WO2019230756A1 - Method for screening substances that affect formation, structure, or function of human blood vessels, and method for producing human blood vessels - Google Patents

Method for screening substances that affect formation, structure, or function of human blood vessels, and method for producing human blood vessels Download PDF

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WO2019230756A1
WO2019230756A1 PCT/JP2019/021215 JP2019021215W WO2019230756A1 WO 2019230756 A1 WO2019230756 A1 WO 2019230756A1 JP 2019021215 W JP2019021215 W JP 2019021215W WO 2019230756 A1 WO2019230756 A1 WO 2019230756A1
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human
tissue
blood vessels
human blood
human tissue
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PCT/JP2019/021215
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French (fr)
Japanese (ja)
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伸幸 ▲高▼倉
陽平 塚田
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国立大学法人大阪大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a method for screening a substance that affects the formation, structure or function of human blood vessels, and a method for producing human blood vessels.
  • Therapeutic agents for malignant tumors include drugs that inhibit DNA replication and synthesis, drugs that induce destruction of cell membranes and cytoskeletons, and anti-tumor agents that have a so-called cytotoxic action in order to suppress cell growth of tumor cells.
  • many molecular targeted drugs and the like based on the blockage of cell survival signals have been widely developed. While these drugs directly act on tumor cells, drugs that target cells present in the stroma of tumor tissue have also been developed.
  • a typical example is an angiogenesis inhibitor developed for the purpose of inhibiting the survival of a tumor by suppressing angiogenesis in the tumor.
  • Vascular Endothelial Growth Factor acts on vascular endothelial cells in tumors to induce angiogenesis such as proliferation of vascular endothelial cells, lumen formation, and matrix remodeling. Supplies oxygen and nutrients to the tumor and induces tumor growth.
  • VEGF is secreted not only by tumor cells but also by fibroblasts and immune cells that have entered the tumor, and acts on vascular endothelial cells in the tumor.
  • Drugs that suppress the function of VEGF and VEGF receptors have been developed and applied clinically by many pharmaceutical companies. Initially, such angiogenesis inhibitors were expected to have an anti-tumor effect even with angiogenesis inhibitors alone, by inhibiting angiogenesis by inhibiting VEGF signals.
  • angiogenesis inhibitors have no effect on destroying the blood vessels in the tumor and causing the tumor to regress, and inhibit excessive angiogenesis-promoting factor (in this case, VEGF) It was thought that by suppressing the progression process and diverting to the maturation process of the blood vessels, the blood vessels in the tumor were normalized, leading to a vascular state with improved drug delivery. Due to such reverse translation from clinical medicine to basic medicine, the concept of normalization of tumor blood vessels by angiogenesis inhibitors has recently been proven.
  • a xenograft model (patient-derived xenograft model; human PDX model) of human tumor tissue to an immunodeficient animal has been considered. That is, this is a model in which a human tumor tissue piece collected from a patient is transplanted into an immunodeficient mouse and the proliferation of human tumor cells is induced for each tumor tissue.
  • this conventional model even when oxygen and nutrients are transported to the human tumor tissue by the bloodstream of the mouse, the human tumor tissue does not become necrotic and does not become settled. No human blood vessels remain, and human cancer is supplied with nutrients by mouse blood vessels.
  • an angiogenesis inhibitor affects angiogenesis during the process of blood vessel growth.
  • an angiogenic agent is administered to PDX model mice without knowing whether human blood vessels are formed or remain in the tumor, and how the blood vessels are affected after multiple days. was compared with the angiogenesis inhibitor non-administered group to compare the degree of remaining blood vessels.
  • Short-Term Human Prostate Primary Xenografts An in Vivo Model of Human Prostate Cancer Vasculature and Angiogenesis., Danny R. Gray et al., CANCER RESEARCH 64, 1712-1721, March 1, 2004 Differential transplantability of human endothelial cells in colorectal cancer and renal cell carcinoma primary xenografts., Laura Sanz et al., Laboratory Investigation (2009) 89, 91-97
  • a screening method for a substance that promotes or suppresses the formation of human blood vessels, or a substance that normalizes the structure or function of human blood vessels comprising the following steps (1) to (7): (1) a step of incising a part of the skin of a non-human immunodeficient animal to expose a subcutaneous tissue or a muscle layer; (2) placing a human tissue on the exposed subcutaneous tissue or muscle layer; (3) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air; (4) engrafting human tissue in a non-human immunodeficient animal; (5) A step of administering a test substance to a non-human immunodeficient animal engrafted with human tissue obtained by steps (1) to (4), (6) observing the shape or structure of a human blood vessel in the engrafted human tissue and / or evaluating the function of the human blood vessel, and (7) engrafting the human tissue not administered with the test substance.
  • a method for screening for a substance that promotes or suppresses the formation of human blood vessels, or a substance that normalizes the structure or function of human blood vessels comprising the following steps (I) to (VI): (I) a step of incising a part of the skin of a non-human immunodeficient animal to expose the subcutaneous tissue or muscle layer, (II) placing a mixture of human tissue and a test substance on the exposed subcutaneous tissue or muscle layer, (III) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air; (IV) engrafting human tissue in a non-human immunodeficient animal; (V) observing the shape or structure of a human blood vessel in the engrafted human tissue and / or evaluating the function of the human blood vessel,
  • the human tissue is a human tissue to which an agent or cell that promotes angiogenesis is added.
  • the step (3) includes sealing human tissue placed using a dorsal skin fold chamber.
  • a method for producing a human blood vessel using a non-human immunodeficient animal comprising the following steps (A) to (E): (A) incising a part of the skin of a non-human immunodeficient animal to expose the subcutaneous tissue or muscle layer, (B) placing human tissue on exposed subcutaneous tissue or muscle layer; (C) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air; (D) engrafting human tissue in a non-human immunodeficient animal to proliferate human blood vessels, and (E) collecting engrafted human tissue.
  • the human tissue is a human tissue to which an agent or cell that promotes angiogenesis is added.
  • step (C) includes sealing human tissue placed using a dorsal skin fold chamber.
  • step (C) includes sealing human tissue placed using a dorsal skin fold chamber.
  • substances that affect the formation, structure or function of human blood vessels can be obtained.
  • the substance obtained by the screening method of the present invention is useful as a therapeutic agent for ischemic disease, a therapeutic agent for a disease that develops or worsens due to angiogenesis, or a therapeutic agent for a disease that involves abnormal or structural abnormalities of blood vessels.
  • the human blood vessel production method of the present invention is an epoch-making method that can proliferate and produce human blood vessels using non-human immunodeficient animals.
  • the manufactured human blood vessel is useful as a blood vessel for transplantation into a patient having an ischemic disease or the like.
  • a broken line indicates a cluster of mesenchymal stem cells transplanted simultaneously. It is a figure which shows the result of having observed the human CD31 positive human blood vessel in the tissue transplantation site
  • the present invention provides a method of screening for a substance that promotes or suppresses the formation of human blood vessels, or a substance that normalizes the structure or function of human blood vessels.
  • the screening method of the present invention may be any method including the following steps (1) to (7).
  • the screening method of the present invention may be a method including the following steps (I) to (VI).
  • (I) a step of incising a part of the skin of a non-human immunodeficient animal to expose the subcutaneous tissue or muscle layer,
  • (II) placing a mixture of human tissue and a test substance on the exposed subcutaneous tissue or muscle layer,
  • (III) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air;
  • IV engrafting human tissue in a non-human immunodeficient animal;
  • (VI) engrafting the human tissue not in contact with the test substance
  • a part of the skin of the non-human immunodeficient animal is incised to expose the subcutaneous tissue or muscle layer.
  • the non-human immunodeficient animal any animal that can engraft transplanted human tissue may be used, and a mammal having a reduced immune function may be used.
  • the mammal is not particularly limited, and may be a mouse, rat, monkey, pig, sheep, cow, dog, cat, or rabbit.
  • the mammal whose immune function is reduced may be a mammal administered with an immunosuppressant, or may be a mammal whose immune function is reduced due to a gene mutation.
  • immunodeficient mammal having a gene mutation examples include Nude mouse, Scid mouse, NOD / Scid mouse, NOG mouse, NSG mouse, NOJ mouse, Rag1 / Rag2 KO mouse, Nude rat, and Scid rat.
  • non-human immunodeficient animals may be depilated. Hair removal may be performed the day before the day of transplanting human tissue into a non-human immunodeficient animal. Hair removal may be performed in a range including the skin portion to be incised. Hair removal may be performed under anesthesia. Although the hair removal method is not particularly limited, the hair removal using a hair clipper and the hair removal using a hair removal agent may be combined.
  • ⁇ A part of the skin of an anesthetized non-human immunodeficient animal is incised to expose the subcutaneous tissue or muscle layer.
  • the subcutaneous tissue is in the lower layers of the epidermis and dermis, and the muscle layer is in the lower layers of the subcutaneous tissue.
  • the epidermis and dermis may be excised to expose the subcutaneous tissue
  • the epidermis, dermis and subcutaneous tissue may be excised to expose the muscle layer. Only the subcutaneous tissue may be exposed in the area where a part of the skin is incised, or only the muscle layer may be exposed in the area where the part of the skin is incised.
  • the muscle layer may be exposed with the tissue remaining.
  • human tissue is placed on the exposed subcutaneous tissue or muscle layer.
  • the human tissue may be a tissue collected from a human. Examples include adipose tissue, muscle tissue, gastrointestinal tissue, nerve tissue, skin tissue, placenta tissue, and the like.
  • the human tissue may be a tissue at a disease site such as a malignant tumor tissue, a benign tumor tissue, an inflammatory disease tissue, or an ischemic disease tissue.
  • the human tissue may be a human tissue with a history of being transplanted into a non-human immunodeficient animal or a human tissue without such a history.
  • a fresh tissue collected from a human or a tissue obtained by thawing a tissue that has been cryopreserved immediately after collection can be used.
  • Transplant fresh tissue into non-human immunodeficient animals within 12 hours, within 10 hours, within 8 hours, and within 6 hours after collection, assuming that they are immersed in an appropriate storage solution and transported under appropriate conditions To do.
  • Cryopreserved tissues are transplanted into non-human immunodeficient animals within 6 hours, 4 hours, and 3 hours after thawing.
  • an appropriate tissue cryopreservation solution such as CELLBANKER (trade name, Nippon Zenyaku Kogyo Co., Ltd.) as soon as possible after collection.
  • Tissue soaked in a suitable tissue cryopreservation solution may be slowly frozen overnight at -80 ° C. and then stored in a liquid nitrogen gas phase.
  • the minced human tissue may be placed on the subcutaneous tissue or muscle layer of the cut skin of the host animal.
  • the size of the minced human tissue is not particularly limited, but may be about 4 to 5 mm square, about 3 to 4 mm square, about 2 to 3 mm square, or about 1 to 2 mm. It may be about a corner.
  • the minced human tissue may be placed on the subcutaneous tissue or muscle layer of the incised skin while suspended in an appropriate physiological buffer.
  • the physiological buffer include cell culture media such as DMEM and RPMI1640, physiological saline, phosphate buffer, phosphate buffered saline (PBS), and the like. These physiological buffers may not be added with phenol red.
  • Drugs or cells that promote angiogenesis may be added to human tissue transplanted into a minced human tissue suspension.
  • the agent that promotes angiogenesis is not particularly limited, and may be a low molecular compound, a medium molecular compound, or a high molecular compound.
  • the drug that promotes angiogenesis may be a natural product or a synthetic product.
  • growth factors such as PlGF (placental growth factor), Angiopoietin-1, PDGF (platelet-derived growth factor), EGF (epidermal growth factor), HGF (hepatocyte growth factor), basic fibroblast growth factor (bFGF), VEGF; And lipids such as lysophosphatidic acid and sphingosine-1-phosphate; bioactive peptides such as apelin; nucleic acids such as aptamer and microRNA; and the like.
  • Examples of cells that promote angiogenesis include human mesenchymal stem cells, vascular endothelial stem cells, vascular endothelial progenitor cells, pericytes, and macrophages. One type or two or more types of drugs or cells may be added.
  • Drugs and cells may be added in combination.
  • the addition amount of the agent or cell that promotes angiogenesis is not particularly limited, and an addition amount by which a desired human blood vessel is formed may be added to the transplanted human tissue.
  • the addition amount may be determined by conducting a preliminary study.
  • the human tissue may be transplanted after infecting a virus expressing a fluorescent protein.
  • the fluorescent protein is not particularly limited, and can be appropriately selected from known fluorescent proteins.
  • viruses lentiviruses and retroviruses that are viruses that stably express a transgene can be preferably used.
  • a virus that expresses a fluorescent protein can be prepared using a known genetic recombination technique, and can infect human tissue before transplantation by a known method.
  • the human tissue is placed in such an amount that the entire subcutaneous tissue or muscle layer of the cut skin of the host animal is covered.
  • the amount of human tissue is increased or decreased depending on the exposed subcutaneous tissue or muscle layer area.
  • the present inventors suspended 50 mg of minced human tissue in 50 ⁇ L of physiological buffer and placed it on the muscle layer. . Therefore, human tissue may be increased or decreased according to the exposed subcutaneous tissue or muscle layer area based on this amount.
  • step (II) the mixture of human tissue and test substance is placed on the exposed subcutaneous tissue or muscle layer of the host animal.
  • the test substance is not particularly limited, and the test substance exemplified in the description of the subsequent step (5) can be preferably used.
  • the method for mixing the human tissue and the test substance is not particularly limited.
  • the test substance may be applied to human tissue, the human tissue may be immersed in the test substance solution, the test substance may be added to a suspension of minced human tissue, and the host animal is exposed.
  • the test substance may be added to human tissue placed on the subcutaneous tissue or muscle layer.
  • the addition amount of the test substance is not particularly limited, and can be appropriately set by conducting a preliminary test.
  • the placed human tissue is sealed so that the placed human tissue does not come into contact with air.
  • the method for sealing the placed human tissue include a method in which a slide glass, a synthetic resin thin plate, a synthetic resin film, or the like is brought into close contact with the surface of the placed human tissue, and its periphery is fixed to the skin of the host animal. It is done.
  • a dorsal skinfold chamber hereinafter referred to as “DSC” may be used.
  • DSC a DSC having a size suitable for the host animal may be selected from commercially available DSCs. Use DSC that has been cleaned, disinfected, and sterilized according to standard methods.
  • the exposed subcutaneous tissue or muscle layer may be the subcutaneous tissue or muscle layer of the skin on the near side, or may be the subcutaneous tissue or muscle layer of the skin on the back side (side to which the back frame is attached).
  • the epidermis and dermis of the near skin or the epidermis, dermis and subcutaneous tissue are excised.
  • the muscle layer of the back skin the epidermis, dermis, subcutaneous tissue and muscle layer of the near skin are excised, and the connective tissue is further excised.
  • the connective tissue and the muscle layer of the back side skin are further excised (step (1)).
  • the minced human tissue prepared at the time of use is placed on the exposed subcutaneous tissue or muscle layer (steps (2) and (II)).
  • the amount of human tissue to be placed is an appropriate amount that covers the entire exposed subcutaneous tissue or muscle layer as described above and that does not generate a gap when the DSC front frame is attached.
  • a front frame with a DSC cover glass is attached and fixed to the back frame (steps (3) and (III)). At this time, be careful not to let air enter the chamber.
  • steps (4) and (IV) human tissues are engrafted in non-human immunodeficient animals.
  • the human tissue transplanted non-human immunodeficient animal prepared in steps (1) to (3) or steps (I) to (III) is bred and transplanted.
  • the human blood vessels in the engrafted human tissue are allowed to survive for a sufficient period of time until they become connected to the host animal's blood vessels.
  • Breeding conditions are not particularly limited, and breeding may be performed under the same conditions as those suitable for breeding non-human immunodeficient animals in which no human tissue has been transplanted.
