the number of blood cells to be administered to the model animal of cytopenia is not particularly limited, and can be appropriately set according to, for example, the type and body weight of the model animal. As a specific example, when the blood cells are platelets, the dose of platelets in the administration step may be, for example, 1 × 10 7 to 1 × 10 9 platelets / animal or 1 to 3 × 10 8 platelets / animal. If the blood cells in the red blood cells, the dose of red blood cells in the administering step may be, for example, 300 × 10 7 ~ 500 × 10 9 erythrocytes / animal, or 300 ~ 500 × 10 8 red blood cells / mouse and the like. When the blood cells are leukocytes, the dose of leukocytes in the administration step is, for example, 1 × 10 1 to 1 × 10 7 leukocytes / animal, 2 × 10 1 to 5 × 10 3 leukocytes / animal, or 2 to 5 ×. 10 2 white blood cells / animal can be given. In the administration step, the type of the model animal and the type of animal from which the blood cells to be administered to the model animal are derived may be the same or different. As a specific example, when the model animal is a rodent such as a mouse, the blood cell to be evaluated may be a rodent blood cell or a blood cell other than a rodent (for example, a human blood cell). The blood cell to be evaluated is, for example, human platelet.
　前記評価工程では、前記血球の機能を評価する。前記血球の機能の評価方法は、例えば、前記血球の種類に応じて適宜決定できる。 In the evaluation step, the function of the blood cell is evaluated. The method for evaluating the function of blood cells can be appropriately determined, for example, according to the type of blood cells.
　前記血球が血小板の場合、前記評価工程は、例えば、前記モデル動物を出血させること、または前記モデル動物の止血時間に基づき、前記血小板の機能、より具体的には、止血機能を評価できる。前記モデル動物の出血および止血時間は、前述の説明を援用できる。前記評価工程では、例えば、予め定めた基準に基づき、前記血小板の機能を評価できる。前記基準は、例えば、動物から単離した血小板を用いて得られた止血時間に基づき、設定してもよいし、評価者がその目的に応じて所望の時間に設定してもよい。具体例として、前記基準の止血時間は、例えば、５分以内である、好ましくは、２分以内である。この場合、前記評価工程において、前記血球の止血時間が、５分以内の場合、好ましくは、２分以内の場合、前記血小板は、前記基準を満たすと判断できる。 When the blood cells are platelets, the evaluation step can evaluate the function of the platelets, more specifically, the hemostatic function, based on, for example, bleeding the model animal or the hemostatic time of the model animal. The bleeding and hemostasis times of the model animal can be incorporated into the above description. In the evaluation step, for example, the function of the platelets can be evaluated based on a predetermined standard. The criteria may be set based on, for example, the hemostasis time obtained using platelets isolated from animals, or may be set by the evaluator at a desired time according to the purpose. As a specific example, the standard hemostasis time is, for example, 5 minutes or less, preferably 2 minutes or less. In this case, in the evaluation step, if the hemostasis time of the blood cells is 5 minutes or less, preferably 2 minutes or less, it can be determined that the platelets satisfy the criteria.
　前記血球が赤血球の場合、前記評価工程は、例えば、粘膜または皮膚の蒼白度、活動性の低下、頻呼吸等の貧血症状に基づき、赤血球の機能、より具体的には、酸素運搬機能を評価できる。 When the blood cells are red blood cells, the evaluation step evaluates the function of red blood cells, more specifically, the oxygen carrying function, based on anemia symptoms such as pallor of mucous membrane or skin, decreased activity, and tachypnea. it can.
　前記血球が白血球の場合、前記評価工程は、例えば、抗原、細菌またはウイルス等の標的を前記モデル動物に投与し、前記標的に対する免疫応答（例えば、抗体産生、炎症性サイトカインの産生、特異的なＢ細胞またはＴ細胞の増殖等）に基づき、白血球の機能、より具体的には、生物的防御機能を評価できる。 When the blood cell is a leukocyte, the evaluation step administers a target such as, for example, an antigen, a bacterium or a virus to the model animal and an immune response to the target (eg, antibody production, production of inflammatory cytokines, specific). Based on the proliferation of B cells or T cells, etc.), the function of leukocytes, more specifically, the biological defense function can be evaluated.
＜血球の製造方法＞
　本発明の血球の製造方法は、前述のように、被検血球について、血球の機能を評価する評価工程と、前記評価工程において、基準を満たす血球を機能性の血球として選抜する選抜工程とを含み、前記評価工程は、前記本発明の血球機能の評価方法により実施される。本発明の血球の製造方法は、前記評価工程を前記本発明の血球機能の評価方法により実施することが特徴であり、その他の工程および条件は、特に制限されない。本発明の血球の製造方法によれば、前記本発明の評価方法により血球の機能を評価するため、感度よく血球の機能を評価できる。このため、本発明の血球の製造方法によれば、例えば、より機能性のよい血球を製造できる。本発明の血球の製造方法は、前記本発明のモデル動物の製造方法、第１のモデル動物、第２のモデル動物、および評価方法の説明を援用できる。 <Blood cell manufacturing method>
As described above, the method for producing blood cells of the present invention comprises an evaluation step of evaluating the function of blood cells for a test blood cell and a selection step of selecting blood cells satisfying the criteria as functional blood cells in the evaluation step. Including, the evaluation step is carried out by the method for evaluating blood cell function of the present invention. The method for producing blood cells of the present invention is characterized in that the evaluation step is carried out by the method for evaluating blood cell function of the present invention, and other steps and conditions are not particularly limited. According to the method for producing a blood cell of the present invention, the function of the blood cell is evaluated by the evaluation method of the present invention, so that the function of the blood cell can be evaluated with high sensitivity. Therefore, according to the method for producing blood cells of the present invention, for example, more functional blood cells can be produced. As the method for producing blood cells of the present invention, the description of the method for producing a model animal of the present invention, the first model animal, the second model animal, and the evaluation method can be incorporated.
　前記被検血球は、例えば、動物から単離した血球でもよいし、多能性細胞、幹細胞等の前駆細胞から誘導した血球でもよい。前記多能性細胞は、例えば、胚性幹細胞（ＥＳ）細胞、人工多能性幹（ｉＰＳ）細胞等があげられる。前記被検血球が単離した血球の場合、本発明の血球の製造方法は、例えば、動物から被検血球を単離する工程を含む。前記血球は、例えば、単離した全血でもよいし、その血球画分でもよい。前記動物は、例えば、ヒトおよび前記非ヒト動物があげられる。前記被検血球が誘導した血球の場合、本発明の血球の製造方法は、例えば、前駆細胞から被検血球を誘導する工程を含む。前記被検細胞が血小板の場合、前記血小板の誘導は、例えば、国際公開第２０１１／０３４０７３号公報および国際公開第２０１２／１５７５８６号公報を参照できる。また、前記血球は、製剤化された血球でもよく、例えば、血球製剤等の血液製剤；血球機能模倣剤；等があげられる。前記血球製剤は、例えば、血小板製剤、赤血球製剤、白血球製剤等があげられる。前記血球機能模倣剤は、例えば、血球の機能を模倣する代替物を含む製剤であり、具体例として、血小板機能模倣剤（Platelet functional mimetics）、赤血球機能模倣剤（Erythrocyte functional mimetics）、白血球機能模倣剤（Leukocyte functional mimetics）等があげられる。 The blood cell to be tested may be, for example, a blood cell isolated from an animal, or a blood cell derived from a progenitor cell such as a pluripotent cell or a stem cell. Examples of the pluripotent cells include embryonic stem cell (ES) cells and induced pluripotent stem (iPS) cells. When the blood cell to be tested is an isolated blood cell, the method for producing a blood cell of the present invention includes, for example, a step of isolating the blood cell to be tested from an animal. The blood cell may be, for example, isolated whole blood or a blood cell fraction thereof. Examples of the animal include humans and the non-human animals. In the case of blood cells induced by the blood cells to be tested, the method for producing blood cells of the present invention includes, for example, a step of inducing blood cells to be tested from progenitor cells. When the test cell is a platelet, for the induction of the platelet, for example, International Publication No. 2011/034073 and International Publication No. 2012/157586 can be referred to. Further, the blood cells may be formulated blood cells, and examples thereof include blood products such as blood cell preparations; blood cell function mimics; and the like. Examples of the blood cell preparation include platelet preparations, red blood cell preparations, leukocyte preparations and the like. The blood cell function mimetic agent is, for example, a preparation containing an alternative that mimics the function of blood cells, and specific examples thereof include a platelet function mimetic agent (Platelet functional mimetics), an erythrocyte function mimetic agent (Erythrocyte functional mimetics), and a leukocyte function mimicry. Agents (Leukocyte functional mimetics) and the like can be mentioned.
　前記評価工程は、例えば、前記本発明の評価方法と同様にして実施できる。 The evaluation step can be carried out, for example, in the same manner as the evaluation method of the present invention.
　前記選抜工程では、前記評価工程において基準を満たす血球を機能性の血球として選抜する。前記選抜工程では、例えば、前記血小板減少症のモデル動物に投与した血球を前記血小板減少症のモデル動物から回収することにより、機能性の血球を選抜してもよい。また、前記被検血球が前述の血球群である場合、前記選抜工程では、例えば、前記基準を満たす血球を抜き取った血球群を機能性の血球として選抜してもよい。 In the selection step, blood cells that meet the criteria in the evaluation step are selected as functional blood cells. In the selection step, functional blood cells may be selected, for example, by collecting blood cells administered to the model animal for thrombocytopenia from the model animal for thrombocytopenia. When the blood cell to be tested is the blood cell group described above, in the selection step, for example, a blood cell group obtained by extracting blood cells satisfying the criteria may be selected as a functional blood cell.
　本発明の血球の製造方法は、前記選抜された機能性の血球から血球製剤を製造する製剤工程を含んでもよい。前記製剤工程は、例えば、公知の血液製剤の製造方法と同様にして実施できる。 The method for producing blood cells of the present invention may include a formulation step of producing a blood cell preparation from the selected functional blood cells. The formulation step can be carried out in the same manner as, for example, a known method for producing a blood product.
＜スクリーニング方法＞
　本発明の血球減少症の治療薬候補物質のスクリーニング方法は、前述のように、被検物質を血球減少症のモデル動物に投与する工程と、前記モデル動物に血液中の血球数が増加する被検物質を、血球減少症の治療薬の候補物質として選択する選択工程とを含み、前記モデル動物は、本発明の血球減少症のモデル動物である。本発明のスクリーニング方法は、前記血球減少症のモデル動物として、前記本発明の第１のモデル動物および／または第２のモデル動物であることが特徴であり、その他の工程および条件は、特に制限されない。本発明のスクリーニング方法では、Ｘ線照射による血球減少症モデル動物と比較して、血球数が減少した前記本発明の第１のモデル動物および／または第２のモデル動物を用いるため、血球の機能をより感度よく評価できる。このため、本発明のスクリーニング方法によれば、例えば、より有効性が高い血球減少症の治療薬の候補物質をスクリーニングできる。本発明の血球の製造方法は、前記本発明のモデル動物の製造方法、第１のモデル動物、第２のモデル動物、および評価方法の説明を援用できる。 <Screening method>
As described above, the method for screening a candidate substance for a therapeutic agent for blood cell hypoplasia of the present invention includes a step of administering a test substance to a model animal of blood cell hypoplasia and a subject in which the number of blood cells in the blood increases in the model animal. The model animal includes a selection step of selecting a test substance as a candidate substance for a therapeutic agent for blood cell hypoplasia, and the model animal is a model animal for blood cell hypoplasia of the present invention. The screening method of the present invention is characterized by being the first model animal and / or the second model animal of the present invention as the model animal of the hemocytopenia, and other steps and conditions are particularly limited. Not done. Since the screening method of the present invention uses the first model animal and / or the second model animal of the present invention in which the number of blood cells is reduced as compared with the blood cell hypoplasia model animal by X-ray irradiation, the function of blood cells. Can be evaluated more sensitively. Therefore, according to the screening method of the present invention, for example, a more effective candidate substance for a therapeutic agent for cytopenia can be screened. As the method for producing blood cells of the present invention, the description of the method for producing a model animal of the present invention, the first model animal, the second model animal, and the evaluation method can be incorporated.
　前記血球減少症のモデル動物は、前述のように、本発明の第１のモデル動物および／または第２のモデル動物である。また、本発明の第１のモデル動物および／または第２のモデル動物は、例えば、前記本発明のモデル動物の製造方法により得ることができる。このため、本発明のスクリーニング方法は、前記本発明のモデル動物の製造方法により、血球減少症のモデル動物を製造する製造工程を含んでもよい。前記製造工程は、例えば、前記投与工程に先立ち実施する。 As described above, the model animal for cytopenia is the first model animal and / or the second model animal of the present invention. Further, the first model animal and / or the second model animal of the present invention can be obtained by, for example, the method for producing the model animal of the present invention. Therefore, the screening method of the present invention may include a production step of producing a model animal for hemocytopenia by the method for producing a model animal of the present invention. The manufacturing step is carried out, for example, prior to the administration step.
　前記投与工程では、被検物質を血球減少症のモデル動物に投与する。前記被検物質は、特に制限されず、例えば、低分子化合物；核酸分子；タンパク質；ペプチド；化合物ライブラリー；遺伝子ライブラリーの発現産物；細胞抽出物；細胞培養上清；発酵微生物産生物；海洋生物抽出物；植物抽出物等があげられる。前記ペプチドは、特殊環状ペプチド等の環状ペプチドでもよい。 In the administration step, the test substance is administered to a model animal of cytopenia. The test substance is not particularly limited, and is, for example, low molecular weight compound; nucleic acid molecule; protein; peptide; compound library; expression product of gene library; cell extract; cell culture supernatant; fermented microbial product; ocean. Biological extracts; plant extracts and the like. The peptide may be a cyclic peptide such as a special cyclic peptide.