  • a shield may be attached around the DSCs to prevent accidents such as breakage of the DSC cover glass.
  • the number of animals bred in the same cage may be reduced in order to prevent accidents such as breakage of the attached DSC cover glass.
  • the time for confirming the engraftment of human tissue is not particularly limited, but may be performed on or after the 10th day after transplantation of human tissue.
  • the fluorescence signal of the fluorescent protein expressed by the virus infected with the human tissue is detected by observing the inside of the DSC of the animal transplanted with the human tissue infected with the virus expressing the fluorescent protein with a fluorescent microscope. This can be done.
  • the observation of blood vessels of human tissue engrafted in the host can be performed at the same time as confirmation of engraftment of human tissue.
  • fluorescently labeled antibodies that bind to molecules expressed by vascular endothelial cells can be used.
  • antibodies for imaging human blood vessels include anti-human CD31 antibody, anti-human CD34 antibody, anti-human VE-cadherin antibody, anti-human VCAM-1 antibody, and anti-human vWF antibody. Can be used. The same applies to antibodies for imaging blood vessels of host animals.
  • anti-mouse CD31 antibody When the host animal is a mouse, anti-mouse CD31 antibody, anti-mouse CD34 antibody, anti-mouse VE-cadherin antibody, anti-mouse VCAM-1 antibody, anti-mouse vWF antibody Etc. can be used.
  • a fluorescent molecule emitting a fluorescent color distinguishable from the fluorescent color emitted from the fluorescent molecule labeled on the human blood vessel imaging antibody is used.
  • the optimal dose is set by preliminary examination according to the antibody to be used, and is administered intravenously to the host animal. In the case of mice, 5 to 20 ⁇ g is usually administered. The inside of the DSC is observed with a fluorescence microscope 12-24 hours after administration of the imaging antibody. If human blood vessel antibodies can be administered intravenously to a host animal and the human blood vessels can be observed, it can be confirmed that the blood vessels of the host animal are connected to the human blood vessels, and at the same time, human tissue is produced in the host animal. I can reasonably infer that I am wearing it.
  • test substance is administered to the non-human immunodeficient animal engrafted with human tissue obtained in steps (1) to (4).
  • test substances include nucleic acids, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cells, cell extracts, cell culture supernatants, plant extracts, mammalian tissue extracts, plasma, etc. It can be preferably used. However, it is not limited to these.
  • the test substance may be a novel substance or a known substance. These test substances may form a salt.
  • a salt of the test substance a salt with a physiologically acceptable acid or base may be used.
  • the timing of administration of the test substance may be after confirming the engraftment of human tissue in step (4), and may be 14 to 21 days after human tissue transplantation.
  • the dose, administration route, and number of administrations of the test substance are preferably set according to the test substance.
  • the solvent used for the preparation of the test substance is administered by the same administration route and the same administration frequency.
  • the administration route is not particularly limited, but systemic administration such as oral administration, intravenous administration and intraperitoneal administration is preferable.
  • step (6) or (V) the shape or structure of the human blood vessel in the engrafted human tissue is observed and / or the function of the human blood vessel is evaluated. Observation and functional evaluation of human blood vessels may be performed at any time after administration of the test substance, and may be performed 14 to 21 days after human tissue transplantation.
  • Observation of the shape or structure of a human blood vessel can be performed by intravenously administering an antibody for imaging a human blood vessel to a host animal as described above.
  • Examples of the shape or structure of the human blood vessel include the area of the human blood vessel (the area occupied by the human blood vessel), the length of the human blood vessel, the number of branches of the human blood vessel, the tortuous nature of the human blood vessel, the expandability of the human blood vessel, and the human blood vessel.
  • the image in the observation field may be analyzed using image analysis software (for example, AngioTool).
  • the above observation items may be quantified by analysis.
  • the functions of human blood vessels include permeability, blood flow, and oxygen delivery. Permeability can be evaluated by, for example, administering a fluorescently labeled dextran or an autofluorescent agent (such as doxorubicin) into the host animal's vein and measuring the area of the fluorescent region leaking around the human blood vessel. Good.
  • a fluorescently labeled dextran or an autofluorescent agent such as doxorubicin
  • the blood flow is collected from the host animal after the fluorescently labeled lectin is administered intravenously to the host animal, and the tissue specimen stained with the anti-human CD31 antibody is observed to detect lectin positive in human CD31 positive cells.
  • the proportion of blood vessels with blood flow may be evaluated by calculating the proportion of cells.
  • the oxygen delivery degree may be evaluated using, for example, a known hypoxic probe (eg, pimonidazole).
  • the test substance can be selected as a test substance that promotes the formation of human blood vessels when the area of human blood vessels, the length of human blood vessels, or the number of branches of human blood vessels is increased.
  • the area of the human blood vessel, the length of the human blood vessel, and the number of branches of the human blood vessel can be quantified using image analysis software (for example, AngioTool) (see Example 4 and FIG. 5).
  • the degree to which the test substance increases the area, length, or number of branches is not particularly limited, for example, 1.2 times or more, 1.4 times or more, 1.6 times or more, 1.8 times or more, 2 times or more, 2.2 times or more, 2.4 times or more, Test substances that increase 2.6 times or more, 2.8 times or more, or 3 times or more can be selected.
  • the comparison with the animal not administered with the test substance may be performed based on accumulated data (background data) of the test substance non-administered animal accumulated in the same experiment in the past.
  • a substance selected as a test substance that promotes the formation of human blood vessels is useful as an active ingredient of a therapeutic agent for ischemic diseases.
  • ischemic diseases include ischemic heart diseases (myocardial infarction, coronary atherosclerosis, etc.), ischemic brain diseases (cerebral infarction, cerebral arteriosclerosis, dementia, etc.), osteoporosis, and aging organ hypofunction , Buerger's disease, chronic obstructive arteriosclerosis, pressure ulcer, various organ transplants and the like.
  • the test substance can be selected as a test substance that suppresses the formation of human blood vessels when the area, the length of human blood vessels, or the number of branches of human blood vessels is reduced.
  • the area of the human blood vessel, the length of the human blood vessel, and the number of branches of the human blood vessel can be quantified using image analysis software (for example, AngioTool) (see Example 4 and FIG. 5).
  • the degree to which the test substance reduces the area, length or number of branches is not particularly limited, for example, 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, A test substance that can be reduced to 0.2 times or less or 0.1 times or less can be selected.
  • the comparison with the animal not administered with the test substance may be performed based on accumulated data (background data) of the test substance non-administered animal accumulated in the same experiment in the past.
  • a substance selected as a test substance that suppresses the formation of human blood vessels is useful as an active ingredient of a therapeutic agent for a disease that develops or worsens due to angiogenesis.
  • diseases include malignant tumors, infections, arteriosclerosis, autoimmune diseases (eg, rheumatoid arthritis, scleroderma, etc.), diabetic retinopathy, age-related macular degeneration, retina of prematurity Symptom, glaucoma, vascular malformation (eg capillary malformation, arteriovenous malformation, etc.), hemangioma, osteoarthritis, keloid, psoriasis, allergic dermatitis, obesity, pulmonary hypertension, asthma, emphysema, chronic bronchitis, Examples include cirrhosis and ascites.
  • test substance when the test substance is administered and normalized as compared to the structure or function of human blood vessels in an animal not administered with the test substance, Can be selected as a test substance that normalizes the structure or function.
  • normalization is determined according to the evaluation item. Normalizing the structure or function of a human blood vessel means, for example, that the tortuousness of a blood vessel increased in the tissue at the disease site returns to the level of the normal tissue, or the blood vessel expandability in the tissue at the disease site is the level of the normal tissue , Increased blood vessel occlusion in diseased tissue returns to normal tissue level, increased blood vessel permeability in diseased tissue returns to normal tissue level, decreased in diseased tissue For example, returning the blood flow to the normal tissue level.
  • the normal level of human blood vessel structure or function may refer to previously accumulated data (background data).
  • Substances selected as test substances that normalize the structure or function of human blood vessels include, for example, malignant tumors, infections, arteriosclerosis, autoimmune diseases (eg, rheumatoid arthritis, scleroderma, etc.), diabetic retina Disease, age-related macular degeneration, retinopathy of prematurity, glaucoma, vascular malformations (eg capillary malformations, arteriovenous malformations, etc.), hemangiomas, osteoarthritis, keloid, psoriasis, allergic dermatitis, obesity, It is useful as an active ingredient of therapeutic agents for pulmonary hypertension, asthma, emphysema, chronic bronchitis, cirrhosis, ascites, renal diseases (eg, glomerulonephropathy, diabetic nephropathy, etc.).
  • autoimmune diseases eg, rheumatoid arthritis, scleroderma, etc.
  • diabetic retina Disease age-related ma
  • the present invention provides a method for producing a human blood vessel using a non-human immunodeficient animal.
  • the production method of the present invention includes the following steps (A) to (E).
  • D engrafting human tissue in a non-human immunodeficient animal to proliferate human blood vessels, and
  • E collecting engrafted human tissue.
  • Steps (A) to (C) are the same as steps (1) to (3) of the screening method of the present invention already described, and are the same as steps (1) to (3) of the screening method of the present invention. Can be implemented. However, since the production method of the present invention can be used for transplantation to humans, it is preferable to use human tissue that is not infected with a virus expressing a fluorescent protein. For the same reason, it is preferable not to use a human tissue at a disease site (for example, a human tissue containing a malignant tumor).
  • step (D) human tissues are engrafted in non-human immunodeficient animals and human blood vessels are increased.
  • the human tissue transplanted non-human immunodeficient animal produced in the steps (A) to (C) is bred, and the transplanted human tissue is engrafted in the host animal and engrafted.
  • Breeding conditions are not particularly limited, and breeding may be performed under the same conditions as those suitable for breeding non-human immunodeficient animals in which no human tissue has been transplanted.
  • a shield may be attached around the DSCs to prevent accidents such as breakage of the DSC cover glass.
  • animals equipped with DSC are bred, the number of animals bred in the same cage may be reduced in order to prevent accidents such as breakage of the attached DSC cover glass.
  • the breeding period of the step (D) is not particularly limited, and may be 8 days or more after human tissue transplantation, 10 days or more, 12 days or more, 14 days or more, 28 days or less after human tissue transplantation, 25 days or less, It may be 21 days or less, 18 days or less, or 14 days or less.
  • Human tissue engraftment is confirmed by transplanting human tissue infected with a virus expressing a fluorescent protein into a small number of host animals, observing the human tissue of the transplanted host animal with a fluorescence microscope, You may carry out by detecting the fluorescence signal of the fluorescent protein to express. If the engraftment of human tissue can be confirmed in a small number of host animals transplanted with human tissues infected with a virus expressing a fluorescent protein, in a host animal transplanted with human tissues not infected with a virus expressing a fluorescent protein on the same day. It can be reasonably inferred that human tissue is engrafted.
  • human tissue engraftment and human blood vessel growth may be simultaneously confirmed using an antibody for imaging human blood vessels without using human tissue infected with a virus expressing a fluorescent protein.
  • Imaging of human blood vessels can be performed by the procedure described in step (4) of the screening method of the present invention. If human blood vessel imaging antibody can be intravenously administered to a host animal to confirm the growth of human blood vessels, it can be reasonably assumed that human tissue has been engrafted in the host animal.
  • step (E) engrafted human tissue is collected.
  • the method of collecting human normal tissue transplanted from a non-human immunodeficient animal is not particularly limited, and human tissue engrafted in the host animal may be collected using a surgical instrument such as a scalpel. Since the collected human tissue contains increased human blood vessels, the human tissue can be immediately immersed in an appropriate preservation solution and transported to a transplantation site to be used for blood vessel transplantation. When transplantation is not performed immediately, the tissue can be cryopreserved by dipping in an appropriate tissue cryopreservation solution. In the case of cryopreservation, slow freezing may be performed overnight at -80 ° C. and then stored in a liquid nitrogen gas phase.
  • diseases that can be expected to be improved by transplanting human blood vessels produced by the production method of the present invention include ischemic heart diseases (myocardial infarction, coronary atherosclerosis, etc.), ischemic brain diseases (cerebral infarction, Cerebrospinal vein sclerosis, dementia, etc.), osteoporosis, aging organ hypofunction, Buerger's disease, chronic obstructive arteriosclerosis, pressure ulcer, various organ transplants, hemophilia, von Willebrand disease, etc. .
  • ischemic heart diseases myocardial infarction, coronary atherosclerosis, etc.
  • ischemic brain diseases cerebral infarction, Cerebrospinal vein sclerosis, dementia, etc.
  • osteoporosis aging organ hypofunction
  • Buerger's disease chronic obstructive arteriosclerosis
  • pressure ulcer various organ transplants
  • hemophilia von Willebrand disease, etc.
  • Example 1 Preparation and observation of mouse transplanted with human tumor tissue ⁇ Experimental method> (1) Mice treatment the day before transplantation 8-week-old female NOD / ShiJic-scid mice (CLEA Japan) were mixed with three types of mixed anesthesia (6 ⁇ g medetomidine hydrochloride (trade name: Domitor, Nippon Zenyaku Kogyo), 80 ⁇ g midazolam (Sand) 200 ⁇ L of butorphanol tartrate (trade name: Betorfal, Meiji Seika Pharma) diluted 200 ⁇ L of Otsuka raw food (Otsuka Pharmaceutical Factory) was intraperitoneally administered.
  • medetomidine hydrochloride trade name: Domitor, Nippon Zenyaku Kogyo
  • Sand 80 ⁇ g midazolam
  • SB butorphanol tartrate
  • Betorfal Betorfal, Meiji Seika Pharma
  • the back was shaved with a clipper and further depilated using a depilatory epirat (Kracie).
  • Karlinsky remove the hair remover with lukewarm water and administer 200 ⁇ L of medetomidine antagonist (diluted with 6 ⁇ g atipamezole hydrochloride (trade name: Antisedan, Nippon Zenyaku Kogyo Co., Ltd.) in 200 ⁇ L Otsuka raw food injection (Otsuka Pharmaceutical Factory). Then, the mouse was awakened on a 37 ° C. hot plate.
  • Lentiviral transfection solution 14 ⁇ L Lentiviral High Titer Packaging Mix (Takara Bio), 2.75 ⁇ g tdTomato gene inserted pLVSIN-EF1 ⁇ Neo Vector (Takara Bio), 27.5 ⁇ L P3000 Reagent (Thermo Fisher Scientific), 25 ⁇ L Lipofectamine 3000 ( Thermo Fisher Scientific) and 1250 ⁇ L of Opti-MEM (Thermo Fisher Scientific) were prepared and allowed to stand at room temperature for 15 minutes. The culture solution of Lenti-X293T cells grown to 80-90% confluent was replaced with a new culture solution (5 ml), and the above lentiviral transfection solution was added. Transfection was performed at 37 ° C.
  • the culture supernatant was centrifuged at 3000 rpm, 4 ° C. for 5 minutes to precipitate cell debris.
  • the supernatant was filtered through Millex-HP 0.45 ⁇ m (polyethersulfone, 33 mm, radiation sterilized filter, MERCK) to remove cell debris, and a filtrate was obtained.
  • the filtrate was ultracentrifuged for 2 hours at 19400 rpm, 4 ° C using an Optima L-100XP ultracentrifuge (BECKMAN COULTER and SW28 swing rotor (BECKMAN COULTER). The supernatant was removed and a precipitate containing virus particles was obtained.
  • DMEM high glucose, no glutamine, no phenol red, Thermo Fisher Scientific
  • the human tumor tissue was washed 4 times with Otsuka raw food injection (Otsuka Pharmaceutical Factory) (50 mL per time). About 1 cm of human tumor tissue in a 10 cm dish containing DMEM (high glucose, no glutamine, no phenol red) (Thermo Fisher Scientific) containing 10% FBS (Sigma), 100 units / mL penicillin / 100 ⁇ g streptomycin (Sigma) Cut into corner sizes. Transfer human tumor tissue to a 1.5 mL tube, add 150 units of HBSS (calcium / magnesium / no phenol red) with 100 units / mL penicillin / 100 ⁇ g / mL streptomycin (Sigma) added, and mince the human tumor tissue with scissors.