　前記投与工程において、前記被検物質を本発明の血球減少症のモデル動物に投与する方法は、特に制限されない。前記投与方法は、例えば、経口投与および非経口投与があげられる。前記非経口投与は、例えば、経静脈内投与、筋肉内投与、腹腔内投与、皮下投与、経鼻投与、経肺投与および経皮投与があげられる。前記被検物質がタンパク質およびペプチドである場合、例えば、前記タンパク質等をコードする遺伝子を有するウイルスベクターを構築し、その感染力を利用して、前記遺伝子を本発明の血球減少症のモデル動物に投与し、その体内で発現させてもよい。 In the administration step, the method of administering the test substance to the model animal for cytopenia of the present invention is not particularly limited. Examples of the administration method include oral administration and parenteral administration. Examples of the parenteral administration include intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, nasal administration, pulmonary administration and transdermal administration. When the test substance is a protein and a peptide, for example, a viral vector having a gene encoding the protein or the like is constructed, and the gene is used as a model animal for hemocytopenia of the present invention by utilizing its infectivity. It may be administered and expressed in the body.
　前記選択工程では、前記モデル動物に血液中の血球数が増加する被検物質を、血球減少症の治療薬の候補物質として選択する。前記血液中の血球数の増加は、例えば、前記血球数に基づき判断してもよいし、比較対象と比較して判断してもよい。前者の場合、前記血球数が、例えば、前記血球減少症の定義の血球数を満たさない場合、前記選択工程では、前記血液中の血球数が増加していると判断できる。後者の場合、前記比較対象は、前記被検物質投与前のモデル動物の血液中の血球数、被検物質を投与していないコントロールのモデル動物の血球数等があげられる。前記比較対象が前記被検物質投与前のモデル動物の血液中の血球数の場合、前記被検物質投与前のモデル動物の血液中の血球数と比較して、前記血球数が有意に増加している場合、前記選択工程では、前記血液中の血球数が増加していると判断できる。また、前記比較対象が被検物質を投与していないコントロールのモデル動物の血球数の場合、前記被検物質を投与していないコントロールのモデル動物と比較して、前記血球数が有意に増加している場合、前記選択工程では、前記血液中の血球数が増加していると判断できる。 In the selection step, a test substance that increases the number of blood cells in the blood of the model animal is selected as a candidate substance for a therapeutic agent for hypocytopenia. The increase in the number of blood cells in the blood may be determined based on, for example, the number of blood cells, or may be determined in comparison with the comparison target. In the former case, when the blood cell count does not satisfy, for example, the blood cell count defined as the hypocytopenia, it can be determined that the blood cell count in the blood is increased in the selection step. In the latter case, the comparison target includes the blood cell count in the blood of the model animal before administration of the test substance, the blood cell count of the control model animal to which the test substance is not administered, and the like. When the comparison target is the blood cell count in the blood of the model animal before the administration of the test substance, the blood cell count is significantly increased as compared with the blood cell count in the blood of the model animal before the administration of the test substance. If so, it can be determined that the number of blood cells in the blood is increasing in the selection step. Further, when the comparison target is the blood cell count of the control model animal to which the test substance is not administered, the blood cell count is significantly increased as compared with the control model animal to which the test substance is not administered. If so, it can be determined that the number of blood cells in the blood is increasing in the selection step.
＜治療薬の候補物質の製造方法＞
　本発明の血球減少症の治療薬の候補物質の製造方法は、前述のように、被検物質から血球減少症の治療薬の候補物質を選抜する選抜工程を含み、前記選抜工程は、前記本発明のスクリーニング方法で実施される。本発明の治療薬の候補物質の製造方法は、前記選抜工程を、前記本発明のスクリーニング方法で実施することが特徴であり、その他の工程および条件は、特に制限されない。本発明の治療薬の候補物質の製造方法は、前記選抜工程を、前記本発明のスクリーニング方法で実施するため、例えば、より有効性が高い血球減少症の治療薬の候補物質を製造できる。本発明の血球の製造方法は、前記本発明のモデル動物の製造方法、第１のモデル動物、第２のモデル動物、評価方法、およびスクリーニング方法の説明を援用できる。 <Manufacturing method of candidate substances for therapeutic agents>
As described above, the method for producing a candidate substance for a therapeutic agent for blood cell hypoplasia of the present invention includes a selection step of selecting a candidate substance for a therapeutic agent for blood cell hypoplasia from a test substance, and the selection step includes the present invention. It is carried out by the screening method of the present invention. The method for producing a candidate substance for a therapeutic agent of the present invention is characterized in that the selection step is carried out by the screening method of the present invention, and other steps and conditions are not particularly limited. In the method for producing a candidate substance for a therapeutic agent of the present invention, since the selection step is carried out by the screening method of the present invention, for example, a more effective candidate substance for a therapeutic agent for hemocytopenia can be produced. As the method for producing blood cells of the present invention, the description of the method for producing a model animal of the present invention, the first model animal, the second model animal, the evaluation method, and the screening method can be incorporated.
　以下、実施例を用いて本発明を詳細に説明するが、本発明は実施例に記載された態様に限定されるものではない。なお、特に示さない限り、市販の試薬およびキット等は、そのプロトコルに従い使用した。 Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the embodiments described in the Examples. Unless otherwise specified, commercially available reagents and kits were used according to the protocol.
［実施例１］
　本発明の血小板減少症のモデル動物の製造方法により、血小板が減少した血小板減少症のモデル動物が作成できることを確認した。 [Example 1]
It was confirmed that the method for producing a model animal for thrombocytopenia of the present invention can produce a model animal for thrombocytopenia in which platelets are reduced.
（１）不死化巨核球細胞の作製
　不死化巨核球は、以下の手順で作製した。 (1) Preparation of Immortalized Megakaryocytes The immortalized megakaryocytes were prepared by the following procedure.
（１－１）iPS細胞からの造血前駆細胞の調製
　ヒトiPS細胞（TKDN SeV2およびNIH5：センダイウイルスを用いて樹立されたヒト胎児皮膚繊維芽細胞由来iPS細胞）から、下記参考文献１に記載の方法に従って、血球細胞への分化培養を実施した。具体的には、ヒトES/iPS細胞コロニーを20ng/mL VEGF（R&D SYSTEMS社製）存在下でC3H10T1/2フィーダ細胞と１４日間共培養して造血前駆細胞（Hematopoietic Progenitor Cells；HPC）を作製した。培養条件は、３７℃、２０％Ｏ２、５％ＣＯ２で実施した（特に記載がない限り、以下同条件）。
参考文献１：Takayama N. et al., “Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells”, J. Exp. Med., 2010, vo.13, pages 2817-2830 (1-1) Preparation of hematopoietic progenitor cells from iPS cells From human iPS cells (TKDN SeV2 and NIH5: human fetal skin fibroblast-derived iPS cells established using Sendai virus), described in Reference 1 below. According to the method, differentiation culture into blood cells was carried out. Specifically, human ES / iPS cell colonies were co-cultured with C3H10T1 / 2 feeder cells in the presence of 20 ng / mL VEGF (manufactured by R & D SYSTEMS) for 14 days to prepare hematopoietic progenitor cells (HPC). .. Culture conditions, 37 ℃, 20% O 2 , 5% was carried out in CO 2 (unless otherwise noted, the following same conditions).
Reference 1: Takayama N. et al., “Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells”, J. Exp. Med., 2010, vo.13, pages 2817-2830
（１－２）遺伝子導入システム
　遺伝子導入システムは、レンチウイルスベクターシステムを利用した。レンチウイルスベクターは、Tetracycline制御性のTet-on（登録商標）遺伝子発現誘導システムベクターである。LV-TRE-mOKS-Ubc-tTA-I2G（下記参考文献２）のmOKSカセットをc-MYC、BMI1、またはBCL-xLに組み替えることで作製した。c-MYC、BMI1、またはBCL-xLが導入されたベクターを、それぞれ、LV-TRE-c-Myc-Ubc-tTA-I2G、LVTRE-BMI1-Ubc-tTA-I2G、およびLV-TRE-BCL-xL-Ubc-tTA-I2Gとした。c-MYC、BMI1、およびBCL-xLウイルスは、293T細胞へ前記レンチウイルスベクターで遺伝子導入することにより作製した。得られたウイルスを目的の細胞に感染させることによって、c-MYC、BMI1、およびBCL-xL遺伝子が目的の細胞のゲノム配列に導入される。安定的にゲノム配列に導入されたこれらの遺伝子は、培地にドキシサイクリン（clontech#631311）を加えることによって強制発現させることができる。
参考文献２：Kobayashi, T.et al., “Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells.”, Cell, 2010, vol.142, No.5, pages 787-799 (1-2) Gene transfer system The gene transfer system used the lentiviral vector system. The lentiviral vector is a Tetracycline-controlled Tet-on® gene expression induction system vector. It was made by recombining the mOKS cassette of LV-TRE-mOKS-Ubc-tTA-I2G (Reference 2 below) with c-MYC, BMI1, or BCL-xL. Vectors with c-MYC, BMI1, or BCL-xL introduced into LV-TRE-c-Myc-Ubc-tTA-I2G, LVTRE-BMI1-Ubc-tTA-I2G, and LV-TRE-BCL-, respectively. It was set to xL-Ubc-tTA-I2G. The c-MYC, BMI1, and BCL-xL viruses were prepared by gene transfer into 293T cells with the lentiviral vector. By infecting the cells of interest with the resulting virus, the c-MYC, BMI1, and BCL-xL genes are introduced into the genomic sequence of the cells of interest. These genes, which have been stably introduced into the genome sequence, can be forcibly expressed by adding doxycycline (clontech # 631311) to the medium.
Reference 2: Kobayashi, T. et al., “Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells.”, Cell, 2010, vol.142, No.5, pages 787-799
（１－３）造血前駆細胞へのc-MYCおよびBMI1ウイルスの感染
　予めC3H10T1/2フィーダ細胞を播種した6 well plate上に、前記（１－１）の方法で得られたHPCを5×104cells/wellとなるように播種し、BMI1ウイルスおよびc-MYCウイルスを用いたレンチウイルス法にてc-MYCおよびBMI1を強制発現させた。このとき、細胞株１種類につき6 wellずつ使用した。具体的には、それぞれMOI（multiplicity of infection）20となるように培地中にウイルス粒子を添加し、スピンインフェクション（32℃、900rpm、６０分間遠心）で感染させた。前記スピンインフェクションは、１２時間おきに２回実施した。培地は、基本培地（15% Fetal Bovine Serum (GIBCO)、1% Penicillin-Streptomycin-Glutamine (GIBCO)、1% Insulin, Transferrin, Selenium Solution (ITS-G) (GIBCO)、0.45 mmol/L 1-Thioglycerol (Sigma-Aldrich)、50μg/mL L-Ascorbic Acid (Sigma-Aldrich)を含有するIMDM (Iscove’s Modified Dulbecco’s Medium) (Sigma-Aldrich)）に、50 ng/mL Human thrombopoietin (TPO)(R&D SYSTEMS)、50 ng/mL Human Stem Cell Factor (SCF) (R&D SYSTEMS)および2μg/mL Doxycycline (Dox、clontech #631311)となるようにそれぞれを添加した培地（以下、分化培地）に、さらに、Protamineを最終濃度が10 μg/mLとなるように添加した培地を用いた。 (1-3) Infection of hematopoietic progenitor cells with c-MYC and BMI1 virus 5 × 10 HPC obtained by the method (1-1) above was placed on a 6-well plate in which C3H10T1 / 2 feeder cells were previously seeded. The cells were seeded at 4 cells / well, and c-MYC and BMI1 were forcibly expressed by the lentivirus method using BMI1 virus and c-MYC virus. At this time, 6 wells were used for each cell line. Specifically, virus particles were added to the medium so as to have a MOI (multiplicity of infection) of 20, and infection was performed by spin infection (32 ° C., 900 rpm, centrifugation for 60 minutes). The spin infection was performed twice every 12 hours. The medium was basal medium (15% Fetal Bovine Serum (GIBCO), 1% Penicillin-Streptomycin-Glutamine (GIBCO), 1% Insulin, Transferrin, Selenium Solution (ITS-G) (GIBCO), 0.45 mmol / L 1-Thioglycerol). IMDM (Iscove's Modified Dulbecco's Medium) (Sigma-Aldrich) containing (Sigma-Aldrich), 50 μg / mL L-Ascorbic Acid (Sigma-Aldrich), 50 ng / mL Human thrombopoietin (TPO) (R & D SYSTEMS), Protamine was further added to the medium (hereinafter referred to as differentiation medium) to which 50 ng / mL Human Stem Cell Factor (SCF) (R & D SYSTEMS) and 2 μg / mL Doxycycline (Dox, clontech # 631311) were added so as to be the final concentration. The medium was added so that the value was 10 μg / mL.
（１－４）巨核球自己増殖株の作製および維持培養
　前記（１－３）の方法でc-MYCおよびBMI1ウイルスの感染を実施した日を感染0日目として、以下の通り、c-MYC遺伝子およびBMI1遺伝子が導入されたHPCを培養することで、巨核球自己増殖株をそれぞれ作製した。c-MYC遺伝子およびBMI1遺伝子の強制発現は、培地に1μg/mL DoxとなるようにDoxを添加することにより実施した。 (1-4) Preparation and maintenance culture of megakaryocyte autoproliferation strain The day when infection with c-MYC and BMI1 virus was carried out by the method of (1-3) above was set as the 0th day of infection, and c-MYC was as follows. Megakaryocyte self-proliferation strains were prepared by culturing the HPC into which the gene and the BMI1 gene were introduced. Forced expression of the c-MYC gene and BMI1 gene was carried out by adding Dox to the medium to a concentration of 1 μg / mL Dox.