  • Otsuka Pharmaceutical Factory Otsuka Pharmaceutical Factory
  • HBSS-VF HBSS containing 100ng / mL VEGF, 100ng / mL bFGF
  • 50 ⁇ L HBSS-VF 50mg human tumor tissue to prepare a human tumor tissue suspension did.
  • the results are shown in FIG.
  • the scale bar indicates 200 ⁇ m. Since tdTomato positive cells were observed in the DSC (upper right), engraftment of transplanted human tumor tissue-derived cells was shown. Further, since human CD31-positive human blood vessels could be observed with the antibody administered intravenously to the host mouse (upper left), it was confirmed that the mouse blood vessels and human blood vessels were connected. Furthermore, in the composite image (lower right) of the mouse CD31 positive mouse blood vessel image (lower left), human blood vessel image (upper left) and tdTomato positive cell image (upper right), the human blood vessels and mouse blood vessels are fluorescent. It was possible to distinguish by the difference, indicating that human blood vessels and mouse blood vessels existed in close proximity.
  • Example 2 Confirmation that tdTomato positive cells and human CD31 positivity are derived from transplanted human tumor tissue ⁇ Experimental method> (1) Preparation of human tumor tissue-grafted mouse A human colon tumor tissue was transplanted into an 8-week-old female NOD / ShiJic-scid mouse by the same procedure as in Examples 1 (1) to (4). On the 17th day after transplantation, Alexa Fluor 488 anti-human CD31 antibody was intravenously administered to mice in the same procedure as in Example 1 (5).
  • mice were perfused with 10 mL of Phosphate buffered saline (PBS) from the left ventricle under anesthesia with somnopentyl (Kyoritsu Pharmaceutical), and then 4% paraformaldehyde-PBS (pH 7) .4) was perfused and fixed with 10 mL.
  • PBS Phosphate buffered saline
  • Tissue was replaced with sucrose in the order of 15% sucrose (Wako Pure Chemical Industries) / PBS, 30% sucrose / PBS, then embedded in Surgipath FSC22 embedded compound blue (Leica), and frozen in a -80 ° C deep freezer .
  • the reaction was allowed to proceed overnight at 0 ° C. Washing with PBS-T for 10 minutes was performed 3 times.
  • Secondary antibodies are Streptavidin Alexa Fluor 647 Conjugate (Thermo Fisher Scientific) and Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 546 (Thermo Fisher Scientific), diluted 200-fold with blocking solution Then, the solution was dropped on the section and reacted in a wet box for 1.5 hours under light shielding conditions at room temperature. The plate was washed 5 times with PBS-T for 10 minutes. A few drops of ProLong Diamond Antifade Mountant (Thermo Fisher Scientific) were dropped and sealed with a cover glass. The prepared tissue specimen was observed and photographed using a confocal laser microscope Leica TCS SP5 (Leica).
  • the results are shown in FIG.
  • the scale bar indicates 50 ⁇ m.
  • the top arrow indicates the position where the tdTomato positive cells and cells stained with human nucleus-specific antibodies overlap in the Merge image (upper right), confirming that the Tomato positive cells indicated by the arrow are human cells It was done.
  • the arrow at the bottom indicates the position where human CD31 positive cells and cells stained with human nucleus-specific antibodies overlap in the Merge image (lower right), confirming that human CD31 positive cells are human cells It was done. From these results, it was found that tdTomato positive cells and human CD31 positive cells were derived from transplanted human tumor tissue. Furthermore, it was confirmed that cells that are stained with a human nucleus-specific antibody but are negative for human CD31 and negative for tdTomato are present in human tumor tissue.
  • Example 3 Confirmation that human blood vessel in transplanted human tumor tissue is connected to mouse blood vessel ⁇ Experimental method> (1) Preparation of human tumor tissue-grafted mouse A human colon tumor tissue was transplanted into an 8-week-old female NOD / ShiJic-scid mouse by the same procedure as in Examples 1 (1) to (4). On the 20th day after transplantation, Alexa Fluor 488 anti-human CD31 antibody and Alexa Fluor 647 anti-mouse CD31 antibody were intravenously administered to mice in the same procedure as in Example 1 (5).
  • mice were perfusion-fixed by the same procedure as in Example 2 (2).
  • the skin from which DSC was cut from the mouse and peripheral tissues such as fat were removed was fixed in 4% paraformaldehyde-PBS at 4 ° C. for 1 hour with shaking.
  • the plate was washed twice with PBS for 10 minutes. While shaking the tissue, in this order to 25% methanol diluted in PBS (x2), 50% methanol (x2), 75% methanol (x2), 100% methanol (x2),
  • the tissue was dehydrated by immersing once for 5 minutes.
  • the tissue was dipped for 5 minutes in the order of 50% benzylbenzoate / benzylalchol (BABB) ( ⁇ 2 times) and 100% BABB ( ⁇ 2 times) diluted with methanol, and a transparent treatment was performed.
  • BABB 50% benzylbenzoate / benzylalchol
  • the tissue was sealed with a cover glass on a slide glass using BABB, and the prepared tissue specimen was observed and photographed using a multiphoton microscope Leica TCS SP8 (Leica) and a 25 ⁇ water immersion lens (Leica). .
  • FIG. 4 The results are shown in FIG. The scale bar indicates 100 ⁇ m. Three different fields of view of the same specimen were photographed as position 1, position 2, and position 3, respectively. As is apparent from FIG. 4, it was found that the blood vessel lumen formed by human CD31-positive human vascular endothelial cells and the blood vessel lumen formed by mouse CD31-positive mouse vascular endothelial cells were connected.
  • Example 4 Human vascular growth in transplanted human tumor tissue ⁇ Experimental method> Human colon tumor tissue was transplanted into 8-week-old female NOD / ShiJic-scid mice in the same procedure as in Examples 1 (1) to (4). On days 13 and 20 after transplantation, Alexa Fluor 488 anti-human CD31 antibody was intravenously administered to mice in the same procedure as in Example 1 (5). The next day (14th day and 21st day after transplantation), using an all-in-one small animal anesthesia machine (Muromachi Kikai), mice were anesthetized with isoflurane (flow rate 1 L / min, concentration 1%) and a multiphoton microscope Leica TCS SP8 MP ( Leica) observed the DSC and took a photo. The area of the human blood vessel, the number of branches of the human blood vessel, and the length of the human blood vessel were measured from the photographed photos using AngioTool blood vessel structure analysis software.
  • FIG. (A) is a microscopic image of human blood vessels (human CD31 positive) on day 14 (d14) and day 21 (d21) after transplantation.
  • the scale bar indicates 200 ⁇ m.
  • (B) is a time-dependent change of the vascular area
  • (C) is a time-dependent change of the number of branches of the blood vessel
  • (D) is a view showing a time-dependent change of the blood vessel length. It was found that the vessel area, the number of branches, and the vessel length all increased from the 14th day to the 21st day.
  • Example 5 Human vascular augmentation in transplanted human pancreatic cancer tissue
  • Human colon cancer tissue is originally known as a cancer rich in blood vessels, but it is a pancreatic cancer known as oligovascular cancer. In some cases, it was considered more difficult to grow human blood vessels in a graft using the production method of the present invention than in the case of colon cancer tissue.
  • mesenchymal stem cell spheroids (cell mass) Mesenchymal stem cells (Human Mesenchymal Stem Cells, Lonza, hereinafter referred to as “huMSC”) are added to MSCGM SingleQuots additive factor set (Mesenchymal Stem Cell Growth Medium SingleQuots Supplements and Growth Factors, Lonza ) was added to MSCBM basic medium (Mesenchymal Stem Cell Growth Medium, Lonza, hereinafter referred to as “MSC medium”). The huMSC was washed with PBS, and the detached cells were seeded on a 6 cm cell culture dish using trypsin / EDTA (Lonza) and cultured until confluent.
  • MSCGM SingleQuots additive factor set Mesenchymal Stem Cell Growth Medium SingleQuots Supplements and Growth Factors, Lonza
  • MSCBM basic medium Mesenchymal Stem Cell Growth Medium, Lonza, hereinafter referred to as “
  • FAST-DiI medium (6 ⁇ L of 0 ⁇ g / mL FAST-DiI (invtorgen) diluted with 3 mL of MSC medium, the final concentration of DiI is 2 ⁇ M), and use a small shaker in a CO 2 incubator at 37 ° C. (Wakenbeetec) and shaken for 2 hours. Two hours later, after washing twice with PBS, the cells were detached from the culture dish using trypsin / EDTA (Lonza) and collected. A cell suspension of 2 ⁇ 10 4 cells / 30 ⁇ L MSC medium was prepared, and spheroids were prepared using the hanging drop method. Spheroids were collected after 3 days.
  • the human pancreatic cancer tissue was washed 4 times with Otsuka raw food injection (Otsuka Pharmaceutical Factory) (50 mL per time). Human pancreatic cancer tissue in a 10cm dish containing DMEM (high (glucose, no glutamine, no phenol red) (Thermo Fisher Scientific) containing 10% FBS (Sigma), 100units / mL penicillin / 100 ⁇ g streptomycin (Sigma) It was cut into a size of about 1 cm square.
  • HBSS-p / s calcium / magnesium / no phenol red
  • Example 6 Human vascular growth in transplanted human normal tissue ⁇ Experimental method> (1) Mouse treatment on the day before transplantation Mouse treatment on the day before transplantation was performed in the same procedure as in Example 1 (1).
  • the normal human large intestine tissue was washed 4 times with Otsuka raw food injection (Otsuka Pharmaceutical Factory) (50 mL per time). Approximately 10% FBS (Sigma), 100units / mL penicillin / 100 ⁇ g streptomycin (Sigma) in DMEM (high glucose, no glutamine, no phenol red) (Thermo Fisher Scientific) in a 10cm dish Cut into 1 cm square size.
  • the results are shown in FIG.
  • the scale bar indicates 500 ⁇ m.
  • Human CD31-positive human blood vessels could be observed with the antibody administered intravenously to the host mouse.
  • the blood vessels of the mouse were connected to the blood vessels of normal human tissue, and the human blood vessels were stained via the blood flow from the veins of the mouse. From this result, it was shown that the use of the production method of the present invention can produce not only cancer tissues but also human blood vessels of normal tissues.

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Abstract

A method for screening substances that promote or suppress the formation of human blood vessels, or substances that normalize the structure or function of human blood vessels, the method including: (1) a step for cutting part of the skin of a non-human immunodeficient animal and exposing subcutaneous tissue or a muscle layer; (2) a step for placing human tissue on the exposed subcutaneous tissue or muscle layer; (3) a step for sealing the placed human tissue so that the placed human tissue is not in contact with the air; (4) a step for grafting the human tissue to the non-human immunodeficient animal; (5) a step for administering a substance to be tested into the non-human immunodeficient animal to which the human tissue was grafted as obtained in steps (1) to (4); (6) a step for observing the formation or structure of human blood vessels in the human blood tissue, and/or evaluating the function of the human blood vessels; and (7) a step for comparing with human blood vessels in a non-human immunodeficient animal to which human tissue was grafted and in which no substance to be tested was administered, and selecting either a substance to be tested that promotes or suppresses the formation of human blood vessels, or a substance to be tested that normalizes the structure or function of the human blood vessels.

Description

ヒト血管の形成、構造または機能に影響を及ぼす物質のスクリーニング方法、およびヒト血管の製造方法Method for screening for substances affecting the formation, structure or function of human blood vessels, and method for producing human blood vessels
　本発明は、ヒト血管の形成、構造または機能に影響を及ぼす物質のスクリーニング方法、およびヒト血管の製造方法に関するものである。 The present invention relates to a method for screening a substance that affects the formation, structure or function of human blood vessels, and a method for producing human blood vessels.
　悪性腫瘍に対する治療薬としては、腫瘍細胞の細胞増殖を抑制するために、DNA複製および合成を阻害する薬剤や、細胞膜や細胞骨格の破壊を誘導する薬剤、いわゆるサイトトキシックな作用を有する抗腫瘍剤、あるいは細胞生存シグナルの遮断を機序とした多くの分子標的薬などが広く開発されてきた。これらの薬剤が腫瘍細胞に直接的に作用をもたらす一方で、腫瘍組織の間質に存在する細胞を標的とする薬剤も開発されている。その代表例が、腫瘍内の血管形成を抑制して、腫瘍の生存を阻害することを目的として開発された血管新生抑制剤である。 Therapeutic agents for malignant tumors include drugs that inhibit DNA replication and synthesis, drugs that induce destruction of cell membranes and cytoskeletons, and anti-tumor agents that have a so-called cytotoxic action in order to suppress cell growth of tumor cells. In addition, many molecular targeted drugs and the like based on the blockage of cell survival signals have been widely developed. While these drugs directly act on tumor cells, drugs that target cells present in the stroma of tumor tissue have also been developed. A typical example is an angiogenesis inhibitor developed for the purpose of inhibiting the survival of a tumor by suppressing angiogenesis in the tumor.
　血管内皮細胞成長因子（vascular endothelial growth factor: VEGF）は、腫瘍内の血管内皮細胞に作用して、血管内皮細胞の増殖、管腔形成、マトリックスの再構築など血管新生を誘導することで腫瘍内に酸素や養分を供給し、腫瘍の増大を誘導する。VEGFは腫瘍細胞のみならず、腫瘍内に侵入した線維芽細胞や免疫細胞からも分泌されて、腫瘍内の血管内皮細胞に作用する。このようなVEGFやVEGFの受容体の機能を抑制する薬剤が多数の製薬企業によって開発され、臨床的に応用されている。当初、このような血管新生抑制剤は、VEGF系シグナルの抑制により血管新生を抑制することで、血管新生抑制剤単独でも抗腫瘍効果が得られると期待されていた。 Vascular Endothelial Growth Factor (vascular VEGF) acts on vascular endothelial cells in tumors to induce angiogenesis such as proliferation of vascular endothelial cells, lumen formation, and matrix remodeling. Supplies oxygen and nutrients to the tumor and induces tumor growth. VEGF is secreted not only by tumor cells but also by fibroblasts and immune cells that have entered the tumor, and acts on vascular endothelial cells in the tumor. Drugs that suppress the function of VEGF and VEGF receptors have been developed and applied clinically by many pharmaceutical companies. Initially, such angiogenesis inhibitors were expected to have an anti-tumor effect even with angiogenesis inhibitors alone, by inhibiting angiogenesis by inhibiting VEGF signals.
　しかし、臨床応用したところ、血管新生抑制剤によるVEGF系シグナルの遮断単独では抗腫瘍効果が発揮できないことが明らかとなった。ただし、血管新生抑制剤と抗腫瘍剤との併用効果は、抗腫瘍剤単独による抗腫瘍効果に比べて優れており、特に無増悪生存期間の有意な延長を誘導できるとの臨床的な観察が報告されている。このことから、現行の血管新生抑制剤に関しては、腫瘍内の血管を破壊して腫瘍を退縮させるまでの効果はなく、過剰な血管新生促進因子（この場合VEGF）を阻害して、血管新生の進行過程を抑制して、血管の成熟化過程に方向転換させることで、腫瘍内の血管を正常化させ、薬剤の送達性が改善した血管の状態に誘導しているのでないかと考えられた。このような臨床医学から基礎医学へのリバーストランスレーションにより、最近では、血管新生抑制剤による腫瘍血管の正常化の概念も証明されつつある。 However, clinical application revealed that blocking the VEGF signal with an angiogenesis inhibitor alone cannot exert an antitumor effect. However, the combined effect of an angiogenesis inhibitor and an anti-tumor agent is superior to the anti-tumor effect of an anti-tumor agent alone, and there is clinical observation that a significant prolongation of progression-free survival can be induced. It has been reported. Therefore, the current angiogenesis inhibitors have no effect on destroying the blood vessels in the tumor and causing the tumor to regress, and inhibit excessive angiogenesis-promoting factor (in this case, VEGF) It was thought that by suppressing the progression process and diverting to the maturation process of the blood vessels, the blood vessels in the tumor were normalized, leading to a vascular state with improved drug delivery. Due to such reverse translation from clinical medicine to basic medicine, the concept of normalization of tumor blood vessels by angiogenesis inhibitors has recently been proven.