・感染２日目～感染１１日目
　感染２日目に、ピペッティングにて上記の方法で得られたウイルス感染済み血球細胞を回収し、1200rpm、５分間遠心操作を行って上清を除去した後、新しい分化培地で懸濁して新しいC3H10T1/2フィーダ細胞上に播種した(6well plate)。感染９日目に同様の操作をすることによって継代を実施した。前記再播種時は、細胞数を計測後、1×105 cells/2mL/wellとなるようにC3H10T1/2フィーダ細胞上に播種した(6well plate)。 -Days 2 to 11 of infection On the 2nd day of infection, virus-infected blood cells obtained by the above method were collected by pipetting, and the supernatant was removed by centrifugation at 1200 rpm for 5 minutes. It was then suspended in a new differentiation medium and seeded on new C3H10T1 / 2 feeder cells (6 well plate). Subculture was performed by performing the same operation on the 9th day of infection. At the time of re-seeding, after counting the number of cells, the cells were seeded on C3H10T1 / 2 feeder cells so as to have 1 × 10 5 cells / 2 mL / well (6 well plate).
・感染１２日目～感染１３日目
　感染２日目と同様の操作を実施した。細胞数を計測後3×105 cells/10mL/100mm dishとなるように、C3H10T1/2フィーダ細胞上に播種した(100mm dish)。 ・ 12th day of infection to 13th day of infection The same operation as on the 2nd day of infection was performed. After counting the number of cells, seeds were seeded on C3H10T1 / 2 feeder cells so as to be 3 × 10 5 cells / 10 mL / 100 mm dish (100 mm dish).
・感染１４日目
　ウイルス感染済み血球細胞を回収し、細胞1.0×105個あたり、抗ヒトCD41a-APC抗体(BioLegend)、抗ヒトCD42b-PE抗体(eBioscience)、および抗ヒトCD235ab-pacific blue（BioLegend）抗体を、それぞれ2μL、1μL、および1μLを用いて、前記血球細胞と抗体とを反応させた。前記反応後、FACS Verse（商標）（BD Biosciences）を用いて解析した。感染１４日目において、CD41a陽性率が５０%以上である細胞を、巨核球自己増殖株とした。 - infection day 14 infected already blood cells were harvested and the cells 1.0 × 10 5 per anti-human CD41a-APC antibody (BioLegend), anti-human CD42b-PE antibody (eBioscience), and anti-human CD235ab-pacific blue ( BioLegend) Antibodies were reacted with the blood cells using 2 μL, 1 μL, and 1 μL, respectively. After the reaction, analysis was performed using FACS Verse ™ (BD Biosciences). On the 14th day of infection, cells having a CD41a positive rate of 50% or more were designated as megakaryocyte self-proliferating strains.
（１－５）巨核球自己増殖株へのBCL-xLウイルス感染
　前記感染１４日目の巨核球自己増殖株に、BCL-xLウイルスを用いたレンチウイルス法にてBCL-xLを遺伝子導入した。MOI 10になるように培地中にウイルス粒子を添加し、スピンインフェクション（32℃、900rpm、６０分間遠心）で感染させた。BCL-xL遺伝子の強制発現は、培地に1μg/mL DoxとなるようにDoxを添加することにより実施した。 (1-5) BCL-xL virus infection in megakaryocyte autoproliferation strain BCL-xL was introduced into the megakaryocyte autoproliferation strain on the 14th day of infection by the lentivirus method using BCL-xL virus. Virus particles were added to the medium to achieve MOI 10 and infected by spin infection (32 ° C, 900 rpm, centrifuge for 60 minutes). Forced expression of the BCL-xL gene was carried out by adding Dox to the medium to a concentration of 1 μg / mL Dox.
（１－６）巨核球不死化株の作成および維持培養
・感染１４日目～感染１８日目
　前記（１－５）の方法で得られたBCL-xL遺伝子を導入した巨核球自己増殖株を回収し、1200rpm、5分間遠心操作を行った。前記遠心後、沈殿した細胞を新しい分化培地で懸濁した後、新しいC3H10T1/2フィーダ細胞上に2×105cells/2mL/wellとなるように播種した（6well plate）。 (1-6) Preparation and maintenance of megakaryocyte immortalized strain 14th day to 18th day of infection A megakaryocyte self-proliferating strain into which the BCL-xL gene obtained by the above method (1-5) was introduced. The cells were collected and centrifuged at 1200 rpm for 5 minutes. After the centrifugation, the precipitated cells were suspended in a new differentiation medium and then seeded on new C3H10T1 / 2 feeder cells to a concentration of 2 × 10 5 cells / 2 mL / well (6 well plate).
・感染１８日目：継代
　BCL-xL遺伝子を導入後の巨核球自己増殖株を回収し、細胞数を計測後、3×105 cells/10mL/100mm dishとなるように播種した。 -Day 18 of infection: Megakaryocyte self-proliferating strains after introduction of the passaged BCL-xL gene were collected, and after counting the number of cells, seeded so as to be 3 × 10 5 cells / 10 mL / 100 mm dish.
・感染２４日目：継代
　BCL-xL遺伝子を導入後の巨核球自己増殖株を回収し、細胞数を計測後、1×105 cells/10mL/100mm dishとなるように播種した。以後、４－７日毎に同様にして継代を行い、維持培養を行った。なお、継代時には、新たな分化培地に懸濁の上、播種した。 -Day 24 of infection: Megakaryocyte self-proliferating strains after introduction of the passaged BCL-xL gene were collected, and after counting the number of cells, seeded so as to be 1 × 10 5 cells / 10 mL / 100 mm dish. After that, subculture was performed in the same manner every 4-7 days, and maintenance culture was performed. At the time of passage, the cells were suspended in a new differentiation medium and seeded.
　感染２４日目にBCL-xLを遺伝子導入した巨核球自己増殖株を回収し、細胞1.0×105個あたり、抗ヒトCD41a-APC抗体(BioLegend)、抗ヒトCD42b-PE抗体(eBioscience)、および抗ヒトCD235ab-Pacific Blue（Anti-CD235ab-PB; BioLegend）抗体を、それぞれ、2μL、1μL、および1μLを用いて、免疫染色した後にFACS Verse（商標）を用いて解析した。そして、感染２４日目において、CD41a陽性率が50%以上である株を不死化巨核球細胞株とした。感染後２４日以上増殖することができたこれらの細胞を、不死化巨核球細胞株SeV2-MKCLおよびNIH5-MKCLとした。 The BCL-xL infection day 24 were collected megakaryocyte self-propagating strains transformed gene, cells 1.0 × 10 5 per anti-human CD41a-APC antibody (BioLegend), anti-human CD42b-PE antibody (eBioscience), and Anti-human CD235ab-Pacific Blue (Anti-CD235ab-PB; BioLegend) antibodies were immunostained with 2 μL, 1 μL, and 1 μL, respectively, and then analyzed with FACS Verse ™. Then, on the 24th day of infection, a strain having a CD41a positive rate of 50% or more was designated as an immortalized megakaryocyte cell line. These cells, which were able to proliferate for 24 days or more after infection, were designated as immortalized megakaryocyte cell lines SeV2-MKCL and NIH5-MKCL.
　得られたSeV2-MKCLおよびNIH5-MKCLを、10cmディッシュ（10mL/ディッシュ）で静置培養した。培地は、IMDMを基本培地として、以下の成分を加えた（濃度は終濃度）。培養条件は、２７℃、５％ＣＯ２とした。
FBS（Sigma#172012 lot.12E261）15％
L-Glutamin (Gibco #25030-081) 2mmol/L
ITS (Gibco #41400-045) 100倍希釈
MTG (monothioglycerol, sigma #M6145-25ML) 450μmol/L
アスコルビン酸(sigma #A4544) 50μg/mL
Puromycin (sigma #P8833-100MG) 2μg/mL
SCF (和光純薬#193-15513) 50ng/mL
TPO様作用物質200ng/mL The obtained SeV2-MKCL and NIH5-MKCL were statically cultured in a 10 cm dish (10 mL / dish). As the medium, IMDM was used as the basic medium, and the following components were added (concentration is final concentration). The culture conditions were 27 ° C. and 5% CO 2 .
FBS (Sigma # 172012 lot.12E261) 15%
L-Glutamin (Gibco # 25030-081) 2 mmol / L
ITS (Gibco # 41400-045) 100-fold dilution
MTG (monothioglycerol, sigma # M6145-25ML) 450 μmol / L
Ascorbic acid (sigma # A4544) 50 μg / mL
Puromycin (sigma # P8833-100MG) 2 μg / mL
SCF (Wako Pure Medicine # 193-15513) 50ng / mL
TPO-like agent 200 ng / mL
（２）巨核球の培養物の生産
　Doxを含まない培地で培養することで強制発現を解除した。具体的には、前記（１）の方法で得た不死化巨核球細胞株（SeV2-MKCLおよびNIH5-MKCL）を、PBS(-)で2度洗浄し、下記血小板生産培地に懸濁した。細胞の播種密度は、1.0×105 cells/mLとした。 (2) Production of megakaryocyte culture The forced expression was released by culturing in a medium containing no Dox. Specifically, the immortalized megakaryocyte cell lines (SeV2-MKCL and NIH5-MKCL) obtained by the method (1) above were washed twice with PBS (-) and suspended in the following platelet production medium. The seeding density of cells was 1.0 × 10 5 cells / mL.
　前記血小板生産培地は、IMDMを基本培地として、以下の成分を加えた（濃度は、終濃度）。
human plasma 5％
L-Glutamin (Gibco #25030-081) 4mmol/L
ITS (Gibco #41400-045) 100倍希釈
MTG (monothioglycerol, sigma #M6145-25ML) 450μmol/L
アスコルビン酸 (sigma #A4544) 50μg/mL
SCF (和光純薬#193-15513) 50ng/mL
TPO様作用物質200ng/mL
ADAM阻害剤15μmol/L
GNF351（Calbiochem #182707）500nmol/LY39983（Chemscene LLC #CS-0096）500nmol/L
Urokinase 5U/mL
低分子heparin（SANOFI、クレキサン）1U/mL The following components were added to the platelet production medium using IMDM as the basic medium (concentration is the final concentration).
human plasma 5%
L-Glutamin (Gibco # 25030-081) 4 mmol / L
ITS (Gibco # 41400-045) 100-fold dilution
MTG (monothioglycerol, sigma # M6145-25ML) 450 μmol / L
Ascorbic acid (sigma # A4544) 50 μg / mL
SCF (Wako Pure Medicine # 193-15513) 50ng / mL
TPO-like agent 200 ng / mL
ADAM inhibitor 15 μmol / L
GNF351 (Calbiochem # 182707) 500nmol / LY39983 (Chemscene LLC # CS-0096) 500nmol / L
Urokinase 5U / mL
Small molecule heparin (SANOFI, crexane) 1 U / mL
　そして、前記血小板生産培地存在下で６日間培養して、血小板を産生させることにより、巨核球の培養物を生産させた。 Then, the cells were cultured in the presence of the platelet production medium for 6 days to produce platelets, thereby producing a culture of megakaryocytes.
（３）精製血小板の製造
　前記（２）で得られた巨核球の培養物について、以下の手順で、血小板を製造（精製）した。なお、同様の精製を２回実施した。 (3) Production of Purified Platelets Platelets were produced (purified) from the megakaryocyte culture obtained in (2) above by the following procedure. The same purification was carried out twice.
（３－１）巨核球の培養物の濃縮
　前記（２）で得られた巨核球の培養物について、培養物バッグに導入した。そして、前記培養物バッグについて、図１のように、濃縮システムに接続した。図１において、洗浄保存液バッグ１および２は、洗浄保存液を含む。前記洗浄保存液は、ビカネイト輸液（ビカーボン輸液、大塚製薬社製）に２０％(v/v%)ＡＣＤおよび2.5％(w/v%)ヒト血清アルブミンを添加し、ＮａＯＨでｐＨ7.2に調整したものを使用した。そして、下記表１にしたがって、中空糸膜（プラズマフローＯＰ、旭化成メディカル社製）を用いて、前記巨核球の培養物を濃縮し、得られた巨核球の培養物の濃縮液を貯蔵バッグに回収した。 (3-1) Concentration of Megakaryocyte Culture The megakaryocyte culture obtained in (2) above was introduced into a culture bag. Then, the culture bag was connected to the concentration system as shown in FIG. In FIG. 1, the cleaning storage liquid bags 1 and 2 include a cleaning storage liquid. The wash and storage solution was prepared by adding 20% (v / v%) ACD and 2.5% (w / v%) human serum albumin to a bicanate infusion solution (bicarbon infusion solution, manufactured by Otsuka Pharmaceutical Co., Ltd.) and adjusting the pH to 7.2 with NaOH. I used the one that I did. Then, according to Table 1 below, the culture of the megakaryocytes was concentrated using a hollow fiber membrane (Plasmaflow OP, manufactured by Asahi Kasei Medical Co., Ltd.), and the obtained concentrate of the culture of megakaryocytes was placed in a storage bag. Recovered.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
（３－２）血小板の遠心分離
　まず、無菌接合装置を用いて、ACP215ディスポーザブルセット（ヘモネティックス社製）の廃液バッグを回収用バッグに置換した。前記回収用バッグは、ハイカリックＩＶＨバック（テルモ　HC-B3006A）を用いた。つぎに、前記巨核球の培養物の濃縮液に対して10％量のACD-A液（テルモ社製）を添加した。前記添加後、ACD-A液を添加した濃縮液を、細胞バッグに注入した。前記細胞バッグは、ハイカリックIVHバック（テルモ　HC-B3006A）を用いた。 (3-2) Centrifugation of platelets First, a waste liquid bag of ACP215 disposable set (manufactured by Hemonetics) was replaced with a collection bag using a sterile bonding device. A high-calic IVH bag (Terumo HC-B3006A) was used as the collection bag. Next, a 10% amount of ACD-A solution (manufactured by Terumo Corporation) was added to the concentrated solution of the megakaryocyte culture. After the addition, the concentrated solution to which the ACD-A solution was added was injected into the cell bag. As the cell bag, a high-calic IVH bag (Terumo HC-B3006A) was used.
　つぎに、無菌接合装置を用いて、ACD-A液を添加した培養物を含む細胞バッグをACP215ディスポーザブルセットに接合した。そして、ACP215をサービスモードで立ち上げ、回転数を2500rpm（350×g）にセットした。ACP215をスタートさせ、前記細胞バッグ中の培養物を約100mL/minで分離ボウルに導入した。前記分離ボウルより流出する液体成分は、回収バッグに回収した。前記細胞バッグ中の培養物の全量を分離ボウルに導入後、さらに500mLの洗浄保存液を前記分離ボウルに導入した。前記分離ボウルに前記洗浄保存液を導入後、遠心を止めてチューブシーラーを用いて回収液（血小板を含む回収された液体成分）を含む回収バッグを切り離した。 Next, the cell bag containing the culture to which the ACD-A solution was added was bonded to the ACP215 disposable set using a sterile bonding device. Then, I started ACP215 in service mode and set the rotation speed to 2500 rpm (350 x g). ACP215 was started and the culture in the cell bag was introduced into a separation bowl at about 100 mL / min. The liquid component flowing out of the separation bowl was collected in a collection bag. After introducing the entire amount of the culture in the cell bag into the separation bowl, an additional 500 mL of a wash storage solution was introduced into the separation bowl. After introducing the washing and preserving solution into the separation bowl, centrifugation was stopped and a collection bag containing the recovery solution (recovered liquid component containing platelets) was separated using a tube sealer.
　新しいACP215ディスポーザブルセットに、前記無菌接合装置を用いて回収液（血小板を含む）を含んだ回収バッグを接合した。ACP215を通常モードで立ち上げた。プログラム設定はWPCを選択し、機器の指示に従い、前記回収バッグを接合したACP215ディスポーザブルセットをセットした。なお、回収液を含んだ回収バッグはスタンドに設置した。 A recovery bag containing a recovery solution (including platelets) was joined to the new ACP215 disposable set using the aseptic joining device. Launched ACP215 in normal mode. WPC was selected as the program setting, and the ACP215 disposable set to which the collection bag was joined was set according to the instructions of the device. The collection bag containing the collection liquid was installed on the stand.
　つぎに、ACP215の遠心速度を5000rpm（1398.8×g）に変更し、遠心をスタートさせた。前記分離ボウルへ前記回収液が導入され始めたとき、自動注入から手動注入に変更した。具体的には、前記回収液を約100mL/minの導入速度で前記分離ボウルに導入した。前記回収液全量を分離ボウルに添加後、さらに500mLの洗浄保存液を追加した。 Next, the centrifugal speed of ACP215 was changed to 5000 rpm (1398.8 x g), and centrifugation was started. When the recovery liquid began to be introduced into the separation bowl, automatic injection was changed to manual injection. Specifically, the recovered solution was introduced into the separation bowl at an introduction rate of about 100 mL / min. After adding the entire amount of the recovered solution to the separation bowl, an additional 500 mL of a washing and preserving solution was added.
（３－３）血小板の洗浄
　洗浄は、ACP215のプログラムに従って、2000mLの前記洗浄保存液で洗浄した。 (3-3) Washing of platelets Washing was carried out with 2000 mL of the washing preservation solution according to the program of ACP215.
（３－４）血小板の回収
　ACP215のプログラムに従って、200mLの洗浄済み血小板を血小板製剤バッグに回収し、ビカネイト輸液（ビカーボン輸液、大塚製薬社製）に２０％(v/v%)ＡＣＤおよび2.5％(w/v%)ヒト血清アルブミンを添加した洗浄保存液に懸濁したｉＰＳＣ由来血小板製剤（製造ロット：ACP161N5）とした。 (3-4) Recovery of platelets According to the ACP215 program, 200 mL of washed platelets were collected in a platelet preparation bag, and 20% (v / v%) ACD and 2.5% were added to Vicanate infusion (bicarbon infusion, manufactured by Otsuka Pharmaceutical Co., Ltd.). An iPSC-derived platelet preparation (production lot: ACP161N5) suspended in a wash preservation solution containing (w / v%) human serum albumin was used.
（４）血小板減少症のモデル動物
　図２にしたがって、血小板減少モデル動物を作製した。具体的には、実施例のモデル動物として、８週齢の雄のＮＯＧマウス（NOD.Shi-scid,IL-2RγKO Jic（NOG）、公益財団法人　実験動物中央研究所社製）を用い、ケタミン（100mg/kg体重）およびキシラジン（10mg／kg体重）の混合液を腹腔内投与することで、麻酔下においた。つぎに、麻酔下のマウスをＸ線照射装置（MX-80Labo、メディエックステック株式会社）に入れて、５Ｇｙの線量を照射した（０日目）。１日後に再度同様に麻酔下のマウスに対して、５Ｇｙの線量を照射した（１日目）。 (4) Model animal for thrombocytopenia A model animal for thrombocytopenia was prepared according to FIG. Specifically, as a model animal of the example, an 8-week-old male NOG mouse (NOD.Shi-scid, IL-2RγKO Jic (NOG), manufactured by Central Institute for Experimental Animals) was used as ketamine. A mixture of (100 mg / kg body weight) and xylazine (10 mg / kg body weight) was intraperitoneally administered under anesthesia. Next, the anesthetized mouse was placed in an X-ray irradiation device (MX-80Labo, Mediextech Co., Ltd.) and irradiated with a dose of 5 Gy (day 0). One day later, the similarly anesthetized mice were irradiated with a dose of 5 Gy again (day 1).
　つぎに、初回のＸ線照射日を０日目として、７日目および８日目に、リン酸緩衝液（ＰＢＳ）で７μｇ／ｍＬに希釈した抗マウスＣＤ４１抗体（clone：MWReg30、カタログ番号：133910、Biolegend,inc.）を、注射針および注射筒を用いて尾静脈より、２００μＬ投与した（ｎ＝５）。比較例のモデル動物は、７日目および８日目の抗体投与を行わなかった以外は同様の処理を行った（ｎ＝５）。また、参考例のモデル動物として、０日目および１日目のＸ線照射を行わず、７日目および８日目に抗マウスＣＤ４１抗体の投与を行った以外は同様に処理を行った（ｎ＝５）。なお、一般状態の著しい悪化（重篤な痩削、自発運動量の低下、横臥位等）が認められる動物を瀕死動物と判断し、安楽死処分することとしたが（以下、同様）、本実施例、比較例および参考例において該当した動物はいなかった。 Next, the anti-mouse CD41 antibody (clone: MWReg30, catalog number:) diluted to 7 μg / mL with phosphate buffer (PBS) on the 7th and 8th days, with the first X-ray irradiation day as the 0th day. 133910, Biolegend, inc.) Was administered 200 μL from the tail vein using a needle and tube (n = 5). The model animals of the comparative examples were subjected to the same treatment except that the antibody was not administered on the 7th and 8th days (n = 5). In addition, as a model animal of the reference example, X-ray irradiation was not performed on the 0th and 1st days, and the same treatment was performed except that the anti-mouse CD41 antibody was administered on the 7th and 8th days (). n = 5). Animals with marked deterioration of general condition (serious emaciation, decreased locomotor activity, recumbent position, etc.) were judged to be moribund animals and were euthanized (the same applies hereinafter). None of the animals were applicable in the examples, comparative examples and reference examples.
（５）被検物質の投与
　９日目に、前記（４）で作成した各マウスの半数に対し、被検物質として、前記（３）で作成した血小板製剤を、マウス１匹あたり２００μＬ、２×１０８plateletsとなるように、注射針および注射筒を用いて尾静脈内に投与した（血小板製剤投与群、ACP161N5）。残りの半数には、前記血小板製剤に代えて、被検物質として、前記ビカネイト輸液（Vehicle）を２００μＬ投与した（Vehicle投与群）。 (5) Administration of test substance On the 9th day, 200 μL of the platelet preparation prepared in (3) above was added to half of each mouse prepared in (4) above as a test substance, 2 × such that 10 8 platelets, were administered into the tail vein using a needle and syringe (platelet product administration group, ACP161N5). In place of the platelet preparation, 200 μL of the vicanate infusion (Vehicle) was administered to the other half as a test substance (Vehicle administration group).
（６）血小板数の測定
（６－１）採血およびサンプルの固定
　前記各マウスについて、０、７、８、および９日目に、尾静脈より採血した。なお、前記血小板製剤またはVehicle投与後は、尾の識別が困難となるため、各マウスの背部に油性フェルトペンにて識別番号を記入した。また、７日目の採血は、抗体投与前に実施した。前記採血前に、血液固定溶液として、ThromboFix Platelet Stabilizer（Cat. No. 6607130，BECKMAN COULTER社製）を用い、予めＡ液とＢ液とを等量混合してworking solutionを調製し、６μＬずつ必要分のチューブに分注しておいた。前記採血は、キャピラリー（V.ヘマトクリット毛細管EDTA-2K、東京硝子器械株式会社）を用いて、各マウスの尾静脈から血液を約１０μＬ採取し、そのうちの６μＬを、前記分注したworking solutionを含むチューブ中に添加し、タッピングによりよく撹拌して固定した血液サンプルを作成した。前記固定した血液サンプルの保存期間は、室温（約２５℃、以下、同様）、暗所の条件で７日間とした。 (6) Measurement of platelet count (6-1) Blood collection and sample fixation Blood was collected from the tail vein on the 0th, 7th, 8th, and 9th days of each of the above mice. Since it is difficult to identify the tail after administration of the platelet preparation or Vehicle, an identification number was entered on the back of each mouse with an oil-based felt-tip pen. In addition, blood sampling on the 7th day was performed before administration of the antibody. Before collecting blood, use ThromboFix Platelet Stabilizer (Cat. No. 6607130, manufactured by BECKMAN COULTER) as a blood fixation solution to prepare a working solution by mixing equal amounts of solution A and solution B in advance, and 6 μL each is required. It was dispensed into a minute tube. For the blood collection, about 10 μL of blood was collected from the tail vein of each mouse using a capillary (V. hematocrit capillary EDTA-2K, Tokyo Glass Instruments Co., Ltd.), and 6 μL of the blood was collected including the dispensed working solution. Blood samples were prepared by adding them into a tube and stirring well by tapping to fix them. The storage period of the fixed blood sample was 7 days at room temperature (about 25 ° C., hereinafter the same) and in a dark place.
（６－２）血液サンプルの調製
　前記固定した各血液サンプル１０μＬに、前記ビカネイト輸液９０μＬを添加し、ＡＣＤ－Ａ添加血液を調製した。調製した各ＡＣＤ－Ａ添加血液２０μＬに対し、ＰＢＳ（Cat. No. 10010-023，Thermo Fisher Scientific K.K）を９８０μＬ添加し、１０００倍希釈濃度サンプルを調製した。前記各１０００倍希釈濃度サンプルを、BD Trucount Tubes（染色試料用）またはラウンドボトムチューブ（非染色試料（コントロール）用、染色校正用試料用）に、１００μＬずつ分注した。前記非染色試料は、測定毎に１本用意し、染色試料は、校正用に１本と各動物分とを用意した。前記各チューブに、PE/Cy7で標識したRat IgG1, κ Isotype Control抗体およびAPC Mouse IgG1，κ Isotype Control抗体（非染色試料用、染色校正用試料：Cat.No. 400416，400120，Biolegend, Inc.）、またはPE-Cy7で標識した anti-mouse CD41抗体およびAPC anti-human CD41抗体（染色試料用：Cat. No. 133916、303710、Biolegend, Inc.）を５μＬずつ添加して撹拌し、室温、遮光下にて１５分間静置し、反応させた。前記反応後の各チューブに、ＰＢＳを４００μＬ／ｔｕｂｅとなるように添加し、測定に使用した。各サンプルは、測定まで室温、暗所で保存した。 (6-2) Preparation of Blood Samples 90 μL of the vicanate infusion solution was added to 10 μL of each fixed blood sample to prepare ACD-A-added blood. To 20 μL of each ACD-A-added blood prepared, 980 μL of PBS (Cat. No. 10010-023, Thermo Fisher Scientific KK) was added to prepare a 1000-fold diluted concentration sample. 100 μL of each of the 1000-fold diluted samples was dispensed into BD Trucount Tubes (for stained samples) or round bottom tubes (for unstained samples (control) and samples for staining calibration). One unstained sample was prepared for each measurement, and one stained sample and one for each animal were prepared for calibration. Rat IgG1, κ Isotype Control antibody and APC Mouse IgG1, κ Isotype Control antibody labeled with PE / Cy7 and APC Mouse IgG1, κ Isotype Control antibody (for unstained sample, sample for staining calibration: Cat.No. 400416, 400120, Biolegend, Inc. ) Or PE-Cy7 labeled anti-mouse CD41 antibody and APC anti-human CD41 antibody (for stained samples: Cat. No. 133916, 303710, Biolegend, Inc.) were added in 5 μL increments and stirred at room temperature. The reaction was allowed to stand for 15 minutes under shading. PBS was added to each tube after the reaction so as to be 400 μL / tube, and used for the measurement. Each sample was stored at room temperature in the dark until measurement.