　ヒトの腫瘍細胞に対する薬剤については、試験管内でヒトの腫瘍細胞に対する影響を観察することや、ヒトの腫瘍細胞を免疫不全マウスへ移植したマウスモデルでの効果を観察することで、ある程度、臨床的に応用した際の効果判定が可能である。一方、腫瘍環境に対する薬剤、例えば腫瘍血管に対する薬剤の効果を判定する場合、単に試験管内でのヒト血管内皮細胞に対する影響を観察するだけでは不十分であり、ヒトの腫瘍内におけるヒト血管内皮細胞による血管新生がどう制御されるのかを解析することが、血管新生抑制剤の効果を判定する上で重要である。 For drugs against human tumor cells, clinical effects are observed to some extent by observing the effects on human tumor cells in vitro or by observing the effects in mouse models in which human tumor cells are transplanted into immunodeficient mice. It is possible to determine the effect when applied to. On the other hand, when determining the effects of drugs on the tumor environment, such as drugs on tumor blood vessels, it is not sufficient to simply observe the effects on human vascular endothelial cells in vitro, and on human vascular endothelial cells in human tumors. Analyzing how angiogenesis is controlled is important in determining the effects of angiogenesis inhibitors.
　このような解析が動物を用いて可能になる方法として、ヒト腫瘍組織の免疫不全動物への異種移植片モデル（patient derived xenograft model; PDXモデル）が考えられてきた。つまり、患者から採取されたヒト腫瘍組織片を、免疫不全マウスに移植し、ヒトの腫瘍細胞の増殖を腫瘍組織ごと誘導するモデルである。しかし、従来のこのモデルでは、マウスの血流によりヒト腫瘍組織に酸素や栄養が運搬されるまでの間に、ヒト腫瘍組織が壊死をおこして定着しないことや、定着したとしてもすでに、その際にはヒトの血管は残存せず、マウスの血管によりヒトの癌が養分の供給を受けている。 As a method that enables such analysis using animals, a xenograft model (patient-derived xenograft model; human PDX model) of human tumor tissue to an immunodeficient animal has been considered. That is, this is a model in which a human tumor tissue piece collected from a patient is transplanted into an immunodeficient mouse and the proliferation of human tumor cells is induced for each tumor tissue. However, in this conventional model, even when oxygen and nutrients are transported to the human tumor tissue by the bloodstream of the mouse, the human tumor tissue does not become necrotic and does not become settled. No human blood vessels remain, and human cancer is supplied with nutrients by mouse blood vessels.
　このPDXモデルでは、殆どの例で、初回の移植では抗腫瘍剤等の薬剤の効果判定には用いられておらず、１回目で定着したヒト腫瘍組織をさらに細かい腫瘍片として再移植し、複数回の移植後に治療薬の効果判定に用いられてきている。このような複数回の移植により、腫瘍塊の中にはすでにヒト由来の血管は存在しないということが判明している。つまり、複数回の再移植後のPDXモデルでは、ヒトの腫瘍血管に対する、血管新生抑制剤の効果の判定は不可能となる。従来、初回の移植後において、腫瘍内の組織を詳細に検討し、ヒトの血管が残存するということも報告されているが（非特許文献１、２）、これまでの方法では、ヒトの腫瘍内において、ヒトの血管が成長していく過程を観察しているものはなかった。 In this PDX model, in most cases, the initial transplantation was not used to determine the effects of drugs such as antitumor agents, and the human tumor tissue that was established at the first time was re-transplanted as finer tumor pieces. It has been used to determine the effects of therapeutic agents after multiple transplants. It has been proved that human-derived blood vessels are not already present in the tumor mass by such multiple transplants. That is, in the PDX model after multiple reimplantations, it is impossible to determine the effect of an angiogenesis inhibitor on human tumor blood vessels. Conventionally, after the initial transplantation, it has been reported that tissues in the tumor are examined in detail and human blood vessels remain (Non-Patent Documents 1 and 2). None of them observed the process of human blood vessel growth.
　血管が成長していく過程において、血管新生抑制剤がどのように血管新生に影響を与えるのかを観察できて始めて、これらの薬剤の効果の判定が可能となる。従来の方法では、腫瘍内にヒトの血管が形成されているか、残存しているかわからない状態で、PDXモデルマウスに対して血管新生剤を投与し、複数日後に血管がどのように影響があるのかを、血管新生抑制剤非投与群との比較で、血管の残存の程度を比較するに終始していた。このような解析系では、血管新生抑制剤の投与前後における、血管の、特にヒトの血管に対する直接作用を同一個体で観察することは不可能であった。 It is possible to determine the effects of these drugs only after observing how an angiogenesis inhibitor affects angiogenesis during the process of blood vessel growth. In the conventional method, an angiogenic agent is administered to PDX model mice without knowing whether human blood vessels are formed or remain in the tumor, and how the blood vessels are affected after multiple days. Was compared with the angiogenesis inhibitor non-administered group to compare the degree of remaining blood vessels. In such an analysis system, it was impossible to observe the direct action of blood vessels, particularly human blood vessels, before and after administration of an angiogenesis inhibitor in the same individual.
　本発明は、ヒト血管に対する薬剤の作用が評価可能な異種移植片モデル動物を用いて、ヒト血管の形成または機能に影響を及ぼす薬剤をスクリーニングできる方法を提供することを課題とする。また、本発明は、非ヒト免疫不全動物を用いてヒト血管を製造する方法を提供することを課題とする。 An object of the present invention is to provide a method capable of screening for drugs that affect the formation or function of human blood vessels using a xenograft model animal that can evaluate the action of the drugs on human blood vessels. Another object of the present invention is to provide a method for producing a human blood vessel using a non-human immunodeficient animal.
　本発明は、上記課題を解決するために、以下の各発明を包含する。
［１］ヒト血管の形成を促進もしくは抑制する物質、または、ヒト血管の構造もしくは機能を正常化する物質をスクリーニングする方法であって、以下の工程（１）～（７）を含むスクリーニング方法：
（１）非ヒト免疫不全動物の皮膚の一部を切開し皮下組織または筋肉層を露出させる工程、
（２）露出した皮下組織または筋肉層にヒト組織を載置する工程、
（３）載置したヒト組織が空気と接触しないように、載置したヒト組織を封じる工程、
（４）ヒト組織を非ヒト免疫不全動物に生着させる工程、
（５）工程（１）～（４）により得られた、ヒト組織が生着した非ヒト免疫不全動物に被験物質を投与する工程、
（６）生着したヒト組織内のヒト血管の形状もしくは構造を観察、および／または、ヒト血管の機能を評価する工程、および
（７）被験物質を投与していない前記ヒト組織が生着した非ヒト免疫不全動物のヒト血管と比較して、ヒト血管の形成を促進もしくは抑制する被験物質、または、ヒト血管の構造もしくは機能を正常化する被験物質を選択する工程。
［２］ヒト血管の形成を促進もしくは抑制する物質、または、ヒト血管の構造もしくは機能を正常化する物質をスクリーニングする方法であって、以下の工程（Ｉ）～（ＶＩ）を含むスクリーニング方法：
（Ｉ）非ヒト免疫不全動物の皮膚の一部を切開し皮下組織または筋肉層を露出させる工程、
（ＩＩ）露出した皮下組織または筋肉層に、ヒト組織と被験物質の混合物を載置する工程、
（ＩＩＩ）載置したヒト組織が空気と接触しないように、載置したヒト組織を封じる工程、
（ＩＶ）ヒト組織を非ヒト免疫不全動物に生着させる工程、
（Ｖ）生着したヒト組織内のヒト血管の形状もしくは構造を観察、および／または、ヒト血管の機能を評価する工程、および
（ＶＩ）被験物質と接触していない前記ヒト組織が生着した非ヒト免疫不全動物のヒト血管と比較して、ヒト血管の形成を促進もしくは抑制する被験物質、または、ヒト血管の構造もしくは機能を正常化する被験物質を選択する工程。
［３］前記工程（２）または（ＩＩ）において、ヒト組織が血管形成を促進する薬剤または細胞を添加したヒト組織である前記［１］または［２］に記載のスクリーニング方法。
［４］非ヒト免疫不全動物に生着したヒト組織内のヒト血管が宿主動物の血管と連結している、前記［１］～［３］のいずれかに記載のスクリーニング方法。
［５］前記工程（３）は、ドーサルスキンフォールドチャンバーを用いて載置したヒト組織を封じることを含む、前記［１］～［４］のいずれかに記載のスクリーニング方法。
［６］非ヒト免疫不全動物を用いるヒト血管の製造方法であって、以下の工程（Ａ）～（Ｅ）を含む製造方法：
（Ａ）非ヒト免疫不全動物の皮膚の一部を切開し皮下組織または筋肉層を露出させる工程、
（Ｂ）露出した皮下組織または筋肉層にヒト組織を載置する工程、
（Ｃ）載置したヒト組織が空気と接触しないように、載置したヒト組織を封じる工程、
（Ｄ）ヒト組織を非ヒト免疫不全動物に生着させ、ヒト血管を増生させる工程、および
（Ｅ）生着したヒト組織を採取する工程。
［７］前記工程（Ｂ）において、ヒト組織が血管形成を促進する薬剤または細胞を添加したヒト組織である前記［６］に記載の製造方法。
［８］前記工程（Ｃ）は、ドーサルスキンフォールドチャンバーを用いて載置したヒト組織を封じることを含む、前記［６］または［７］に記載の製造方法。
［９］移植用ヒト血管の製造方法である前記［６］～［８］のいずれかに記載の製造方法。 The present invention includes the following inventions in order to solve the above problems.
[1] A screening method for a substance that promotes or suppresses the formation of human blood vessels, or a substance that normalizes the structure or function of human blood vessels, comprising the following steps (1) to (7):
(1) a step of incising a part of the skin of a non-human immunodeficient animal to expose a subcutaneous tissue or a muscle layer;
(2) placing a human tissue on the exposed subcutaneous tissue or muscle layer;
(3) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air;
(4) engrafting human tissue in a non-human immunodeficient animal;
(5) A step of administering a test substance to a non-human immunodeficient animal engrafted with human tissue obtained by steps (1) to (4),
(6) observing the shape or structure of a human blood vessel in the engrafted human tissue and / or evaluating the function of the human blood vessel, and (7) engrafting the human tissue not administered with the test substance. A step of selecting a test substance that promotes or suppresses the formation of human blood vessels, or a test substance that normalizes the structure or function of human blood vessels, compared to human blood vessels of non-human immunodeficient animals.
[2] A method for screening for a substance that promotes or suppresses the formation of human blood vessels, or a substance that normalizes the structure or function of human blood vessels, comprising the following steps (I) to (VI):
(I) a step of incising a part of the skin of a non-human immunodeficient animal to expose the subcutaneous tissue or muscle layer,
(II) placing a mixture of human tissue and a test substance on the exposed subcutaneous tissue or muscle layer,
(III) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air;
(IV) engrafting human tissue in a non-human immunodeficient animal;
(V) observing the shape or structure of a human blood vessel in the engrafted human tissue and / or evaluating the function of the human blood vessel, and (VI) engrafting the human tissue not in contact with the test substance A step of selecting a test substance that promotes or suppresses the formation of human blood vessels, or a test substance that normalizes the structure or function of human blood vessels, compared to human blood vessels of non-human immunodeficient animals.
[3] The screening method according to [1] or [2], wherein in the step (2) or (II), the human tissue is a human tissue to which an agent or cell that promotes angiogenesis is added.
[4] The screening method according to any one of [1] to [3] above, wherein human blood vessels in human tissue engrafted in a non-human immunodeficient animal are connected to blood vessels of the host animal.
[5] The screening method according to any one of [1] to [4], wherein the step (3) includes sealing human tissue placed using a dorsal skin fold chamber.
[6] A method for producing a human blood vessel using a non-human immunodeficient animal, comprising the following steps (A) to (E):
(A) incising a part of the skin of a non-human immunodeficient animal to expose the subcutaneous tissue or muscle layer,
(B) placing human tissue on exposed subcutaneous tissue or muscle layer;
(C) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air;
(D) engrafting human tissue in a non-human immunodeficient animal to proliferate human blood vessels, and (E) collecting engrafted human tissue.
[7] The production method according to [6], wherein in the step (B), the human tissue is a human tissue to which an agent or cell that promotes angiogenesis is added.
[8] The production method according to [6] or [7], wherein the step (C) includes sealing human tissue placed using a dorsal skin fold chamber.
[9] The production method according to any one of [6] to [8], which is a method for producing a human blood vessel for transplantation.
　本発明のスクリーニング方法によれば、ヒト血管の形成、構造または機能に影響を及ぼす物質を取得することができる。本発明のスクリーニング方法により取得した物質は、虚血性疾患の治療薬、血管新生に起因して発症または悪化する疾患の治療薬、または血管の構造異常もしくは機能異常を伴う疾患の治療薬として有用である。また、本発明のヒト血管の製造方法は、非ヒト免疫不全動物を用いてヒト血管を増生し、製造することができる画期的な方法である。製造されたヒト血管は、虚血性疾患等の患者への移植用血管として有用である。 According to the screening method of the present invention, substances that affect the formation, structure or function of human blood vessels can be obtained. The substance obtained by the screening method of the present invention is useful as a therapeutic agent for ischemic disease, a therapeutic agent for a disease that develops or worsens due to angiogenesis, or a therapeutic agent for a disease that involves abnormal or structural abnormalities of blood vessels. is there. The human blood vessel production method of the present invention is an epoch-making method that can proliferate and produce human blood vessels using non-human immunodeficient animals. The manufactured human blood vessel is useful as a blood vessel for transplantation into a patient having an ischemic disease or the like.
ドーサルスキンフォールドチャンバー（dorsal skinfold chamber）を装着したマウスの外観を示した図である。It is the figure which showed the external appearance of the mouse | mouth which equipped with the dorsal skin fold chamber (dorsal | skinfold | chamber). ヒト大腸腫瘍組織移植後27日目の移植組織におけるヒトCD31陽性のヒト血管、マウスCD31陽性のマウス血管、移植組織由来のtdTomato陽性細胞を観察した結果を示す図である。It is a figure which shows the result of having observed the human CD31 positive human blood vessel, mouse CD31 positive mouse blood vessel, and the tdTomato positive cell derived from a transplanted tissue in the transplanted tissue on the 27th day after human colon tumor tissue transplantation. ヒト大腸腫瘍組織移植後18日目の移植組織における移植組織由来のtdTomato陽性細胞、ヒトCD31陽性ヒト血管内皮細胞、およびヒトの核特異的な抗体によって染色される細胞を観察した結果を示す図である。The figure which shows the result of observing cells stained with transplanted tissue-derived tdTomato-positive cells, human CD31-positive human vascular endothelial cells, and human nuclear-specific antibodies in the transplanted tissue 18 days after transplantation of human colon tumor tissue is there. ヒト大腸腫瘍組織移植後21日目の腫瘍組織移植部位の皮膚におけるヒトCD31陽性のヒト血管とマウスCD31陽性のマウス血管を観察した結果を示す図である。It is a figure which shows the result of having observed human CD31 positive human blood vessel and mouse CD31 positive mouse blood vessel in the skin of the tumor tissue transplantation site on the 21st day after human colon tumor tissue transplantation. ヒト大腸腫瘍組織移植後14日目と21日目に、移植組織におけるヒトCD31陽性のヒト血管を観察し（Ａ）、血管面積（Ｂ）、血管の分岐数（Ｃ）および血管長（Ｄ）を定量した結果を示す図である。On day 14 and day 21 after transplantation of human colon tumor tissue, human CD31-positive human blood vessels in the transplanted tissue were observed (A), blood vessel area (B), number of blood vessel branches (C), and blood vessel length (D) It is a figure which shows the result of having quantified. ヒト膵がん組織移植後6日目と12日目のヒト膵がん組織移植部位の皮膚におけるヒトCD31陽性のヒト血管を観察した結果を示す図であり、同じ試験を２回実施した結果を示している。破線は同時に移植した間葉系幹細胞の塊を示す。It is a figure which shows the result of having observed human CD31 positive human blood vessel in the skin of the human pancreatic cancer tissue transplantation site on the 6th day and the 12th day after human pancreatic cancer tissue transplantation, and the result of carrying out the same test twice Show. A broken line indicates a cluster of mesenchymal stem cells transplanted simultaneously. ヒト大腸正常組織移植後19日目の組織移植部位におけるヒトCD31陽性のヒト血管を観察した結果を示す図であり、同じ試験を２回実施した結果を示している。It is a figure which shows the result of having observed the human CD31 positive human blood vessel in the tissue transplantation site | part 19 days after a human colon normal tissue transplant, and has shown the result of having implemented the same test twice.
〔スクリーニング方法〕
　本発明は、ヒト血管の形成を促進もしくは抑制する物質、または、ヒト血管の構造もしくは機能を正常化する物質をスクリーニングする方法を提供する。本発明のスクリーニング方法は以下の工程（１）～（７）を含む方法であればよい。
（１）非ヒト免疫不全動物の皮膚の一部を切開し皮下組織または筋肉層を露出させる工程、
（２）露出した皮下組織または筋肉層にヒト組織を載置する工程、
（３）載置したヒト組織が空気と接触しないように、載置したヒト組織を封じる工程、
（４）ヒト組織を非ヒト免疫不全動物に生着させる工程、
（５）（１）～（４）の工程により得られた、ヒト組織が生着した非ヒト免疫不全動物に被験物質を投与する工程、
（６）生着したヒト組織内のヒト血管の形状もしくは構造を観察、および／または、ヒト血管の機能を評価する工程、および
（７）被験物質を投与していない前記ヒト組織が生着した非ヒト免疫不全動物のヒト血管と比較して、ヒト血管の形成を促進もしくは抑制する被験物質、または、ヒト血管の構造もしくは機能を正常化する被験物質を選択する工程。 [Screening method]
The present invention provides a method of screening for a substance that promotes or suppresses the formation of human blood vessels, or a substance that normalizes the structure or function of human blood vessels. The screening method of the present invention may be any method including the following steps (1) to (7).
(1) a step of incising a part of the skin of a non-human immunodeficient animal to expose a subcutaneous tissue or a muscle layer;
(2) placing a human tissue on the exposed subcutaneous tissue or muscle layer;
(3) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air;
(4) engrafting human tissue in a non-human immunodeficient animal;
(5) A step of administering a test substance to a non-human immunodeficient animal engrafted with human tissue obtained by the steps (1) to (4),
(6) observing the shape or structure of a human blood vessel in the engrafted human tissue and / or evaluating the function of the human blood vessel, and (7) engrafting the human tissue not administered with the test substance. A step of selecting a test substance that promotes or suppresses the formation of human blood vessels, or a test substance that normalizes the structure or function of human blood vessels, compared to human blood vessels of non-human immunodeficient animals.
　また、本発明のスクリーニング方法は、以下の工程（Ｉ）～（ＶＩ）を含む方法であってもよい。
（Ｉ）非ヒト免疫不全動物の皮膚の一部を切開し皮下組織または筋肉層を露出させる工程、
（ＩＩ）露出した皮下組織または筋肉層に、ヒト組織と被験物質の混合物を載置する工程、
（ＩＩＩ）載置したヒト組織が空気と接触しないように、載置したヒト組織を封じる工程、
（ＩＶ）ヒト組織を非ヒト免疫不全動物に生着させる工程、
（Ｖ）生着したヒト組織内のヒト血管の形状もしくは構造を観察、および／または、ヒト血管の機能を評価する工程、および
（ＶＩ）被験物質と接触していない前記ヒト組織が生着した非ヒト免疫不全動物のヒト血管と比較して、ヒト血管の形成を促進もしくは抑制する被験物質、または、ヒト血管の構造もしくは機能を正常化する被験物質を選択する工程。 Further, the screening method of the present invention may be a method including the following steps (I) to (VI).
(I) a step of incising a part of the skin of a non-human immunodeficient animal to expose the subcutaneous tissue or muscle layer,
(II) placing a mixture of human tissue and a test substance on the exposed subcutaneous tissue or muscle layer,
(III) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air;
(IV) engrafting human tissue in a non-human immunodeficient animal;
(V) observing the shape or structure of a human blood vessel in the engrafted human tissue and / or evaluating the function of the human blood vessel, and (VI) engrafting the human tissue not in contact with the test substance A step of selecting a test substance that promotes or suppresses the formation of human blood vessels, or a test substance that normalizes the structure or function of human blood vessels, compared to human blood vessels of non-human immunodeficient animals.
　工程（１）および（Ｉ）では、非ヒト免疫不全動物の皮膚の一部を切開し皮下組織または筋肉層を露出させる。非ヒト免疫不全動物としては、移植されたヒト組織が生着できる動物であればよく、免疫機能が低下している哺乳動物を使用してもよい。哺乳動物は特に限定されず、マウス、ラット、サル、ブタ、ヒツジ、ウシ、イヌ、ネコ、ウサギであってもよい。免疫機能が低下している哺乳動物としては、免疫抑制剤を投与した哺乳動物であってもよく、遺伝子変異により免疫機能が低下した哺乳動物であってもよい。遺伝子変異を有する免疫不全哺乳動物としては、例えば、Nudeマウス、Scidマウス、NOD/Scidマウス、NOGマウス、NSGマウス、NOJマウス、Rag1/Rag2 KO マウス、Nudeラット、Scidラットなどが挙げられる。 In steps (1) and (I), a part of the skin of the non-human immunodeficient animal is incised to expose the subcutaneous tissue or muscle layer. As the non-human immunodeficient animal, any animal that can engraft transplanted human tissue may be used, and a mammal having a reduced immune function may be used. The mammal is not particularly limited, and may be a mouse, rat, monkey, pig, sheep, cow, dog, cat, or rabbit. The mammal whose immune function is reduced may be a mammal administered with an immunosuppressant, or may be a mammal whose immune function is reduced due to a gene mutation. Examples of the immunodeficient mammal having a gene mutation include Nude mouse, Scid mouse, NOD / Scid mouse, NOG mouse, NSG mouse, NOJ mouse, Rag1 / Rag2 KO mouse, Nude rat, and Scid rat.
　皮膚の切開操作を効率よく行うために、非ヒト免疫不全動物を除毛してもよい。除毛は非ヒト免疫不全動物にヒト組織を移植する日の前日に行ってもよい。除毛は切開する皮膚部分を含む範囲で行ってもよい。除毛は麻酔下で行ってもよい。除毛方法は特に限定されないが、通常バリカンによる剃毛と除毛剤による除毛を組み合わせて行ってもよい。 In order to efficiently perform skin incision operations, non-human immunodeficient animals may be depilated. Hair removal may be performed the day before the day of transplanting human tissue into a non-human immunodeficient animal. Hair removal may be performed in a range including the skin portion to be incised. Hair removal may be performed under anesthesia. Although the hair removal method is not particularly limited, the hair removal using a hair clipper and the hair removal using a hair removal agent may be combined.
　麻酔下の非ヒト免疫不全動物の皮膚の一部を切開し、皮下組織または筋肉層を露出させる。皮下組織は表皮および真皮の下層に存在し、筋肉層は皮下組織の下層に存在する。したがって、皮下組織を露出させるために表皮および真皮を切除してもよく、筋肉層を露出させるために、表皮、真皮および皮下組織を切除してもよい。皮膚の一部を切開した領域に皮下組織のみが露出していてもよく、皮膚の一部を切開した領域に筋肉層のみが露出していてもよく、皮膚の一部を切開した領域に皮下組織が残った状態で筋肉層が露出していてもよい。 ¡A part of the skin of an anesthetized non-human immunodeficient animal is incised to expose the subcutaneous tissue or muscle layer. The subcutaneous tissue is in the lower layers of the epidermis and dermis, and the muscle layer is in the lower layers of the subcutaneous tissue. Thus, the epidermis and dermis may be excised to expose the subcutaneous tissue, and the epidermis, dermis and subcutaneous tissue may be excised to expose the muscle layer. Only the subcutaneous tissue may be exposed in the area where a part of the skin is incised, or only the muscle layer may be exposed in the area where the part of the skin is incised. The muscle layer may be exposed with the tissue remaining.
　工程（２）および（ＩＩ）では、露出した皮下組織または筋肉層にヒト組織を載置する。ヒト組織はヒトから採取された組織であればよい。例えば脂肪組織、筋組織、消化管組織、神経組織、皮膚組織、胎盤組織などが挙げられる。ヒト組織は、悪性腫瘍組織、良性腫瘍組織、炎症性疾患組織、虚血疾患組織などの疾患部位の組織であってもよい。ヒト組織は、非ヒト免疫不全動物に移植された経歴があるヒト組織でもよく、そのような経歴がないヒト組織でもよい。 In steps (2) and (II), human tissue is placed on the exposed subcutaneous tissue or muscle layer. The human tissue may be a tissue collected from a human. Examples include adipose tissue, muscle tissue, gastrointestinal tissue, nerve tissue, skin tissue, placenta tissue, and the like. The human tissue may be a tissue at a disease site such as a malignant tumor tissue, a benign tumor tissue, an inflammatory disease tissue, or an ischemic disease tissue. The human tissue may be a human tissue with a history of being transplanted into a non-human immunodeficient animal or a human tissue without such a history.
　ヒト組織は、ヒトから採取した新鮮な組織、または採取後速やかに凍結保存した組織を解凍した組織を用いることができる。新鮮な組織は、適切な保存液に浸漬して適切な条件で輸送されることを前提に、採取後12時間以内、10時間以内、8時間以内、6時間以内に非ヒト免疫不全動物に移植する。凍結保存した組織は、解凍後6時間以内、4時間以内、3時間以内に非ヒト免疫不全動物に移植する。組織を凍結保存する場合は、採取後できるだけ早くCELLBANKER（商品名、日本全薬工業）等の適切な組織凍結保存液に組織を浸漬し、凍結させる。適切な組織凍結保存液に浸漬した組織を、-80℃で一夜緩速凍結を行い、その後液体窒素の気相中で保存してもよい。 As the human tissue, a fresh tissue collected from a human or a tissue obtained by thawing a tissue that has been cryopreserved immediately after collection can be used. Transplant fresh tissue into non-human immunodeficient animals within 12 hours, within 10 hours, within 8 hours, and within 6 hours after collection, assuming that they are immersed in an appropriate storage solution and transported under appropriate conditions To do. Cryopreserved tissues are transplanted into non-human immunodeficient animals within 6 hours, 4 hours, and 3 hours after thawing. When cryopreserving a tissue, immerse the tissue in an appropriate tissue cryopreservation solution such as CELLBANKER (trade name, Nippon Zenyaku Kogyo Co., Ltd.) as soon as possible after collection. Tissue soaked in a suitable tissue cryopreservation solution may be slowly frozen overnight at -80 ° C. and then stored in a liquid nitrogen gas phase.
　移植したヒト組織の中心部が虚血状態になり壊死することを回避するために、細切したヒト組織を宿主動物の切開した皮膚の皮下組織または筋肉層に載置してもよい。細切したヒト組織の大きさは特に限定されないが、4～5mm角程度であってもよく、3～4mm角程度であってもよく、2～3mm角程度であってもよく、1～2mm角程度であってもよい。細切したヒト組織は、適当な生理的緩衝液に懸濁した状態で、切開した皮膚の皮下組織または筋肉層に載置してもよい。生理的緩衝液としては、例えばDMEMやRPMI1640などの細胞培養用培地、生理食塩水、リン酸緩衝液、リン酸緩衝生理食塩水（PBS）などが挙げられる。これらの生理的緩衝液はフェノールレッドが添加されていないものでもよい。 In order to prevent the center of the transplanted human tissue from becoming ischemic and necrotic, the minced human tissue may be placed on the subcutaneous tissue or muscle layer of the cut skin of the host animal. The size of the minced human tissue is not particularly limited, but may be about 4 to 5 mm square, about 3 to 4 mm square, about 2 to 3 mm square, or about 1 to 2 mm. It may be about a corner. The minced human tissue may be placed on the subcutaneous tissue or muscle layer of the incised skin while suspended in an appropriate physiological buffer. Examples of the physiological buffer include cell culture media such as DMEM and RPMI1640, physiological saline, phosphate buffer, phosphate buffered saline (PBS), and the like. These physiological buffers may not be added with phenol red.
　細切したヒト組織懸濁液に移植したヒト組織に血管形成を促進する薬剤または細胞を添加してもよい。血管形成を促進する薬剤は特に限定されず低分子化合物でもよく、中分子化合物でもよく、高分子化合物でもよい。また、血管形成を促進する薬剤は、天然物でもよく、合成物でもよい。例えば、PlGF（placental growth factor）、Angiopoietin-1、PDGF（platelet-derived growth factor）、EGF（epidermal growth factor）、HGF（hepatocyte growth factor）、basic fibroblast growth factor（bFGF）、VEGF等の増殖因子；リゾホスファチジン酸、スフィンゴシン－１－リン酸等の脂質；アペリン等の生理活性ペプチド；アプタマー、microRNA等の核酸；などが挙げられる。血管形成を促進する細胞としては、ヒト間葉系幹細胞、血管内皮幹細胞、血管内皮前駆細胞、ペリサイト、マクロファージなどが挙げられる。添加する薬剤または細胞は１種類でもよく、２種類以上でもよい。薬剤と細胞を組み合わせて添加してもよい。血管形成を促進する薬剤または細胞を添加量は特に限定されず、移植したヒト組織に所望のヒト血管が形成される添加量を添加すればよい。例えば、予備検討を行うことにより添加量を決定してもよい。 Drugs or cells that promote angiogenesis may be added to human tissue transplanted into a minced human tissue suspension. The agent that promotes angiogenesis is not particularly limited, and may be a low molecular compound, a medium molecular compound, or a high molecular compound. The drug that promotes angiogenesis may be a natural product or a synthetic product. For example, growth factors such as PlGF (placental growth factor), Angiopoietin-1, PDGF (platelet-derived growth factor), EGF (epidermal growth factor), HGF (hepatocyte growth factor), basic fibroblast growth factor (bFGF), VEGF; And lipids such as lysophosphatidic acid and sphingosine-1-phosphate; bioactive peptides such as apelin; nucleic acids such as aptamer and microRNA; and the like. Examples of cells that promote angiogenesis include human mesenchymal stem cells, vascular endothelial stem cells, vascular endothelial progenitor cells, pericytes, and macrophages. One type or two or more types of drugs or cells may be added. Drugs and cells may be added in combination. The addition amount of the agent or cell that promotes angiogenesis is not particularly limited, and an addition amount by which a desired human blood vessel is formed may be added to the transplanted human tissue. For example, the addition amount may be determined by conducting a preliminary study.