（６－３）フローサイトメーター測定
　前記各サンプルについて、フローサイトメーター（BD FACSLyric、日本ベクトン・ディッキンソン株式会社）およびBD FACSuite（測定ソフトウェア、日本ベクトン・ディッキンソン株式会社）を用いて、血小板数を測定した。なお、機器の精度管理は、CS&T beads（Cat.No.656505、日本ベクトン・ディッキンソン株式会社）を用いて行った。まず、前記非染色試料（コントロール）をフローサイトメーターにセットし、ＦＳＣおよびＳＳＣで展開した。測定にあたり、赤血球画分および血小板画分が適切に表示されるように検出器の感度を調整した。前記非染色試料中の血小板画分をゲーティングし、PE-Cy7のヒストグラムで展開した。展開にあたり、陰性のピークが適切に表示されるようにPE-Cy7の感度調整を行った。そして、染色校正用試料をフローサイトメーターにセットし、APCのヒストグラムで展開した。展開にあたり、陰性および陽性のピークが適切な位置にくるように、APCの感度調整を行った。前記染色校正用試料については、APC/PE-Cy7の二次元ヒストグラムで展開し、必要に応じて蛍光補正を行った。上記で設定した条件に基づき、染色試料中の血小板数を測定した。測定は、Trucount Tubesビーズを6,000個計測したところで終了した。なお、感度調整は測定毎に１回実施し、試料間で条件は変更しないものとした。 (6-3) Flow cytometer measurement For each of the above samples, platelet count was measured using a flow cytometer (BD FACSLyric, Japan Becton Dickinson Co., Ltd.) and BD FAC Suite (measurement software, Japan Becton Dickinson Co., Ltd.). did. The quality of the equipment was controlled using CS & T beads (Cat.No.656505, Japan Becton Dickinson Co., Ltd.). First, the unstained sample (control) was set in a flow cytometer and developed by FSC and SSC. In the measurement, the sensitivity of the detector was adjusted so that the red blood cell fraction and the platelet fraction were displayed appropriately. The platelet fraction in the unstained sample was gated and developed on a PE-Cy7 histogram. Upon development, the sensitivity of PE-Cy7 was adjusted so that the negative peak was displayed appropriately. Then, the sample for staining calibration was set on the flow cytometer and developed by the histogram of APC. Upon deployment, the sensitivity of the APC was adjusted so that the negative and positive peaks were in the appropriate positions. The staining calibration sample was developed with a two-dimensional histogram of APC / PE-Cy7, and fluorescence correction was performed as necessary. Based on the conditions set above, the number of platelets in the stained sample was measured. The measurement was completed when 6,000 Trucount Tubes beads were measured. The sensitivity was adjusted once for each measurement, and the conditions were not changed between the samples.
（６－４）データ解析
　前記測定データを、FACSuiteまたはFlowJo（FlowJo LLC.）を使用して解析し、Microsoft Excel 2010（日本マイクロソフト株式会社）を使用して、各サンプル中の血小板数を算出した。具体的には、校正用染色試料について上記と同様の方法でPE（Trucount Tubes）およびPE-Cy7（CD41）のヒストグラムを表示し、陽性ピークが陰性側に降り立つところの蛍光強度を求めた。求めた蛍光強度より高い領域に入った細胞を陽性細胞とし、各測定試料における陽性細胞数（血小板測定数：PE-Cy7陽性細胞、Trucount Tubesビーズ測定数：PE陽性細胞）を求めた。前記血小板測定数およびTrucount Tubesビーズ測定数より、下記式（１）に従い、血小板の絶対数を算出した。算出した血小板数を図３および下記表２に示す。
Figure JPOXMLDOC01-appb-M000002
 (6-4) Data analysis The measured data was analyzed using FACSuite or FlowJo (FlowJo LLC.), And the number of platelets in each sample was calculated using Microsoft Excel 2010 (Microsoft Japan Co., Ltd.). .. Specifically, the histograms of PE (Trucount Tubes) and PE-Cy7 (CD41) were displayed for the calibrated stained sample by the same method as described above, and the fluorescence intensity where the positive peak fell on the negative side was determined. The cells that entered the region higher than the obtained fluorescence intensity were regarded as positive cells, and the number of positive cells in each measurement sample (platelet measurement number: PE-Cy7 positive cell, Trucount Tubes bead measurement number: PE positive cell) was determined. From the number of platelets measured and the number of Trucount Tubes beads measured, the absolute number of platelets was calculated according to the following formula (1). The calculated platelet counts are shown in FIG. 3 and Table 2 below.
Figure JPOXMLDOC01-appb-M000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　図３は、各マウスの血液中の血小板数を示すグラフである。図３において、横軸は、サンプルの種類および日付を示し、縦軸は、血小板数を示す。図３および前記表２に示すように、０日目においては、いずれの群においても、血小板数は約２５０×１０４血小板／μＬであった。また、８日目において、実施例のマウスの全例が、Ｘ線照射および１回目の抗ＣＤ４１抗体の投与後において、血液中の血小板数が７．３×１０４血小板／μＬ以下となっており、血小板減少症が生じていることが確認できた。他方、比較例および参考例のマウスでは、０日目と比較して血中の血小板数は減少し、一部は、血液中の血小板数が７．３×１０４血小板／μＬ以下となっており、血小板減少症が生じているが、大半は、７．３×１０４血小板／μＬを超え、血小板減少症が生じておらず、個体差が大きいことがわかった。このため、本発明のモデル動物の製造方法により、血小板減少症のモデル動物を製造できること、ならびに本発明のモデル動物の製造方法により得られるモデル動物は、Ｘ線照射で得られるモデル動物と比較して、血小板数が減少していること、その再現性が高いこと、および個体間のばらつきが少ないことがわかった。また、７日目において、約７６×１０４血小板／μＬであり、０日目の血小板数の約３０％であり、前記血中の血小板数において、抗ＣＤ４１抗体を投与することにより血小板減少症を誘導できている。これらのことから、正常な対象動物の血中の血小板数を基準として、前記マウスの血中の血小板数が、約３０％以下の場合、抗血小板抗体の投与により、血小板減少症を誘導できることがわかった。 FIG. 3 is a graph showing the number of platelets in the blood of each mouse. In FIG. 3, the horizontal axis represents the sample type and date, and the vertical axis represents the platelet count. As shown in FIG. 3 and Table 2 above, on day 0, the platelet count was approximately 250 × 10 4 platelets / μL in all groups. Further, on the eighth day, all of the mouse device of embodiment, after administration of the X-ray irradiation and the first anti-CD41 antibodies, platelet count in blood becomes less 7.3 × 10 4 platelets / [mu] L It was confirmed that thrombocytopenia had occurred. On the other hand, the mice in comparative examples and reference examples, the number of platelets compared to the blood to day 0 is reduced, in part, the number of platelets in the blood becomes less 7.3 × 10 4 platelets / [mu] L cage, although thrombocytopenia occurs, most greater than 7.3 × 10 4 platelets / [mu] L, no thrombocytopenia occurs, it was found that a large individual difference. Therefore, the model animal for platelet hypoplasia can be produced by the method for producing a model animal of the present invention, and the model animal obtained by the method for producing a model animal of the present invention is compared with the model animal obtained by X-ray irradiation. It was found that the platelet count was reduced, its reproducibility was high, and there was little variation among individuals. Further, in 7 days, about 76 × 10 4 platelets / [mu] L, about 30% of the number of platelets day 0, thrombocytopenia by the platelet count of the blood, administering an anti-CD41 antibody Can be induced. From these facts, it is possible to induce thrombocytopenia by administration of an antiplatelet antibody when the blood platelet count of the mouse is about 30% or less based on the blood platelet count of a normal target animal. all right.
［実施例２］
　本発明の製造方法より作成した血小板減少症のモデル動物の出血時間を確認した。 [Example 2]
The bleeding time of a model animal for thrombocytopenia prepared by the production method of the present invention was confirmed.
　前記実施例１（５）の９日目に被検物質を投与したマウスを用い、血小板製剤の投与の有無による各マウスの出血時間（止血時間、Bleeding time）を確認した（実施例、比較例および参考例のいずれもｎ＝４）。前記被検物質の投与から１０分後の採血終了後に、マウスをイソフルラン麻酔による麻酔下においた。そして、各個体の腹側の尾動脈について、尾の先端より２ｃｍの部分に対し、２３Ｇの注射針のエッジ部を用いて切創した。前記切創部からの出血を確認した後、５０ｍｌファルコンチューブ内で３７℃に保温した生理食塩液４５ｍＬに、尾先端から４ｃｍ程度を浸し、出血箇所からの止血までの時間を測定した。測定時間は最大で１０分間（６００秒）とし、１０分を超えても止血しなかった個体については、出血時間を６００秒とした。また、切創は１固体あたり１箇所とした。なお、前記実施例１で用いたマウスのうち、実施例、比較例および参考例から１個体ずつサテライトとしたため、本試験には用いなかった。各マウスの出血時間を、図４および下記表３に示す。 Using the mice to which the test substance was administered on the 9th day of Example 1 (5), the bleeding time (hemostatic time, Bleeding time) of each mouse was confirmed depending on the presence or absence of administration of the platelet preparation (Example, Comparative Example). And all of the reference examples are n = 4). After the completion of blood sampling 10 minutes after the administration of the test substance, the mice were anesthetized with isoflurane anesthesia. Then, the ventral tail artery of each individual was incised at a portion 2 cm from the tip of the tail using the edge portion of a 23 G injection needle. After confirming bleeding from the incision, 45 mL of physiological saline kept at 37 ° C. in a 50 ml falcon tube was immersed about 4 cm from the tip of the tail, and the time from the bleeding site to hemostasis was measured. The maximum measurement time was 10 minutes (600 seconds), and for individuals who did not stop bleeding even after 10 minutes, the bleeding time was 600 seconds. In addition, the number of cuts was one per solid. Of the mice used in Example 1, one individual was designated as a satellite from Examples, Comparative Examples, and Reference Examples, so it was not used in this test. The bleeding time of each mouse is shown in FIG. 4 and Table 3 below.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　図４は、出血時間を示すグラフである。図４において、横軸は、サンプルの種類および投与した被検物質の種類を示し、縦軸は、出血時間を示す。図４および前記表３に示すように、比較例のマウスにおいて、Vehicle投与群は、出血時間の平均値が１７７±２３秒（Mean±S.E.（標準誤差）、以下、同様）であり、血小板製剤投与群の出血時間の平均値が９７±９秒であり、血小板製剤の投与による出血時間の短縮傾向が認められた。また、参考例のマウスにおいて、Vehicle投与群は、出血時間の平均値が１１１±１０秒であり、血小板製剤投与群の出血時間の平均値は８３±１７秒であり、血小板製剤の投与による出血時間の短縮傾向は認められなかった。他方、実施例のマウスにおいて、Vehicle投与群は、全個体の出血時間が６００秒を超え、顕著な出血時間の延長が認められた。また、実施例のマウスにおける血小板製剤投与群の出血時間の平均値は、２１３±３４秒となり、血小板製剤の投与による出血時間の明らかな短縮が認められた。なお、実施例２と独立して同様の試験を行なったところ、同様の傾向を示す結果が得られた。 FIG. 4 is a graph showing the bleeding time. In FIG. 4, the horizontal axis represents the type of sample and the type of test substance administered, and the vertical axis represents the bleeding time. As shown in FIG. 4 and Table 3 above, in the mice of the comparative example, the average value of the bleeding time in the Vehicle-administered group was 177 ± 23 seconds (Mean ± SE (standard error), hereinafter the same), and the platelet preparation. The average bleeding time in the administration group was 97 ± 9 seconds, and there was a tendency for the bleeding time to be shortened by the administration of platelet preparations. In the mouse of the reference example, the average value of the bleeding time in the Vehicle-administered group was 111 ± 10 seconds, and the average value of the bleeding time in the platelet preparation-administered group was 83 ± 17 seconds, and bleeding due to the administration of the platelet preparation. No tendency to shorten the time was observed. On the other hand, in the mice of the example, in the Vehicle-administered group, the bleeding time of all the individuals exceeded 600 seconds, and a remarkable prolongation of the bleeding time was observed. In addition, the average value of the bleeding time in the platelet preparation group in the mice of the examples was 213 ± 34 seconds, and a clear reduction in the bleeding time due to the administration of the platelet preparation was observed. When the same test was conducted independently of Example 2, results showing the same tendency were obtained.
　以上の事から、本発明の製造方法により作成した血小板減少症のモデル動物は、血小板数が減少しており、顕著な出血時間の延長が認められることから、血小板の機能の評価に適していることが分かった。 From the above, the model animal for thrombocytopenia prepared by the production method of the present invention has a decreased platelet count and a remarkable prolongation of bleeding time, and is therefore suitable for evaluation of platelet function. It turned out.
［参考例１］
　異なる強度のＸ線照射による、マウスへの影響（一般状態、体重の変化、血小板数の変化、および出血時間）を確認した。 [Reference Example 1]
The effects on mice (general condition, changes in body weight, changes in platelet count, and bleeding time) due to different intensity X-ray irradiation were confirmed.
　７週齢の雄のＮＯＧマウス（公益財団法人　実験動物中央研究所社製）を用い、０および１日目のＸ線照射時の線量を、４Ｇｙ、５Ｇｙまたは６Ｇｙ（４Ｇｙ照射群、５Ｇｙ照射群、６Ｇｙ照射群）とした以外は前記実施例１における比較例のマウスと同様の処理を行った（ｎ＝４）。なお、初回のＸ線照射時を０日目としている。また、本参考例においては、未処理（Ｘ線非照射群、Ｎｏｒｍａｌ）のマウスも試験に加えた。そして、０、５、および９日目に、各マウスの体重を記録した。 Using a 7-week-old male NOG mouse (manufactured by Central Institute for Experimental Animals), the dose at the time of X-ray irradiation on the 0th and 1st days was 4Gy, 5Gy or 6Gy (4Gy irradiation group, 5Gy irradiation group). , 6 Gy irradiation group), and the same treatment as that of the mouse of the comparative example in Example 1 was performed (n = 4). The time of the first X-ray irradiation is set as the 0th day. In addition, in this reference example, untreated (X-ray non-irradiated group, Normal) mice were also added to the test. Then, on days 0, 5, and 9, the weight of each mouse was recorded.
　血小板数の測定のため、５、７、および９日目に、キャピラリー（V.ヘマトクリット毛細管EDTA-2K、東京硝子器械株式会社）を用いて、各マウスの尾静脈より血液を１０μＬ採取した。採取した血液のうち、５μＬを、５％のＡＣＤ－Ａを添加したビカーボン輸液９５μＬ中に添加して、ＡＣＤ－Ａ添加血液サンプルを調製した。なお、ＡＣＤ－Ａ添加血液サンプルは、必要に応じてＰＢＳ（Cat. No. 10010-023，Thermo Fisher Scientific K.K）で希釈した。 For the measurement of platelet count, 10 μL of blood was collected from the tail vein of each mouse on days 5, 7, and 9 using a capillary (V. hematocrit capillary EDTA-2K, Tokyo Glass Instruments Co., Ltd.). Of the collected blood, 5 μL was added to 95 μL of a bicarbon infusion solution supplemented with 5% ACD-A to prepare an ACD-A-added blood sample. The ACD-A-added blood sample was diluted with PBS (Cat. No. 10010-023, Thermo Fisher Scientific K.K) as needed.
　前記調製した血液サンプルを使用し、サンプルに添加する抗体をPE/Cy7で標識したRat IgG1, κ Isotype Control抗体（非染色試料および染色校正用試料：Cat. No. 400416，Biolegend, Inc.）、またはPE-Cy7で標識したanti-mouse CD41抗体（染色試料：Cat. No. 133916，Biolegend, Inc.）５μＬとした以外は、前記実施例１（６－３）および（６－４）と同様にして、血小板数の測定、フローサイトメーター測定、およびデータ解析を行った。また、９日目の前記採血後、各マウスについて、被検物質を投与しなかった以外は前記実施例２と同様にして、出血時間の測定を行った。なお、６Ｇｙ照射群のマウスのうち１個体は、止血確認後に再出血したため、出血時間の測定を再開し、再度出血が認められなくなった時点を測定終了とした。 Using the prepared blood sample, the antibody to be added to the sample was labeled with PE / Cy7, Rat IgG1, κ Isotype Control antibody (unstained sample and stained calibration sample: Cat. No. 400416, Biolegend, Inc.), Alternatively, the same as in Examples 1 (6-3) and (6-4) except that the anti-mouse CD41 antibody labeled with PE-Cy7 (stained sample: Cat. No. 133916, Biolegend, Inc.) was 5 μL. Then, the platelet count was measured, the flow cytometer was measured, and the data was analyzed. In addition, after the blood sampling on the 9th day, the bleeding time was measured in the same manner as in Example 2 except that the test substance was not administered to each mouse. Since one of the mice in the 6 Gy irradiation group rebleeded after confirming hemostasis, the measurement of the bleeding time was restarted, and the measurement was terminated when no bleeding was observed again.
　血小板数の変化および出血時間を、下記表４～表５に示す。なお、Ｘ線非照射群、４、５、および６Ｇｙ照射群のマウスにおいて、一般状態に変化は見られなかったため、データの詳細は示していない。 The changes in platelet count and bleeding time are shown in Tables 4 to 5 below. The details of the data are not shown in the mice in the X-ray non-irradiation group, 4, 5, and 6 Gy irradiation group because no change was observed in the general state.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　前記表４に示すように、Ｘ線照射強度が強くなるに従い、マウスの血小板数が大きく減少する傾向を示した。具体的には、Ｘ線非照射群のマウスは、９日目の血小板数の平均値が２２４．２±４．７（×104 platelets/μL、mean±S.E.、n=4）であったのに対し、４Ｇｙ照射群のマウスは、２１．１±２．４（×104 platelets/μL、mean±S.E.、n=4）、５Ｇｙ照射群のマウスは、１２．１±２．３（×104 platelets/μL、mean±S.E.、n=4）、６Ｇｙ照射群のマウスは、１１．７±１．９（×104 platelets/μL、mean±S.E.、n=2）であった。前記実施例１で示しているように、約７６×１０４血小板／μＬに減少していれば、抗血小板抗体の投与により、血小板数が７．３×１０４血小板／μＬ以下に減少する。このため、Ｘ線を２回照射する場合、１回あたり４～６Ｇｙの照射後、抗血小板抗体を投与することで、血小板減少症のモデル動物が製造できることが示唆された。 As shown in Table 4, the platelet count of the mouse tended to decrease significantly as the X-ray irradiation intensity increased. Specifically, the mice in the X-ray non-irradiated group had an average platelet count of 224.2 ± 4.7 (× 10 4 platelets / μL, mean ± SE, n = 4) on the 9th day. On the other hand, the mice in the 4 Gy irradiation group were 21.1 ± 2.4 (× 10 4 platelets / μL, mean ± SE, n = 4), and the mice in the 5 Gy irradiation group were 12.1 ± 2.3 (12.1 ± 2.3). The mice in the × 10 4 platelets / μL, mean ± SE, n = 4) and 6 Gy irradiation groups were 11.7 ± 1.9 (× 10 4 platelets / μL, mean ± SE, n = 2). As shown in Example 1, if the number of platelets is reduced to about 76 × 10 4 platelets / μL, the number of platelets is reduced to 7.3 × 10 4 platelets / μL or less by administration of the antiplatelet antibody. Therefore, it was suggested that a model animal for thrombocytopenia can be produced by administering an antiplatelet antibody after irradiation of 4 to 6 Gy each time when X-rays are irradiated twice.
　前記表５に示すように、Ｘ線非照射群のマウスは、出血時間が１５７±２３秒（ｎ＝４）であった。これに対し、４Ｇｙ照射群のマウスは、３３９±９４秒（ｎ＝４）、５Ｇｙ照射群のマウスは、３７０±９２秒（ｎ＝４）、６Ｇｙ照射群のマウスは、５２７秒（ｎ＝２）であり、Ｘ線を照射したマウスは、Ｘ線非照射群のマウスと比較して、Ｘ線の照射量に応じて出血時間が延長する傾向が見られるものの、個体間のばらつきが非常に大きかった。 As shown in Table 5 above, the mice in the X-ray non-irradiated group had a bleeding time of 157 ± 23 seconds (n = 4). On the other hand, the mouse in the 4 Gy irradiation group was 339 ± 94 seconds (n = 4), the mouse in the 5 Gy irradiation group was 370 ± 92 seconds (n = 4), and the mouse in the 6 Gy irradiation group was 527 seconds (n = 4). In 2), the mice irradiated with X-rays tended to prolong the bleeding time depending on the amount of X-ray irradiation as compared with the mice in the X-ray non-irradiated group, but the variation among individuals was very large. It was big.
　以上の事から、放射線照射量を多くするに従い、血小板数を減少させることができることが分かった。また、Ｘ線を２回照射する場合、１回あたり４～６Ｇｙの照射後、抗血小板抗体を投与することで、血小板減少症のモデル動物が製造できると推定された。 From the above, it was found that the platelet count can be reduced as the irradiation dose is increased. In addition, when X-rays were irradiated twice, it was estimated that a model animal for thrombocytopenia could be produced by administering an antiplatelet antibody after each irradiation with 4 to 6 Gy.
［参考例２］
　異なる強度のＸ線照射による、血小板数の変化を確認した。 [Reference example 2]
Changes in platelet count due to X-ray irradiation of different intensities were confirmed.
　６週齢の雄のＮＯＧマウス（公益財団法人　実験動物中央研究所社製）を用い、０日目のＸ線照射時の線量を、１．５Ｇｙ、２．０Ｇｙまたは２．５Ｇｙ（１．５Ｇｙ照射群、２．０Ｇｙ照射群、または２．５Ｇｙ照射群）とした以外は前記実施例１における比較例のマウスと同様の処理を行った（ｎ＝４）。なお、初回のＸ線照射時を０日目としている。 Using a 6-week-old male NOG mouse (manufactured by Central Institute for Experimental Animals), the dose at the time of X-ray irradiation on the 0th day was 1.5 Gy, 2.0 Gy or 2.5 Gy (1.5 Gy). The same treatment as that of the mouse of the comparative example in Example 1 was carried out except that the mouse was designated as an irradiation group, a 2.0 Gy irradiation group, or a 2.5 Gy irradiation group (n = 4). The time of the first X-ray irradiation is set as the 0th day.
　血小板数の測定のため、Ｘ線処理前（０日）、７、および９日目に、キャピラリー（V.ヘマトクリット毛細管EDTA-2K、東京硝子器械株式会社）を用いて、各マウスの尾静脈より血液を１０μＬ採取した以外は、前記参考例１と同様にして、血小板数の測定、フローサイトメーター測定、およびデータ解析を行った。これらの結果を下記表６に示す。 From the tail vein of each mouse using a capillary (V. hematocrit capillary EDTA-2K, Tokyo Glass Instruments Co., Ltd.) before X-ray treatment (day 0), 7 and 9 days to measure the platelet count. Platelet count measurement, flow cytometer measurement, and data analysis were performed in the same manner as in Reference Example 1 except that 10 μL of blood was collected. These results are shown in Table 6 below.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　前記表６に示すように、７日目および９日目のいずれにおいても、Ｘ線照射強度が強くなるに従い、マウスの血小板数が大きく減少する傾向を示した。具体的には、７日目において、１．５Ｇｙ照射群のマウスは、７１．１±５．９（×104 platelets/μL、mean±S.E、n=4）、２．０Ｇｙ照射群のマウスは、６０．２±２．０（×104 platelets/μL、mean±S.E、n=4）、２．５Ｇｙ照射群のマウスは、５５．７±２．５（×104 platelets/μL、mean±S.E、n=4）であった。また、９日目において、１．５Ｇｙ照射群のマウスは、５１．７±２．１（×104 platelets/μL、mean±S.E、n=4）、２．０Ｇｙ照射群のマウスは、５２．９±３．６（×104 platelets/μL、mean±S.E、n=4）、２．５Ｇｙ照射群のマウスは、５０．６±１．９（×104 platelets/μL、mean±S.E、n=4）であった。前記実施例１で示しているように、約７６×１０４血小板／μLに減少していれば、抗血小板抗体の投与により、血小板数が７．３×１０４血小板／μＬ以下に減少する。このため、Ｘ線を１回照射する場合、１回あたり１．５Ｇｙ以上のＸ線照射後、抗血小板抗体を投与することで、血小板減少症のモデル動物が製造できることが示唆された。 As shown in Table 6, on both the 7th and 9th days, the platelet count of the mice tended to decrease significantly as the X-ray irradiation intensity increased. Specifically, on the 7th day, the mice in the 1.5 Gy irradiation group were 71.1 ± 5.9 (× 10 4 platelets / μL, mean ± SE, n = 4), and the mice in the 2.0 Gy irradiation group. 60.2 ± 2.0 (× 10 4 platelets / μL, mean ± SE, n = 4), 55.7 ± 2.5 (× 10 4 platelets / μL) for mice in the 2.5 Gy irradiation group. It was mean ± SE, n = 4). On the 9th day, the mice in the 1.5 Gy irradiation group had 51.7 ± 2.1 (× 10 4 platelets / μL, mean ± SE, n = 4), and the mice in the 2.0 Gy irradiation group had 52. Mice in the 9.9 ± 3.6 (× 10 4 platelets / μL, mean ± SE, n = 4), 2.5 Gy irradiation group were 50.6 ± 1.9 (× 10 4 platelets / μL, mean ± SE). , N = 4). As shown in Example 1, if reduced to about 76 × 10 4 platelets / [mu] L, administration of anti-platelet antibodies, platelet count is reduced to below 7.3 × 10 4 platelets / [mu] L. Therefore, it was suggested that a model animal for thrombocytopenia can be produced by administering an antiplatelet antibody after X-ray irradiation of 1.5 Gy or more each time when X-ray is irradiated once.
　以上のことから、放射線照射量を大きくするに従い、血小板数を減少させることができることが分かった。また、Ｘ線を１回照射する場合、１回あたり１．５Ｇｙ以上のＸ線照射後、抗血小板抗体を投与することで、血小板減少症のモデル動物が製造できると推定された。 From the above, it was found that the platelet count can be reduced as the irradiation dose is increased. In addition, when X-rays are irradiated once, it is estimated that a model animal for thrombocytopenia can be produced by administering an antiplatelet antibody after each irradiation with 1.5 Gy or more of X-rays.