　移植したヒト組織の生着確認を容易にするために、蛍光タンパク質を発現するウイルスをヒト組織に感染させてから移植してもよい。蛍光タンパク質は特に限定されず、公知の蛍光タンパク質から適宜選択することができる。ウイルスとしては導入遺伝子を安定発現させるウイルスであるレンチウイルス、レトロウイルスを好適に用いることができる。蛍光タンパク質を発現するウイルスは、公知の遺伝子組み換え技術を用いて作製することができ、公知の方法で移植前のヒト組織に感染させることができる。 In order to facilitate engraftment confirmation of the transplanted human tissue, the human tissue may be transplanted after infecting a virus expressing a fluorescent protein. The fluorescent protein is not particularly limited, and can be appropriately selected from known fluorescent proteins. As viruses, lentiviruses and retroviruses that are viruses that stably express a transgene can be preferably used. A virus that expresses a fluorescent protein can be prepared using a known genetic recombination technique, and can infect human tissue before transplantation by a known method.
　ヒト組織は、宿主動物の切開した皮膚の皮下組織または筋肉層の全体が覆われる量を載置する。したがって、ヒト組織量は、露出した皮下組織または筋肉層の面積に応じて増減される。例えば、本発明者らは、マウスの皮膚を約7mm×7mmの大きさで切除した場合、細切したヒト組織50mgを50μLの生理的緩衝液に懸濁して筋肉層上に載置している。したがって、この量を基準に、露出した皮下組織または筋肉層の面積に応じてヒト組織を増減させてもよい。 The human tissue is placed in such an amount that the entire subcutaneous tissue or muscle layer of the cut skin of the host animal is covered. Thus, the amount of human tissue is increased or decreased depending on the exposed subcutaneous tissue or muscle layer area. For example, when the skin of a mouse is excised with a size of about 7 mm × 7 mm, the present inventors suspended 50 mg of minced human tissue in 50 μL of physiological buffer and placed it on the muscle layer. . Therefore, human tissue may be increased or decreased according to the exposed subcutaneous tissue or muscle layer area based on this amount.
　工程（ＩＩ）では、ヒト組織と被験物質の混合物を宿主動物の露出した皮下組織または筋肉層に載置する。被験物質は特に限定されず、後段の工程（５）の説明で例示した被験物質を好ましく用いることができる。ヒト組織と被験物質を混合する方法は特に限定されない。例えば、ヒト組織に被験物質を塗布してもよく、被験物質溶液にヒト組織を浸漬してもよく、細切したヒト組織の懸濁液に被験物質を添加してもよく、宿主動物の露出した皮下組織または筋肉層に載置したヒト組織に被験物質を添加してもよい。被験物質の添加量は特に限定されず、予備試験を行って適宜設定することができる。 In step (II), the mixture of human tissue and test substance is placed on the exposed subcutaneous tissue or muscle layer of the host animal. The test substance is not particularly limited, and the test substance exemplified in the description of the subsequent step (5) can be preferably used. The method for mixing the human tissue and the test substance is not particularly limited. For example, the test substance may be applied to human tissue, the human tissue may be immersed in the test substance solution, the test substance may be added to a suspension of minced human tissue, and the host animal is exposed. The test substance may be added to human tissue placed on the subcutaneous tissue or muscle layer. The addition amount of the test substance is not particularly limited, and can be appropriately set by conducting a preliminary test.
　工程（３）および（ＩＩＩ）では、載置したヒト組織が空気と接触しないように、載置したヒト組織を封じる。載置したヒト組織を封じる方法としては、例えば、載置したヒト組織の表面にスライドグラス、合成樹脂薄板、合成樹脂フィルムなどを密着させ、その周囲を宿主動物の皮膚に固定する方法などが挙げられる。載置したヒト組織を封じる一つの手段として、ドーサルスキンフォールドチャンバー（dorsal skinfold chamber、以下「DSC」と記す）を使用してもよい。DSCは市販のDSCの中から宿主動物に適したサイズのDSCを選択して使用すればよい。DSCは定法に従い洗浄、消毒、滅菌したものを使用する。 In steps (3) and (III), the placed human tissue is sealed so that the placed human tissue does not come into contact with air. Examples of the method for sealing the placed human tissue include a method in which a slide glass, a synthetic resin thin plate, a synthetic resin film, or the like is brought into close contact with the surface of the placed human tissue, and its periphery is fixed to the skin of the host animal. It is done. As one means for sealing the placed human tissue, a dorsal skinfold chamber (hereinafter referred to as “DSC”) may be used. As the DSC, a DSC having a size suitable for the host animal may be selected from commercially available DSCs. Use DSC that has been cleaned, disinfected, and sterilized according to standard methods.
　以下、マウスを宿主動物として、DSCを用いて移植するヒト組織を封じる手順を説明するが、これは一例でありこの手順に限定されるものではない。マウス以外の非ヒト動物を宿主動物として使用する場合も、同様の手順で行うことができる。 Hereinafter, a procedure for sealing a human tissue to be transplanted using DSC using a mouse as a host animal will be described, but this is an example and the present invention is not limited to this procedure. The same procedure can be used when a non-human animal other than a mouse is used as a host animal.
　マウスを麻酔し、除毛した背部の皮膚を牽引して二重となった皮膚にDSCのバックフレームを装着する。バックフレームにはフレームを固定するためのチューブが付いているので、このチューブをマウスの皮膚に貫通させる。続いて、手前側（バックフレームを装着していない側）の皮膚をチャンバーの形状および大きさに合わせて切開・切除し、皮下組織または筋肉層を露出させる。露出させる皮下組織または筋肉層は、手前側の皮膚の皮下組織または筋肉層でもよく、奥側（バックフレームを装着した側）の皮膚の皮下組織または筋肉層でもよい。手前側の皮膚の皮下組織または筋肉層を露出させる場合は、手前側の皮膚の表皮および真皮、または、表皮、真皮および皮下組織を切除する。奥側の皮膚の筋肉層を露出させる場合は、手前側の皮膚の表皮、真皮、皮下組織および筋肉層を切除し、さらに結合組織を切除する。奥側の皮膚の皮下組織を露出させる場合は、手前側の皮膚の表皮、真皮、皮下組織および筋肉層を切除し、さらに結合組織および奥側の皮膚の筋肉層を切除する（工程（１）および（Ｉ））。次に、露出させた皮下組織または筋肉層の上に、用時調製した細切したヒト組織を載置する（工程（２）および（ＩＩ））。載置するヒト組織量は、上記のとおり露出した皮下組織または筋肉層の全体が覆われ、かつ、DSCのフロントフレームを装着したときに隙間を生じない量が適量である。次に、DSCのカバーグラス付きフロントフレームを装着し、バックフレームに固定する（工程（３）および（ＩＩＩ））。この際、チャンバー内に空気が入らないよう注意する。 Anesthetize the mouse and attach the DSC back frame to the doubled skin by pulling the skin on the back where the hair has been removed. Since the back frame has a tube for fixing the frame, this tube is passed through the skin of the mouse. Subsequently, the skin on the front side (the side on which the back frame is not attached) is cut and excised according to the shape and size of the chamber to expose the subcutaneous tissue or muscle layer. The exposed subcutaneous tissue or muscle layer may be the subcutaneous tissue or muscle layer of the skin on the near side, or may be the subcutaneous tissue or muscle layer of the skin on the back side (side to which the back frame is attached). When the subcutaneous tissue or muscle layer of the near skin is exposed, the epidermis and dermis of the near skin or the epidermis, dermis and subcutaneous tissue are excised. When exposing the muscle layer of the back skin, the epidermis, dermis, subcutaneous tissue and muscle layer of the near skin are excised, and the connective tissue is further excised. When exposing the subcutaneous tissue of the back side skin, the epidermis, dermis, subcutaneous tissue and muscle layer of the near side skin are excised, and the connective tissue and the muscle layer of the back side skin are further excised (step (1)). And (I)). Next, the minced human tissue prepared at the time of use is placed on the exposed subcutaneous tissue or muscle layer (steps (2) and (II)). The amount of human tissue to be placed is an appropriate amount that covers the entire exposed subcutaneous tissue or muscle layer as described above and that does not generate a gap when the DSC front frame is attached. Next, a front frame with a DSC cover glass is attached and fixed to the back frame (steps (3) and (III)). At this time, be careful not to let air enter the chamber.
　工程（４）および（ＩＶ）では、ヒト組織を非ヒト免疫不全動物に生着させる。具体的には、工程（４）および（ＩＶ）では、工程（１）～（３）または工程（Ｉ）～（ＩＩＩ）で作製したヒト組織移植非ヒト免疫不全動物を飼育し、移植したヒト組織が宿主動物に生着すると共に、生着したヒト組織内のヒト血管が宿主動物の血管と連結した状態になるまでの十分な期間生存させる。飼育条件は特に限定されず、ヒト組織が移植されていない非ヒト免疫不全動物の飼育に適した条件と同じ条件で飼育を行えばよい。なお、DSCを装着した動物を飼育する場合、装着したDSCのカバーグラスが破損する等の事故を防ぐために、DSCの周囲にシールドを取り付けてもよい。また、DSCを装着した動物を飼育する場合、装着したDSCのカバーグラスが破損する等の事故を防ぐために、同一ケージ内で飼育する動物の匹数を少なくしてもよい。 In steps (4) and (IV), human tissues are engrafted in non-human immunodeficient animals. Specifically, in steps (4) and (IV), the human tissue transplanted non-human immunodeficient animal prepared in steps (1) to (3) or steps (I) to (III) is bred and transplanted. As the tissue engrafts in the host animal, the human blood vessels in the engrafted human tissue are allowed to survive for a sufficient period of time until they become connected to the host animal's blood vessels. Breeding conditions are not particularly limited, and breeding may be performed under the same conditions as those suitable for breeding non-human immunodeficient animals in which no human tissue has been transplanted. When animals with DSCs are reared, a shield may be attached around the DSCs to prevent accidents such as breakage of the DSC cover glass. When animals equipped with DSC are bred, the number of animals bred in the same cage may be reduced in order to prevent accidents such as breakage of the attached DSC cover glass.
　ヒト組織の生着確認を行う時期は特に限定されないが、ヒト組織移植後10日目以降に行ってもよい。ヒト組織の生着確認は、蛍光タンパク質を発現するウイルスを感染させたヒト組織を移植した動物のDSC内を蛍光顕微鏡で観察し、ヒト組織に感染したウイルスが発現する蛍光タンパク質の蛍光シグナルを検出することにより行うことができる。 The time for confirming the engraftment of human tissue is not particularly limited, but may be performed on or after the 10th day after transplantation of human tissue. For confirmation of engraftment of human tissue, the fluorescence signal of the fluorescent protein expressed by the virus infected with the human tissue is detected by observing the inside of the DSC of the animal transplanted with the human tissue infected with the virus expressing the fluorescent protein with a fluorescent microscope. This can be done.
　宿主に生着したヒト組織の血管の観察は、ヒト組織の生着確認と同時期に行うことができる。血管のイメージングには、血管内皮細胞が発現する分子に結合する蛍光標識抗体を用いることができる。ヒト血管をイメージングするための抗体としては、抗ヒトCD31抗体、抗ヒトCD34抗体、抗ヒトVE-カドヘリン抗体、抗ヒトVCAM-1抗体、抗ヒトvWF抗体などが挙げられ、市販の蛍光標識抗体を使用することができる。宿主動物の血管をイメージングするための抗体も同様であり、宿主動物がマウスの場合、抗マウスCD31抗体、抗マウスCD34抗体、抗マウスVE-カドヘリン抗体、抗マウスVCAM-1抗体、抗マウスvWF抗体などを用いることができる。宿主動物の血管のイメージング用抗体の蛍光標識には、ヒト血管のイメージング用抗体に標識された蛍光分子が発する蛍光色と区別可能な蛍光色を発する蛍光分子を用いる。 The observation of blood vessels of human tissue engrafted in the host can be performed at the same time as confirmation of engraftment of human tissue. For imaging of blood vessels, fluorescently labeled antibodies that bind to molecules expressed by vascular endothelial cells can be used. Examples of antibodies for imaging human blood vessels include anti-human CD31 antibody, anti-human CD34 antibody, anti-human VE-cadherin antibody, anti-human VCAM-1 antibody, and anti-human vWF antibody. Can be used. The same applies to antibodies for imaging blood vessels of host animals. When the host animal is a mouse, anti-mouse CD31 antibody, anti-mouse CD34 antibody, anti-mouse VE-cadherin antibody, anti-mouse VCAM-1 antibody, anti-mouse vWF antibody Etc. can be used. For the fluorescent labeling of the host animal blood vessel imaging antibody, a fluorescent molecule emitting a fluorescent color distinguishable from the fluorescent color emitted from the fluorescent molecule labeled on the human blood vessel imaging antibody is used.
　イメージング用の抗体は、用いる抗体に応じて最適な投与量を予備検討により設定し、宿主動物の静脈内に投与する。マウスの場合、通常5～20μgを投与する。イメージング用抗体の投与から12～24時間後にDSC内を蛍光顕微鏡で観察する。ヒト血管のイメージング用抗体を宿主動物に静脈内投与してヒト血管を観察することができれば、宿主動物の血管とヒト血管が連結していることが確認できると同時に、ヒト組織が宿主動物に生着していることが合理的に推認できる。 For the antibody for imaging, the optimal dose is set by preliminary examination according to the antibody to be used, and is administered intravenously to the host animal. In the case of mice, 5 to 20 μg is usually administered. The inside of the DSC is observed with a fluorescence microscope 12-24 hours after administration of the imaging antibody. If human blood vessel antibodies can be administered intravenously to a host animal and the human blood vessels can be observed, it can be confirmed that the blood vessels of the host animal are connected to the human blood vessels, and at the same time, human tissue is produced in the host animal. I can reasonably infer that I am wearing it.
　工程（５）では、工程（１）～（４）により得られた、ヒト組織が生着した非ヒト免疫不全動物に被験物質を投与する。被験物質としては、例えば、核酸、ペプチド、タンパク、非ペプチド性化合物、合成化合物、発酵生産物、細胞、細胞抽出液、細胞培養上清、植物抽出液、哺乳動物の組織抽出液、血漿等を好ましく用いることができる。ただし、これらに限定されない。被験物質は、新規な物質であってもよいし、公知の物質であってもよい。これら被験物質は塩を形成していてもよい。被験物質の塩としては、生理学的に許容される酸や塩基との塩を用いてもよい。 In step (5), the test substance is administered to the non-human immunodeficient animal engrafted with human tissue obtained in steps (1) to (4). Examples of test substances include nucleic acids, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cells, cell extracts, cell culture supernatants, plant extracts, mammalian tissue extracts, plasma, etc. It can be preferably used. However, it is not limited to these. The test substance may be a novel substance or a known substance. These test substances may form a salt. As a salt of the test substance, a salt with a physiologically acceptable acid or base may be used.
　被験物質を投与する時期は、工程（４）でヒト組織の生着を確認した後であればよく、ヒト組織移植後14～21日目であってもよい。被験物質の投与量、投与経路、投与回数は、被験物質に応じて設定することが好ましい。対照群には、例えば同じ投与経路および同じ投与回数で、被験物質の調製に使用した溶媒を投与する。投与経路は特に限定されないが、経口投与、静脈内投与、腹腔内投与等の全身投与が好ましい。 The timing of administration of the test substance may be after confirming the engraftment of human tissue in step (4), and may be 14 to 21 days after human tissue transplantation. The dose, administration route, and number of administrations of the test substance are preferably set according to the test substance. In the control group, for example, the solvent used for the preparation of the test substance is administered by the same administration route and the same administration frequency. The administration route is not particularly limited, but systemic administration such as oral administration, intravenous administration and intraperitoneal administration is preferable.
　工程（６）または（Ｖ）では、生着したヒト組織内のヒト血管の形状もしくは構造を観察、および／または、ヒト血管の機能を評価する。ヒト血管の観察および機能評価は、被験物質投与以後であればどの時期に行ってもよく、ヒト組織移植後14～21日目であってもよい。 In step (6) or (V), the shape or structure of the human blood vessel in the engrafted human tissue is observed and / or the function of the human blood vessel is evaluated. Observation and functional evaluation of human blood vessels may be performed at any time after administration of the test substance, and may be performed 14 to 21 days after human tissue transplantation.