　以上、実施形態および実施例を参照して本発明を説明したが、本発明は、上記実施形態および実施例に限定されるものではない。本発明の構成や詳細には、本発明のスコープ内で当業者が理解し得る様々な変更をすることができる。 Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the structure and details of the present invention within the scope of the present invention.
　この出願は、２０１９年３月１３日に出願された日本出願特願２０１９－０４６５６２を基礎とする優先権を主張し、その開示のすべてをここに取り込む。 This application claims priority based on Japanese application Japanese Patent Application No. 2019-046562 filed on March 13, 2019, and incorporates all of its disclosures herein.
＜付記＞
　上記の実施形態および実施例の一部または全部は、以下の付記のように記載されうるが、以下には限られない。
（付記１）
非ヒト動物に放射線を照射する照射工程と、
前記非ヒト動物に抗血球抗体を投与する投与工程とを含む、血球減少症のモデル動物の製造方法。
（付記２）
前記抗血球抗体は、血球の細胞膜表面抗原に対する抗体である、付記１記載の製造方法。
（付記３）
前記抗血球抗体は、血小板の細胞膜表面抗原に対する抗体である、付記１または２記載の製造方法。
（付記４）
前記血小板の細胞膜表面抗原に対する抗体は、抗ＣＤ４１抗体、抗ＣＤ４２ａ抗体、抗ＣＤ４２ｂ抗体、抗ＣＤ４２ｃ抗体、抗ＣＤ４２ｄ抗体、抗ＣＤ４９ｂ抗体、抗ＣＤ６１抗体、抗ＣＤ１０９抗体、および抗ＧＰＶＩ抗体からなる群から選択された少なくとも一つを含む、付記３記載の製造方法。
（付記５）
前記抗血球抗体は、細胞除去機能性抗体である、付記１から４のいずれかに記載の製造方法。
（付記６）
前記照射工程における放射線照射量は、１．５～１２Ｇｙの範囲である、付記１から５のいずれかに記載の製造方法。
（付記７）
前記照射工程を複数回実施する、付記１から６のいずれかに記載の製造方法。
（付記８）
前記放射線は、Ｘ線およびγ線の少なくとも一方である、付記１から７のいずれかに記載の製造方法。
（付記９）
前記照射工程後に前記投与工程を実施する、付記１から８のいずれかに記載の製造方法。
（付記１０）
前記照射工程後、最初の放射線照射時を基準として、５～１３日に前記投与工程を実施する、付記１から９のいずれかに記載の製造方法。
（付記１１）
前記非ヒト動物は、齧歯類である、付記１から１０のいずれかに記載の製造方法。
（付記１２）
前記非ヒト動物は、マウスである、付記１から１１のいずれかに記載の製造方法。
（付記１３）
前記マウスは、免疫不全マウスまたは細網内皮系が破壊されたマウスである、付記１２記載の製造方法。
（付記１４）
前記免疫不全マウスは、ＮＯＧマウスである、付記１３記載の製造方法。
（付記１５）
前記血球は、血小板、赤血球、および白血球からなる群から選択された少なくとも一つである、付記１から１４のいずれかに記載の製造方法。
（付記１６）
付記１から１５のいずれかに記載の血球減少症のモデル動物の製造方法により得られる、血球減少症のモデル動物。
（付記１７）
下記条件（１）～（３）のいずれか一つ以上を満たす、付記１６記載の血球減少症のモデル動物：
（１）血液中の血小板数が、７．３×１０４個／μＬ以下である。
（２）血液中の赤血球数が、５００×１０４個／μＬ以下である。
（３）血液中の白血球数が、３×１０２個／μＬ以下である。
（付記１８）
下記条件（１）～（３）のいずれか一つ以上を満たす、血球減少症のモデル動物：
（１）前記モデル動物の血液中の血小板数が、７．３×１０４個／μＬ以下である。
（２）血液中の赤血球数が、５００×１０４個／μＬ以下である。
（３）血液中の白血球数が、３×１０２個／μＬ以下である。
（付記１９）
下記条件（４）および（５）の少なくとも一方を満たす、付記１６から１８のいずれかに記載のモデル動物：
（４）出血開始から止血までの時間が５分を超える；
（５）前記モデル動物の血液は、抗血球抗体を含む。
（付記２０）
血球減少症のモデル動物に血球を投与する工程と
前記血球の機能を評価する評価工程とを含み、
前記血球減少症のモデル動物は、付記１６から１９のいずれかに記載の血球減少症のモデル動物である、血球機能の評価方法。
（付記２１）
前記血球は、血小板であり、
前記評価工程において、前記モデル動物を出血させて前記血小板の機能を評価する、付記２０記載の評価方法。
（付記２２）
前記血球は、血小板であり、
前記評価工程において、止血時間に基づき、前記血小板の機能を評価する、付記２０または２１記載の評価方法。
（付記２３）
前記血球は、血小板であり、
前記血小板の機能は、止血機能である、付記２０から２２のいずれかに記載の評価方法。
（付記２４）
前記血球は、血小板であり、
前記血小板は、血小板製剤または血小板機能模倣剤（Platelet functional mimetics）である、付記２０から２３のいずれかに記載の評価方法。
（付記２５）
前記血球は、血小板であり、
前記血小板は、ヒト血小板である、付記２０から２４のいずれかに記載の評価方法。
（付記２６）
付記１から１５のいずれかに記載の血球減少症のモデル動物の製造方法により、血球減少症のモデル動物を製造する製造工程を含む、付記２０から２５のいずれかに記載の評価方法。
（付記２７）
被検血球について、血球の機能を評価する評価工程と、
前記評価工程において、基準を満たす血球を機能性の血球として選抜する選抜工程とを含み、
前記評価工程は、付記２０から２６のいずれかに記載の血球機能の評価方法により実施される、血球の製造方法。
（付記２８）
前記血球は、血小板である、付記２７記載の製造方法。
（付記２９）
被検物質を血球減少症のモデル動物に投与する工程と、
前記モデル動物に血液中の血球数が増加する被検物質を、血球減少症の治療薬の候補物質として選択する選択工程とを含み、
前記モデル動物は、付記１６から１９のいずれかに記載の血球減少症のモデル動物である、血球減少症の治療薬候補物質のスクリーニング方法。
（付記３０）
前記選択工程において、前記被検物質を投与していないコントロールのモデル動物と比較して、前記血球数が増加している被検物質を、前記治療用候補物質として選択する、付記２９記載のスクリーニング方法。
（付記３１）
前記被検物質が、低分子化合物、ペプチド、タンパク質および核酸からなる群から選択された少なくとも１つである、付記２９または３０記載のスクリーニング方法。
（付記３２）
前記血球は、血小板である、付記２９から３１のいずれかに記載のスクリーニング方法。
（付記３３）
付記１から１５のいずれかに記載の血球減少症のモデル動物の製造方法により、血球減少症のモデル動物を製造する製造工程を含む、付記２９から３２のいずれかに記載のスクリーニング方法。
（付記３４）
被検物質から血球減少症の治療薬の候補物質を選抜する選抜工程を含み、
前記選抜工程は、付記２９から３３のいずれかに記載の血球減少症の治療薬候補物質のスクリーニング方法で実施される、血球減少症の治療薬の候補物質の製造方法。 <Appendix>
Some or all of the above embodiments and examples may be described as, but not limited to, the following appendices.
(Appendix 1)
Irradiation process to irradiate non-human animals and
A method for producing a model animal for cytopenia, which comprises an administration step of administering an anti-blood cell antibody to the non-human animal.
(Appendix 2)
The production method according to Appendix 1, wherein the anti-blood cell antibody is an antibody against a cell membrane surface antigen of blood cells.
(Appendix 3)
The production method according to Appendix 1 or 2, wherein the anti-blood cell antibody is an antibody against a cell membrane surface antigen of platelets.
(Appendix 4)
The antibody against the cell membrane surface antigen of the platelets comprises a group consisting of an anti-CD41 antibody, an anti-CD42a antibody, an anti-CD42b antibody, an anti-CD42c antibody, an anti-CD42d antibody, an anti-CD49b antibody, an anti-CD61 antibody, an anti-CD109 antibody, and an anti-GPVI antibody. The manufacturing method according to Appendix 3, which comprises at least one selected.
(Appendix 5)
The production method according to any one of Supplementary note 1 to 4, wherein the anti-blood cell antibody is a cell-removing functional antibody.
(Appendix 6)
The production method according to any one of Appendix 1 to 5, wherein the radiation irradiation amount in the irradiation step is in the range of 1.5 to 12 Gy.
(Appendix 7)
The production method according to any one of Supplementary note 1 to 6, wherein the irradiation step is carried out a plurality of times.
(Appendix 8)
The production method according to any one of Supplementary note 1 to 7, wherein the radiation is at least one of X-ray and γ-ray.
(Appendix 9)
The production method according to any one of Supplementary note 1 to 8, wherein the administration step is carried out after the irradiation step.
(Appendix 10)
The production method according to any one of Supplementary note 1 to 9, wherein the administration step is carried out on 5 to 13 days after the irradiation step with reference to the time of the first irradiation.
(Appendix 11)
The production method according to any one of Appendix 1 to 10, wherein the non-human animal is a rodent.
(Appendix 12)
The production method according to any one of Appendix 1 to 11, wherein the non-human animal is a mouse.
(Appendix 13)
The production method according to Appendix 12, wherein the mouse is an immunodeficient mouse or a mouse in which the reticuloendothelial system is destroyed.
(Appendix 14)
The production method according to Appendix 13, wherein the immunodeficient mouse is a NOG mouse.
(Appendix 15)
The production method according to any one of Supplementary note 1 to 14, wherein the blood cell is at least one selected from the group consisting of platelets, erythrocytes, and white blood cells.
(Appendix 16)
A model animal for cytopenia obtained by the method for producing a model animal for cytopenia according to any one of Appendix 1 to 15.
(Appendix 17)
The model animal for cytopenia according to Appendix 16, which satisfies any one or more of the following conditions (1) to (3):
(1) The number of platelets in the blood is 7.3 × 10 4 / μL or less.
(2) The number of red blood cells in the blood is 500 × 10 4 cells / μL or less.
(3) The number of white blood cells in the blood is 3 × 10 2 cells / μL or less.
(Appendix 18)
A model animal for cytopenia that satisfies any one or more of the following conditions (1) to (3):
(1) The number of platelets in the blood of the model animal is 7.3 × 10 4 cells / μL or less.
(2) The number of red blood cells in the blood is 500 × 10 4 cells / μL or less.
(3) The number of white blood cells in the blood is 3 × 10 2 cells / μL or less.
(Appendix 19)
The model animal according to any one of Appendix 16 to 18, which satisfies at least one of the following conditions (4) and (5):
(4) The time from the start of bleeding to hemostasis exceeds 5 minutes;
(5) The blood of the model animal contains an anti-blood cell antibody.
(Appendix 20)
It includes a step of administering blood cells to a model animal of cytopenia and an evaluation step of evaluating the function of the blood cells.
The method for evaluating blood cell function, wherein the model animal for cytopenia is the model animal for cytopenia according to any one of Appendix 16 to 19.
(Appendix 21)
The blood cells are platelets and
The evaluation method according to Appendix 20, wherein in the evaluation step, the model animal is bleeding to evaluate the function of the platelets.
(Appendix 22)
The blood cells are platelets and
The evaluation method according to Appendix 20 or 21, wherein in the evaluation step, the function of the platelets is evaluated based on the hemostasis time.
(Appendix 23)
The blood cells are platelets and
The evaluation method according to any one of Appendix 20 to 22, wherein the function of the platelet is a hemostatic function.
(Appendix 24)
The blood cells are platelets and
The evaluation method according to any one of Appendix 20 to 23, wherein the platelet is a platelet preparation or a platelet functional mimetics.
(Appendix 25)
The blood cells are platelets and
The evaluation method according to any one of Appendix 20 to 24, wherein the platelet is a human platelet.
(Appendix 26)
The evaluation method according to any one of Annex 20 to 25, which comprises a manufacturing step of producing a model animal for cytopenia by the method for producing a model animal for cytopenia according to any one of Supplements 1 to 15.
(Appendix 27)
An evaluation process for evaluating the function of blood cells to be tested and
The evaluation step includes a selection step of selecting blood cells satisfying the criteria as functional blood cells.
The method for producing blood cells, wherein the evaluation step is carried out by the method for evaluating blood cell function according to any one of Appendix 20 to 26.
(Appendix 28)
The production method according to Appendix 27, wherein the blood cells are platelets.
(Appendix 29)
The process of administering the test substance to a model animal for cytopenia,
The model animal includes a selection step of selecting a test substance that increases the number of blood cells in the blood as a candidate substance for a therapeutic agent for hypocytopenia.
The method for screening a candidate substance for a therapeutic agent for cytopenia, wherein the model animal is a model animal for cytopenia according to any one of Supplementary note 16 to 19.
(Appendix 30)
The screening according to Appendix 29, wherein in the selection step, the test substance having an increased blood cell count is selected as the therapeutic candidate substance as compared with the control model animal to which the test substance is not administered. Method.
(Appendix 31)
The screening method according to Appendix 29 or 30, wherein the test substance is at least one selected from the group consisting of low molecular weight compounds, peptides, proteins and nucleic acids.
(Appendix 32)
The screening method according to any of Supplementary note 29 to 31, wherein the blood cells are platelets.
(Appendix 33)
The screening method according to any one of Annex 29 to 32, which comprises a manufacturing step of producing a model animal for cytopenia by the method for producing a model animal for cytopenia according to any one of Supplements 1 to 15.