　ヒト血管の形状または構造の観察は、上記のようにヒト血管をイメージングするための抗体を宿主動物に静脈内投与することにより行うことができる。ヒト血管の形状または構造としては、例えばヒト血管の面積（ヒト血管が視野に占める面積）、ヒト血管の長さ、ヒト血管の分岐数、ヒト血管の蛇行性、ヒト血管の拡張性、ヒト血管の閉塞性等が挙げられる。観察視野の画像を、画像解析ソフト（例えば、AngioTool等）を用いて解析してもよい。解析により、上記の観察項目を数値化してもよい。 Observation of the shape or structure of a human blood vessel can be performed by intravenously administering an antibody for imaging a human blood vessel to a host animal as described above. Examples of the shape or structure of the human blood vessel include the area of the human blood vessel (the area occupied by the human blood vessel), the length of the human blood vessel, the number of branches of the human blood vessel, the tortuous nature of the human blood vessel, the expandability of the human blood vessel, and the human blood vessel. For example. The image in the observation field may be analyzed using image analysis software (for example, AngioTool). The above observation items may be quantified by analysis.
　ヒト血管の機能としては、透過性、血流、酸素送達度などが挙げられる。透過性は、例えば、蛍光標識デキストランや自家蛍光を発する薬剤（例えばドキソルビシン等）を宿主動物の静脈内に投与し、ヒト血管の周囲に漏れている蛍光領域面積を測定することで評価してもよい。血流は、例えば、蛍光標識レクチンを宿主動物の静脈内に投与した後生着したヒト組織を宿主動物から採取し、抗ヒトCD31抗体で染色した組織標本を観察してヒトCD31陽性細胞におけるレクチン陽性細胞の割合を算出することにより、血流のある血管の割合を評価してもよい。酸素送達度は、例えば、公知の低酸素プローブ（例えばピモニダゾール等）を用いて評価してもよい。 The functions of human blood vessels include permeability, blood flow, and oxygen delivery. Permeability can be evaluated by, for example, administering a fluorescently labeled dextran or an autofluorescent agent (such as doxorubicin) into the host animal's vein and measuring the area of the fluorescent region leaking around the human blood vessel. Good. For example, the blood flow is collected from the host animal after the fluorescently labeled lectin is administered intravenously to the host animal, and the tissue specimen stained with the anti-human CD31 antibody is observed to detect lectin positive in human CD31 positive cells. The proportion of blood vessels with blood flow may be evaluated by calculating the proportion of cells. The oxygen delivery degree may be evaluated using, for example, a known hypoxic probe (eg, pimonidazole).
　工程（７）および（ＶＩ）では、被験物質を投与していない動物のヒト血管の面積、ヒト血管の長さ、またはヒト血管の分岐数と比較して、被験物質を投与した動物のヒト血管の面積、ヒト血管の長さ、またはヒト血管の分岐数が増加している場合に、当該被験物質をヒト血管の形成を促進する被験物質として選択することができる。ヒト血管の面積、ヒト血管の長さおよびヒト血管の分岐数は、画像解析ソフト（例えば、AngioTool等）を用いて数値化することができる（実施例４、図５参照）。被験物質が面積、長さまたは分岐数を増加させる程度は特に限定されないが、例えば、1.2倍以上、1.4倍以上、1.6倍以上、1.8倍以上、2倍以上、2.2倍以上、2.4倍以上、2.6倍以上、2.8倍以上、3倍以上に増加させる被験物質を選択することができる。なお、被験物質を投与していない動物との比較は、過去の同じ実験により蓄積された被験物質非投与動物の蓄積データ（背景データ）に基づいて行ってもよい。 In steps (7) and (VI), compared to the area of human blood vessels, the length of human blood vessels, or the number of branches of human blood vessels in animals not administered with the test substance, The test substance can be selected as a test substance that promotes the formation of human blood vessels when the area of human blood vessels, the length of human blood vessels, or the number of branches of human blood vessels is increased. The area of the human blood vessel, the length of the human blood vessel, and the number of branches of the human blood vessel can be quantified using image analysis software (for example, AngioTool) (see Example 4 and FIG. 5). The degree to which the test substance increases the area, length, or number of branches is not particularly limited, for example, 1.2 times or more, 1.4 times or more, 1.6 times or more, 1.8 times or more, 2 times or more, 2.2 times or more, 2.4 times or more, Test substances that increase 2.6 times or more, 2.8 times or more, or 3 times or more can be selected. The comparison with the animal not administered with the test substance may be performed based on accumulated data (background data) of the test substance non-administered animal accumulated in the same experiment in the past.
　ヒト血管の形成を促進する被験物質として選択された物質は、虚血性疾患の治療薬の有効成分として有用である。虚血性疾患としては、例えば、虚血性心疾患（心筋梗塞、冠状動脈硬化症等）、虚血性脳疾患（脳梗塞、脳総脈硬化症、痴呆症等）、骨粗鬆症、老化性臓器機能低下症、バージャー病、慢性閉塞性動脈硬化症、褥瘡、種々の臓器移植などが挙げられる。 A substance selected as a test substance that promotes the formation of human blood vessels is useful as an active ingredient of a therapeutic agent for ischemic diseases. Examples of ischemic diseases include ischemic heart diseases (myocardial infarction, coronary atherosclerosis, etc.), ischemic brain diseases (cerebral infarction, cerebral arteriosclerosis, dementia, etc.), osteoporosis, and aging organ hypofunction , Buerger's disease, chronic obstructive arteriosclerosis, pressure ulcer, various organ transplants and the like.
　工程（７）および（ＶＩ）では、被験物質を投与していない動物のヒト血管の面積、ヒト血管の長さ、またはヒト血管の分岐数と比較して、被験物質を投与した動物のヒト血管の面積、ヒト血管の長さ、またはヒト血管の分岐数が減少している場合に、当該被験物質をヒト血管の形成を抑制する被験物質として選択することができる。ヒト血管の面積、ヒト血管の長さおよびヒト血管の分岐数は、画像解析ソフト（例えば、AngioTool等）を用いて数値化することができる（実施例４、図５参照）。被験物質が面積、長さまたは分岐数を減少させる程度は特に限定されないが、例えば、0.9倍以下、0.8倍以下、0.7倍以下、0.6倍以下、0.5倍以下、0.4倍以下、0.3倍以下、0.2倍以下、0.1倍以下に減少させる被験物質を選択することができる。なお、被験物質を投与していない動物との比較は、過去の同じ実験により蓄積された被験物質非投与動物の蓄積データ（背景データ）に基づいて行ってもよい。 In steps (7) and (VI), compared to the area of human blood vessels, the length of human blood vessels, or the number of branches of human blood vessels in animals not administered with the test substance, The test substance can be selected as a test substance that suppresses the formation of human blood vessels when the area, the length of human blood vessels, or the number of branches of human blood vessels is reduced. The area of the human blood vessel, the length of the human blood vessel, and the number of branches of the human blood vessel can be quantified using image analysis software (for example, AngioTool) (see Example 4 and FIG. 5). The degree to which the test substance reduces the area, length or number of branches is not particularly limited, for example, 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, A test substance that can be reduced to 0.2 times or less or 0.1 times or less can be selected. The comparison with the animal not administered with the test substance may be performed based on accumulated data (background data) of the test substance non-administered animal accumulated in the same experiment in the past.
　ヒト血管の形成を抑制する被験物質として選択された物質は、血管新生に起因して発症または悪化する疾患の治療薬の有効成分として有用である。このような疾患としては、例えば、悪性腫瘍、感染症、動脈硬化症、自己免疫疾患（例えば、慢性関節リウマチ、強皮症等）、糖尿病性網膜症、加齢性黄斑変性症、未熟児網膜症、緑内障、血管奇形（例えば、毛細血管奇形、動静脈奇形等）、血管腫、変形性関節症、ケロイド、乾癬、アレルギー性皮膚炎、肥満、肺高血圧症、喘息、肺気腫、慢性気管支炎、肝硬変、腹水症などが挙げられる。 A substance selected as a test substance that suppresses the formation of human blood vessels is useful as an active ingredient of a therapeutic agent for a disease that develops or worsens due to angiogenesis. Examples of such diseases include malignant tumors, infections, arteriosclerosis, autoimmune diseases (eg, rheumatoid arthritis, scleroderma, etc.), diabetic retinopathy, age-related macular degeneration, retina of prematurity Symptom, glaucoma, vascular malformation (eg capillary malformation, arteriovenous malformation, etc.), hemangioma, osteoarthritis, keloid, psoriasis, allergic dermatitis, obesity, pulmonary hypertension, asthma, emphysema, chronic bronchitis, Examples include cirrhosis and ascites.
　工程（７）および（ＶＩ）では、被験物質を投与していない動物のヒト血管の構造または機能と比較して、被験物質を投与した場合に正常化している場合に、当該被験物質をヒト血管の構造または機能を正常化する被験物質として選択することができる。ここで正常化は評価項目に応じて判断される。ヒト血管の構造または機能を正常化とは、例えば、疾患部位の組織において亢進した血管の蛇行性が正常組織のレベルに戻ること、疾患部位の組織において亢進した血管の拡張性が正常組織のレベルに戻ること、疾患部位の組織において亢進した血管の閉塞性が正常組織のレベルに戻ること、疾患部位の組織において亢進した血管の透過性が正常組織のレベルに戻ること、疾患部位の組織において低下した血流が正常組織のレベルに戻ることなどが挙げられる。ヒト血管の構造または機能の正常レベルは、過去に蓄積されたデータ（背景データ）を参照してもよい。 In steps (7) and (VI), when the test substance is administered and normalized as compared to the structure or function of human blood vessels in an animal not administered with the test substance, Can be selected as a test substance that normalizes the structure or function. Here, normalization is determined according to the evaluation item. Normalizing the structure or function of a human blood vessel means, for example, that the tortuousness of a blood vessel increased in the tissue at the disease site returns to the level of the normal tissue, or the blood vessel expandability in the tissue at the disease site is the level of the normal tissue , Increased blood vessel occlusion in diseased tissue returns to normal tissue level, increased blood vessel permeability in diseased tissue returns to normal tissue level, decreased in diseased tissue For example, returning the blood flow to the normal tissue level. The normal level of human blood vessel structure or function may refer to previously accumulated data (background data).
　ヒト血管の構造または機能を正常化する被験物質として選択された物質は、例えば、悪性腫瘍、感染症、動脈硬化症、自己免疫疾患（例えば、慢性関節リウマチ、強皮症等）、糖尿病性網膜症、加齢性黄斑変性症、未熟児網膜症、緑内障、血管奇形（例えば、毛細血管奇形、動静脈奇形等）、血管腫、変形性関節症、ケロイド、乾癬、アレルギー性皮膚炎、肥満、肺高血圧症、喘息、肺気腫、慢性気管支炎、肝硬変、腹水症、腎疾患（例えば、糸球体腎症、糖尿病性腎症等）などの治療薬の有効成分として有用である。 Substances selected as test substances that normalize the structure or function of human blood vessels include, for example, malignant tumors, infections, arteriosclerosis, autoimmune diseases (eg, rheumatoid arthritis, scleroderma, etc.), diabetic retina Disease, age-related macular degeneration, retinopathy of prematurity, glaucoma, vascular malformations (eg capillary malformations, arteriovenous malformations, etc.), hemangiomas, osteoarthritis, keloid, psoriasis, allergic dermatitis, obesity, It is useful as an active ingredient of therapeutic agents for pulmonary hypertension, asthma, emphysema, chronic bronchitis, cirrhosis, ascites, renal diseases (eg, glomerulonephropathy, diabetic nephropathy, etc.).
〔ヒト血管の製造方法〕
　本発明は、非ヒト免疫不全動物を用いるヒト血管の製造方法を提供する。本発明の製造方法は以下の工程（Ａ）～（Ｅ）を含む。
（Ａ）非ヒト免疫不全動物の皮膚の一部を切開し皮下組織または筋肉層を露出させる工程、
（Ｂ）露出した皮下組織または筋肉層にヒト組織を載置する工程、
（Ｃ）載置したヒト組織が空気と接触しないように、載置したヒト組織を封じる工程、
（Ｄ）ヒト組織を非ヒト免疫不全動物に生着させ、ヒト血管を増生させる工程、および
（Ｅ）生着したヒト組織を採取する工程。 [Method for producing human blood vessel]
The present invention provides a method for producing a human blood vessel using a non-human immunodeficient animal. The production method of the present invention includes the following steps (A) to (E).
(A) incising a part of the skin of a non-human immunodeficient animal to expose the subcutaneous tissue or muscle layer,
(B) placing human tissue on exposed subcutaneous tissue or muscle layer;
(C) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air;
(D) engrafting human tissue in a non-human immunodeficient animal to proliferate human blood vessels, and (E) collecting engrafted human tissue.
　工程（Ａ）～（Ｃ）は、既に説明した本発明のスクリーニング方法の工程（１）～（３）と同じであり、本発明のスクリーニング方法の工程（１）～（３）と同じように実施することができる。ただし、本発明の製造方法は、ヒトへの移植に利用され得るので、蛍光タンパク質を発現するウイルスを感染させていないヒト組織を用いることが好ましい。また、同じ理由から、疾患部位のヒト組織（例えば悪性腫瘍を含むヒト組織）を使用しないことが好ましい。 Steps (A) to (C) are the same as steps (1) to (3) of the screening method of the present invention already described, and are the same as steps (1) to (3) of the screening method of the present invention. Can be implemented. However, since the production method of the present invention can be used for transplantation to humans, it is preferable to use human tissue that is not infected with a virus expressing a fluorescent protein. For the same reason, it is preferable not to use a human tissue at a disease site (for example, a human tissue containing a malignant tumor).
　工程（Ｄ）では、ヒト組織を非ヒト免疫不全動物に生着させ、ヒト血管を増生させる。具体的には、工程（Ｄ）では、工程（Ａ）～（Ｃ）で作製したヒト組織移植非ヒト免疫不全動物を飼育し、移植したヒト組織が宿主動物に生着すると共に、生着したヒト組織内のヒト血管が増生するまでの十分な期間生存させる。飼育条件は特に限定されず、ヒト組織が移植されていない非ヒト免疫不全動物の飼育に適した条件と同じ条件で飼育を行えばよい。なお、DSCを装着した動物を飼育する場合、装着したDSCのカバーグラスが破損する等の事故を防ぐために、DSCの周囲にシールドを取り付けてもよい。また、DSCを装着した動物を飼育する場合、装着したDSCのカバーグラスが破損する等の事故を防ぐために、同一ケージ内で飼育する動物の匹数を少なくしてもよい。 In step (D), human tissues are engrafted in non-human immunodeficient animals and human blood vessels are increased. Specifically, in the step (D), the human tissue transplanted non-human immunodeficient animal produced in the steps (A) to (C) is bred, and the transplanted human tissue is engrafted in the host animal and engrafted. Survive for a sufficient period of time until human blood vessels in the human tissue grow. Breeding conditions are not particularly limited, and breeding may be performed under the same conditions as those suitable for breeding non-human immunodeficient animals in which no human tissue has been transplanted. When animals with DSCs are reared, a shield may be attached around the DSCs to prevent accidents such as breakage of the DSC cover glass. When animals equipped with DSC are bred, the number of animals bred in the same cage may be reduced in order to prevent accidents such as breakage of the attached DSC cover glass.
　工程（Ｄ）の飼育期間は特に限定されず、ヒト組織移植後8日間以上、10日間以上、12日間以上、14日間以上であってもよく、ヒト組織移植後28日間以下、25日間以下、21日間以下、18日間以下、14日間以下であってもよい。 The breeding period of the step (D) is not particularly limited, and may be 8 days or more after human tissue transplantation, 10 days or more, 12 days or more, 14 days or more, 28 days or less after human tissue transplantation, 25 days or less, It may be 21 days or less, 18 days or less, or 14 days or less.