(Appendix 34)
Includes a selection process that selects candidate substances for the treatment of cytopenia from the test substances.
The selection step is a method for producing a candidate substance for a therapeutic drug for cytopenia, which is carried out by the screening method for a candidate substance for a therapeutic drug for cytopenia according to any one of Supplementary notes 29 to 33.
　以上のように、本発明によれば、前記Ｘ線照射によるモデル動物の製造方法と比較して、得られたモデル動物の血中の血球数を減少させることができる。また、本発明によれば、例えば、Ｘ線照射による血球減少症モデル動物と比較して、得られたモデル動物の血中の血球数を減少させることができるため、前記血球が血小板の場合、出血時の止血時間（出血時間）が延長できる。このため、本発明によれば、例えば、血球の機能の評価、血小板減少症を含む血球減少症の治療用候補物質を取得に適した血球減少症のモデル動物を製造できる。したがって、本発明は、例えば、血球を使用する細胞医薬分野、血液製剤分野、医療分野等において極めて有用である。 As described above, according to the present invention, the number of blood cells in the blood of the obtained model animal can be reduced as compared with the method for producing a model animal by X-ray irradiation. Further, according to the present invention, for example, the number of blood cells in the blood of the obtained model animal can be reduced as compared with the blood cell hypoplasia model animal by X-ray irradiation. Therefore, when the blood cells are platelets, Hemostasis time (bleeding time) at the time of bleeding can be extended. Therefore, according to the present invention, it is possible to produce a model animal for cytopenia suitable for, for example, evaluation of blood cell function and acquisition of a candidate substance for treatment of cytopenia including thrombocytopenia. Therefore, the present invention is extremely useful in, for example, the field of cell medicine using blood cells, the field of blood products, the field of medical treatment, and the like.

Claims (34)

  1. 非ヒト動物に放射線を照射する照射工程と、
    前記非ヒト動物に抗血球抗体を投与する投与工程とを含む、血球減少症のモデル動物の製造方法。     Irradiation process to irradiate non-human animals and
    A method for producing a model animal for cytopenia, which comprises an administration step of administering an anti-blood cell antibody to the non-human animal.
  2. 前記抗血球抗体は、血球の細胞膜表面抗原に対する抗体である、請求項１記載の製造方法。 The production method according to claim 1, wherein the anti-blood cell antibody is an antibody against a cell membrane surface antigen of blood cells.
  3. 前記抗血球抗体は、血小板の細胞膜表面抗原に対する抗体である、請求項１または２記載の製造方法。 The production method according to claim 1 or 2, wherein the anti-blood cell antibody is an antibody against a cell membrane surface antigen of platelets.
  4. 前記血小板の細胞膜表面抗原に対する抗体は、抗ＣＤ４１抗体、抗ＣＤ４２ａ抗体、抗ＣＤ４２ｂ抗体、抗ＣＤ４２ｃ抗体、抗ＣＤ４２ｄ抗体、抗ＣＤ４９ｂ抗体、抗ＣＤ６１抗体、抗ＣＤ１０９抗体、および抗ＧＰＶＩ抗体からなる群から選択された少なくとも一つを含む、請求項３記載の製造方法。 The antibody against the cell membrane surface antigen of the platelets comprises a group consisting of an anti-CD41 antibody, an anti-CD42a antibody, an anti-CD42b antibody, an anti-CD42c antibody, an anti-CD42d antibody, an anti-CD49b antibody, an anti-CD61 antibody, an anti-CD109 antibody, and an anti-GPVI antibody. The production method according to claim 3, which comprises at least one selected.
  5. 前記抗血球抗体は、細胞除去機能性抗体である、請求項１から４のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 4, wherein the anti-blood cell antibody is a cell-removing functional antibody.
  6. 前記照射工程における放射線照射量は、１．５～１２Ｇｙの範囲である、請求項１から５のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 5, wherein the radiation irradiation amount in the irradiation step is in the range of 1.5 to 12 Gy.
  7. 前記照射工程を複数回実施する、請求項１から６のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 6, wherein the irradiation step is carried out a plurality of times.
  8. 前記放射線は、Ｘ線およびγ線の少なくとも一方である、請求項１から７のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 7, wherein the radiation is at least one of X-ray and γ-ray.
  9. 前記照射工程後に前記投与工程を実施する、請求項１から８のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 8, wherein the administration step is carried out after the irradiation step.
  10. 前記照射工程後、最初の放射線照射時を基準として、５～１３日に前記投与工程を実施する、請求項１から９のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 9, wherein the administration step is carried out on 5 to 13 days after the irradiation step with reference to the time of the first irradiation.
  11. 前記非ヒト動物は、齧歯類である、請求項１から１０のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 10, wherein the non-human animal is a rodent.
  12. 前記非ヒト動物は、マウスである、請求項１から１１のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 11, wherein the non-human animal is a mouse.
  13. 前記マウスは、免疫不全マウスまたは細網内皮系が破壊されたマウスである、請求項１２記載の製造方法。 The production method according to claim 12, wherein the mouse is an immunodeficient mouse or a mouse in which the reticuloendothelial system is destroyed.
  14. 前記免疫不全マウスは、ＮＯＧマウスである、請求項１３記載の製造方法。 The production method according to claim 13, wherein the immunodeficient mouse is a NOG mouse.
  15. 前記血球は、血小板、赤血球、および白血球からなる群から選択された少なくとも一つである、請求項１から１４のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 14, wherein the blood cell is at least one selected from the group consisting of platelets, red blood cells, and white blood cells.
  16. 請求項１から１５のいずれか一項に記載の血球減少症のモデル動物の製造方法により得られる、血球減少症のモデル動物。 A model animal for cytopenia obtained by the method for producing a model animal for cytopenia according to any one of claims 1 to 15.
  17. 下記条件（１）～（３）のいずれか一つ以上を満たす、請求項１６記載の血球減少症のモデル動物：
    （１）血液中の血小板数が、７．３×１０４個／μＬ以下である。
    （２）血液中の赤血球数が、５００×１０４個／μＬ以下である。
    （３）血液中の白血球数が、３×１０２個／μＬ以下である。     The model animal for cytopenia according to claim 16, which satisfies any one or more of the following conditions (1) to (3):
    (1) The number of platelets in the blood is 7.3 × 10 4 / μL or less.
    (2) The number of red blood cells in the blood is 500 × 10 4 cells / μL or less.
    (3) The number of white blood cells in the blood is 3 × 10 2 cells / μL or less.
  18. 下記条件（１）～（３）のいずれか一つ以上を満たす、血球減少症のモデル動物：
    （１）前記モデル動物の血液中の血小板数が、７．３×１０４個／μＬ以下である。
    （２）血液中の赤血球数が、５００×１０４個／μＬ以下である。
    （３）血液中の白血球数が、３×１０２個／μＬ以下である。     A model animal for cytopenia that satisfies any one or more of the following conditions (1) to (3):
    (1) The number of platelets in the blood of the model animal is 7.3 × 10 4 cells / μL or less.
    (2) The number of red blood cells in the blood is 500 × 10 4 cells / μL or less.
    (3) The number of white blood cells in the blood is 3 × 10 2 cells / μL or less.
  19. 下記条件（４）および（５）の少なくとも一方を満たす、請求項１６から１８のいずれか一項に記載のモデル動物：
    （４）出血開始から止血までの時間が５分を超える；
    （５）前記モデル動物の血液は、抗血球抗体を含む。     The model animal according to any one of claims 16 to 18, which satisfies at least one of the following conditions (4) and (5):
    (4) The time from the start of bleeding to hemostasis exceeds 5 minutes;
    (5) The blood of the model animal contains an anti-blood cell antibody.
  20. 血球減少症のモデル動物に血球を投与する工程と
    前記血球の機能を評価する評価工程とを含み、
    前記血球減少症のモデル動物は、請求項１６から１９のいずれか一項に記載の血球減少症のモデル動物である、血球機能の評価方法。     It includes a step of administering blood cells to a model animal of cytopenia and an evaluation step of evaluating the function of the blood cells.
    The method for evaluating blood cell function, wherein the model animal for cytopenia is the model animal for cytopenia according to any one of claims 16 to 19.
  21. 前記血球は、血小板であり、
    前記評価工程において、前記モデル動物を出血させて前記血小板の機能を評価する、請求項２０記載の評価方法。     The blood cells are platelets and
    The evaluation method according to claim 20, wherein in the evaluation step, the model animal is bleeding to evaluate the function of the platelets.
  22. 前記血球は、血小板であり、
    前記評価工程において、止血時間に基づき、前記血小板の機能を評価する、請求項２０または２１記載の評価方法。     The blood cells are platelets and
    The evaluation method according to claim 20 or 21, wherein in the evaluation step, the function of the platelets is evaluated based on the hemostasis time.
  23. 前記血球は、血小板であり、
    前記血小板の機能は、止血機能である、請求項２０から２２のいずれか一項に記載の評価方法。     The blood cells are platelets and
    The evaluation method according to any one of claims 20 to 22, wherein the function of the platelet is a hemostatic function.
  24. 前記血球は、血小板であり、
    前記血小板は、血小板製剤または血小板機能模倣剤（Platelet functional mimetics）である、請求項２０から２３のいずれか一項に記載の評価方法。     The blood cells are platelets and
    The evaluation method according to any one of claims 20 to 23, wherein the platelet is a platelet preparation or a platelet functional mimetics.
  25. 前記血球は、血小板であり、
    前記血小板は、ヒト血小板である、請求項２０から２４のいずれか一項に記載の評価方法。     The blood cells are platelets and
    The evaluation method according to any one of claims 20 to 24, wherein the platelet is a human platelet.
  26. 請求項１から１５のいずれか一項に記載の血球減少症のモデル動物の製造方法により、血球減少症のモデル動物を製造する製造工程を含む、請求項２０から２５のいずれか一項に記載の評価方法。 The method according to any one of claims 20 to 25, which comprises a manufacturing process for producing a model animal for blood cell deficiency by the method for producing a model animal for blood cell deficiency according to any one of claims 1 to 15. Evaluation method.
  27. 被検血球について、血球の機能を評価する評価工程と、
    前記評価工程において、基準を満たす血球を機能性の血球として選抜する選抜工程とを含み、
    前記評価工程は、請求項２０から２６のいずれか一項に記載の血球機能の評価方法により実施される、血球の製造方法。     An evaluation process for evaluating the function of blood cells to be tested and
    The evaluation step includes a selection step of selecting blood cells satisfying the criteria as functional blood cells.
    The method for producing blood cells, wherein the evaluation step is carried out by the method for evaluating blood cell function according to any one of claims 20 to 26.
  28. 前記血球は、血小板である、請求項２７記載の製造方法。 The production method according to claim 27, wherein the blood cells are platelets.
  29. 被検物質を血球減少症のモデル動物に投与する工程と、
    前記モデル動物に血液中の血球数が増加する被検物質を、血球減少症の治療薬の候補物質として選択する選択工程とを含み、
    前記モデル動物は、請求項１６から１９のいずれか一項に記載の血球減少症のモデル動物である、血球減少症の治療薬候補物質のスクリーニング方法。     The process of administering the test substance to a model animal for cytopenia,
    The model animal includes a selection step of selecting a test substance that increases the number of blood cells in the blood as a candidate substance for a therapeutic agent for hypocytopenia.
    The method for screening a candidate substance for a therapeutic agent for cytopenia, wherein the model animal is the model animal for cytopenia according to any one of claims 16 to 19.
  30. 前記選択工程において、前記被検物質を投与していないコントロールのモデル動物と比較して、前記血球数が増加している被検物質を、前記治療用候補物質として選択する、請求項２９記載のスクリーニング方法。 29. The method of claim 29, wherein in the selection step, the test substance having an increased blood cell count is selected as the therapeutic candidate substance as compared with the control model animal to which the test substance is not administered. Screening method.
  31. 前記被検物質が、低分子化合物、ペプチド、タンパク質および核酸からなる群から選択された少なくとも１つである、請求項２９または３０記載のスクリーニング方法。 The screening method according to claim 29 or 30, wherein the test substance is at least one selected from the group consisting of low molecular weight compounds, peptides, proteins and nucleic acids.
  32. 前記血球は、血小板である、請求項２９から３１のいずれか一項に記載のスクリーニング方法。 The screening method according to any one of claims 29 to 31, wherein the blood cells are platelets.
  33. 請求項１から１５のいずれか一項に記載の血球減少症のモデル動物の製造方法により、血球減少症のモデル動物を製造する製造工程を含む、請求項２９から３２のいずれか一項に記載のスクリーニング方法。 The method according to any one of claims 29 to 32, which comprises a manufacturing process for producing a model animal for blood cell deficiency by the method for producing a model animal for blood cell deficiency according to any one of claims 1 to 15. Screening method.
  34. 被検物質から血球減少症の治療薬の候補物質を選抜する選抜工程を含み、
    前記選抜工程は、請求項２９から３３のいずれか一項に記載の血球減少症の治療薬候補物質のスクリーニング方法で実施される、血球減少症の治療薬の候補物質の製造方法。     Includes a selection process that selects candidate substances for the treatment of cytopenia from the test substances.
    The method for producing a candidate substance for a therapeutic drug for cytopenia, wherein the selection step is carried out by the screening method for a candidate substance for a therapeutic drug for cytopenia according to any one of claims 29 to 33.
PCT/JP2020/010961 2019-03-13 2020-03-12 Method for producing cytopenia model animal, cytopenia model animal, method for evaluating blood cell function, method for producing blood cells, method for screening cytopenia therapeutic drug material candidate, and method for producing cytopenia therapeutic drug material candidate WO2020184685A1 (en)

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