　ヒト組織の生着確認は、蛍光タンパク質を発現するウイルスを感染させたヒト組織を少数の宿主動物に移植し、移植した宿主動物のヒト組織を蛍光顕微鏡で観察し、ヒト組織に感染したウイルスが発現する蛍光タンパク質の蛍光シグナルを検出することにより行ってもよい。蛍光タンパク質を発現するウイルスを感染させたヒト組織を移植した少数の宿主動物においてヒト組織の生着が確認できれば、蛍光タンパク質を発現するウイルスを感染させていないヒト組織を同日に移植した宿主動物においても、ヒト組織が生着していることを合理的に推認できる。 Human tissue engraftment is confirmed by transplanting human tissue infected with a virus expressing a fluorescent protein into a small number of host animals, observing the human tissue of the transplanted host animal with a fluorescence microscope, You may carry out by detecting the fluorescence signal of the fluorescent protein to express. If the engraftment of human tissue can be confirmed in a small number of host animals transplanted with human tissues infected with a virus expressing a fluorescent protein, in a host animal transplanted with human tissues not infected with a virus expressing a fluorescent protein on the same day. It can be reasonably inferred that human tissue is engrafted.
　また、蛍光タンパク質を発現するウイルスを感染させたヒト組織を使用せずに、ヒト血管をイメージングするための抗体を用いて、ヒト組織の生着とヒト血管の増生を同時確認してもよい。ヒト血管のイメージングは、上記本発明のスクリーニング方法の工程（４）で説明した手順で行うことができる。ヒト血管のイメージング用抗体を宿主動物に静脈内投与してヒト血管の増生を確認することができれば、ヒト組織が宿主動物に生着していることが合理的に推認できる。 Alternatively, human tissue engraftment and human blood vessel growth may be simultaneously confirmed using an antibody for imaging human blood vessels without using human tissue infected with a virus expressing a fluorescent protein. Imaging of human blood vessels can be performed by the procedure described in step (4) of the screening method of the present invention. If human blood vessel imaging antibody can be intravenously administered to a host animal to confirm the growth of human blood vessels, it can be reasonably assumed that human tissue has been engrafted in the host animal.
　工程（Ｅ）では、生着したヒト組織を採取する。非ヒト免疫不全動物から移植したヒト正常組織を採取する方法は特に限定されず、宿主動物に生着しているヒト組織をメス等の手術器具を用いて採取すればよい。採取したヒト組織には、増生したヒト血管が含まれているので、直ちに適切な保存液に浸漬して移植現場に輸送し、血管の移植に使用することができる。また、直ちに移植を行わない場合は、適切な組織凍結保存液に浸漬して凍結保存することができる。凍結保存する場合は、-80℃で一夜緩速凍結を行い、その後液体窒素の気相中で保存してもよい。 In step (E), engrafted human tissue is collected. The method of collecting human normal tissue transplanted from a non-human immunodeficient animal is not particularly limited, and human tissue engrafted in the host animal may be collected using a surgical instrument such as a scalpel. Since the collected human tissue contains increased human blood vessels, the human tissue can be immediately immersed in an appropriate preservation solution and transported to a transplantation site to be used for blood vessel transplantation. When transplantation is not performed immediately, the tissue can be cryopreserved by dipping in an appropriate tissue cryopreservation solution. In the case of cryopreservation, slow freezing may be performed overnight at -80 ° C. and then stored in a liquid nitrogen gas phase.
　本発明の製造方法で製造したヒト血管を移植することにより病態の改善が期待できる疾患としては、例えば、虚血性心疾患（心筋梗塞、冠状動脈硬化症等）、虚血性脳疾患（脳梗塞、脳総脈硬化症、痴呆症等）、骨粗鬆症、老化性臓器機能低下症、バージャー病、慢性閉塞性動脈硬化症、褥瘡、種々の臓器移植、血友病、フォン・ヴィレブランド病などが挙げられる。 Examples of diseases that can be expected to be improved by transplanting human blood vessels produced by the production method of the present invention include ischemic heart diseases (myocardial infarction, coronary atherosclerosis, etc.), ischemic brain diseases (cerebral infarction, Cerebrospinal vein sclerosis, dementia, etc.), osteoporosis, aging organ hypofunction, Buerger's disease, chronic obstructive arteriosclerosis, pressure ulcer, various organ transplants, hemophilia, von Willebrand disease, etc. .
　以下、一実施例について説明するが、本発明はこれらに限定されるものではない。 Hereinafter, although an Example is described, this invention is not limited to these.
実施例１：ヒト腫瘍組織片移植マウスの作製と観察
＜実験方法＞
（１）移植前日のマウス処置
　8週齢の雌のNOD/ShiJic-scidマウス（日本クレア）に、三種混合麻酔（6μg塩酸メデトミジン（商品名：ドミトール、日本全薬工業）、80μgミダゾラム（サンド）、100μg酒石酸ブトルファノール（商品名：ベトルファール、Meiji Seika ファルマ）を200μL大塚生食注（大塚製薬工場）にて希釈して調製）を200μL腹腔内投与した。後肢引き込み反射消失を確認後、背部をバリカンで剃毛し、さらに除毛剤エピラット（クラシエ）を用いて除毛した。ぬるま湯にて除毛剤を取り除き、メデトミジン拮抗薬（6μg塩酸アチパメゾール（商品名：アンチセダン、日本全薬工業）を200μL大塚生食注（大塚製薬工場）にて希釈して調製）200μLを腹腔内投与し、37℃ホットプレート上にてマウスを覚醒させた。 Example 1: Preparation and observation of mouse transplanted with human tumor tissue <Experimental method>
(1) Mice treatment the day before transplantation 8-week-old female NOD / ShiJic-scid mice (CLEA Japan) were mixed with three types of mixed anesthesia (6 μg medetomidine hydrochloride (trade name: Domitor, Nippon Zenyaku Kogyo), 80 μg midazolam (Sand) 200 μL of butorphanol tartrate (trade name: Betorfal, Meiji Seika Pharma) diluted 200 μL of Otsuka raw food (Otsuka Pharmaceutical Factory) was intraperitoneally administered. After confirming disappearance of the hind limb reflex, the back was shaved with a clipper and further depilated using a depilatory epirat (Kracie). Remove the hair remover with lukewarm water and administer 200 μL of medetomidine antagonist (diluted with 6 μg atipamezole hydrochloride (trade name: Antisedan, Nippon Zenyaku Kogyo Co., Ltd.) in 200 μL Otsuka raw food injection (Otsuka Pharmaceutical Factory). Then, the mouse was awakened on a 37 ° C. hot plate.
（２）tdTomato発現レンチウイルスの作製
　Lenti-X293T細胞（タカラバイオ）を3×106cells/wellで、Biocoat（商品名）10cmディシュ（Corning）に播種した。培養液には10％FCS（Sigma）、100units/mL penicillin/100μg streptomycin（Sigma）、1×GlutaMax Supplement（Thermo Fisher Scientific）を含むDMEM（Sigma）を用いた。レンチウイルストランスフェクション溶液（14μLのLentiviral High Titer Packaging Mix（タカラバイオ）、2.75μgのtdTomato遺伝子挿入pLVSIN-EF1α Neo Vector（タカラバイオ）、27.5μLのP3000 Reagent（Thermo Fisher Scientific）、25μLのLipofectamine 3000（Thermo Fisher Scientific）、1250μLのOpti-MEM（Thermo Fisher Scientific）を混合）を調製し、室温にて15分間静置した。80～90％コンフレントに増殖したLenti-X293T細胞の培養液を新しい培養液（5ml）に交換し、上記レンチウイルストランスフェクション溶液を添加した。CO2インキュベーターにて37℃で6時間トランスフェクションを行い、その後10mLの新しい培養液に交換した。トランスフェクション開始から48時間後に培養上清を回収し、10mLの培養液を加え、さらに24時間培養後に培養上清を回収し、4℃にて保存した。 (2) Preparation of tdTomato-expressing lentivirus Lenti-X293T cells (Takara Bio) were seeded at 3 × 10 6 cells / well in a Biocoat (trade name) 10 cm dish. As the culture solution, DMEM (Sigma) containing 10% FCS (Sigma), 100 units / mL penicillin / 100 μg streptomycin (Sigma), and 1 × GlutaMax Supplement (Thermo Fisher Scientific) was used. Lentiviral transfection solution (14 μL Lentiviral High Titer Packaging Mix (Takara Bio), 2.75 μg tdTomato gene inserted pLVSIN-EF1α Neo Vector (Takara Bio), 27.5 μL P3000 Reagent (Thermo Fisher Scientific), 25 μL Lipofectamine 3000 ( Thermo Fisher Scientific) and 1250 μL of Opti-MEM (Thermo Fisher Scientific) were prepared and allowed to stand at room temperature for 15 minutes. The culture solution of Lenti-X293T cells grown to 80-90% confluent was replaced with a new culture solution (5 ml), and the above lentiviral transfection solution was added. Transfection was performed at 37 ° C. for 6 hours in a CO 2 incubator and then replaced with 10 mL of fresh culture medium. The culture supernatant was collected 48 hours after the start of transfection, 10 mL of culture solution was added, and the culture supernatant was further collected after 24 hours of culture and stored at 4 ° C.
　培養上清を、3000rpm、4℃、5分間遠心してセルデブリを沈殿させた。上清をMillex-HP 0.45μm（ポリエーテルスルホン、33mm、放射線滅菌済みフィルター、MERCK）にてろ過して細胞片を取り除き、ろ液を得た。ろ液をOptima L-100XP 超遠心分離機（BECKMAN COULTERおよびSW28スウィングロータ（BECKMAN COULTER）を用いて、19400rpm、4℃、2時間超遠心した。上清を除き、ウイルス粒子を含む沈渣を得た。DMEM（high glucose、no glutamine、no phenol red、Thermo Fisher Scientific）2mLを加えて沈渣を懸濁し、SW 55Ti スウィングロータ（BECKMAN COULTER社）を用いて、24000rpm、4℃、2時間超遠心した。上清を除き、ウイルス粒子を含む沈渣にDMEM（high glucose、no glutamine、no phenol red、Thermo Fisher Scientific）200μLを加えて懸濁し、tdTomatoを発現するレンチウイルスの濃縮液を得た。フローサイトメーターを用いて、tdTomatoを発現するレンチウイルスの力価を測定した。 The culture supernatant was centrifuged at 3000 rpm, 4 ° C. for 5 minutes to precipitate cell debris. The supernatant was filtered through Millex-HP 0.45 μm (polyethersulfone, 33 mm, radiation sterilized filter, MERCK) to remove cell debris, and a filtrate was obtained. The filtrate was ultracentrifuged for 2 hours at 19400 rpm, 4 ° C using an Optima L-100XP ultracentrifuge (BECKMAN COULTER and SW28 swing rotor (BECKMAN COULTER). The supernatant was removed and a precipitate containing virus particles was obtained. 2 mL of DMEM (high glucose, no glutamine, no phenol red, Thermo Fisher Scientific) was added to suspend the sediment, followed by ultracentrifugation at 24000 rpm, 4 ℃ for 2 hours using SW 55Ti swing rotor (BECKMANTERCOULTER). The supernatant was removed, and 200 μL of DMEM (high glucose, no glutamine, no phenol red, Thermo Fisher Scientific) was added and suspended in the sediment containing the virus particles to obtain a concentrated solution of lentivirus expressing tdTomato. Was used to measure the titer of lentivirus expressing tdTomato.
（３）移植用ヒト大腸腫瘍組織懸濁液の調製
　国立研究開発法人医薬基盤・健康・栄養研究所、泉南資源研究資源研究施設内より提供を受けたヒト大腸腫瘍組織を実験に用いた。患者から摘出した大腸腫瘍組織を、保存液（100μg/ml Kanamycin Sulfate（和光純薬工業）、0.5μg/ml Amphotericin B（Thermo Fisher Scientific）を希釈したHBSS（Thermo Fisher Scientific））で2度洗浄し、氷上の保存液に浸漬して、医療機関から発明者らの研究室に運搬した。 (3) Preparation of Human Colon Tumor Tissue Suspension for Transplantation Human colon tumor tissue provided by the National Institute of Biomedical Innovation, Health and Nutrition Research Institute, and Sennan Resources Research Resources Laboratory was used for the experiment. The colon tumor tissue removed from the patient was washed twice with a preservation solution (100 μg / ml Kanamycin Sulfate (Wako Pure Chemical Industries), 0.5 μg / ml Amphotericin B (Thermo Fisher Scientific) diluted HBSS (Thermo Fisher Scientific)). The sample was immersed in a storage solution on ice and transported from a medical institution to the inventors' laboratory.
　ヒト腫瘍組織を大塚生食注（大塚製薬工場）で4回洗浄した（1回あたり50mL）。10％FBS（Sigma）、100units/mL penicillin/100μg streptomycin（Sigma）を含むDMEM（high glucose、no glutamine、no phenol red）（Thermo Fisher Scientific）を入れた10cmディシュ内で、ヒト腫瘍組織を約1cm角の大きさに切断した。1.5 mLチューブにヒト腫瘍組織を移し、100 units/mL penicillin/100μg/mL streptomycin（Sigma）を添加したHBSS（calcium/magnesium/no phenol red）150μLを加え、ヒト腫瘍組織をハサミで細切し、200μLワイドボアフィルターバリアピペットチップ（Axygen）で吸える大きさ（約1～2mm角）にした。細切したヒト腫瘍組織と、3×107 TU/mL tdTomato発現レンチウイルス/10％FCS/1×GlutaMax/10μg/mL Polybrene（SANTA CRUZ BIOTECHNOLOGY）/100ng/mL VEGF （PEPROTECH社）/100ng/mL bFGF（PEPROTECH）/DMEM（no phenol red）（Thermo Fisher Scientific）を混合した溶液2mLをスクリューキャップチューブに加え、氷上にて2時間、130rpmで振盪した。HBSS-VF（100ng/mL VEGF、100ng/mL bFGFを含有するHBSS）でヒト腫瘍組織を洗浄した後、50mgのヒト腫瘍組織に50μLのHBSS-VFを添加してヒト腫瘍組織懸濁液を調製した。 The human tumor tissue was washed 4 times with Otsuka raw food injection (Otsuka Pharmaceutical Factory) (50 mL per time). About 1 cm of human tumor tissue in a 10 cm dish containing DMEM (high glucose, no glutamine, no phenol red) (Thermo Fisher Scientific) containing 10% FBS (Sigma), 100 units / mL penicillin / 100 μg streptomycin (Sigma) Cut into corner sizes. Transfer human tumor tissue to a 1.5 mL tube, add 150 units of HBSS (calcium / magnesium / no phenol red) with 100 units / mL penicillin / 100 μg / mL streptomycin (Sigma) added, and mince the human tumor tissue with scissors. 200μL wide bore filter barrier pipette tip (Axygen) was sized to absorb (about 1-2mm square). Minced human tumor tissue and 3 × 10 7 TU / mL tdTomato expression lentivirus / 10% FCS / 1 × GlutaMax / 10 μg / mL Polybrene (SANTA CRUZ BIOTECHNOLOGY) / 100 ng / mL VEGF (PEPROTECH) / 100 ng / mL 2 mL of a mixed solution of bFGF (PEPROTECH) / DMEM (no phenol red) (Thermo Fisher Scientific) was added to the screw cap tube and shaken at 130 rpm on ice for 2 hours. After washing human tumor tissue with HBSS-VF (HBSS containing 100ng / mL VEGF, 100ng / mL bFGF), add 50μL HBSS-VF to 50mg human tumor tissue to prepare a human tumor tissue suspension did.
（４）マウスへのヒト腫瘍組織の移植
　前日に背部除毛処理したNOD/ShiJic-scidマウスに三種混合麻酔を腹腔内投与し、後肢引き込み反射消失を確認後、除毛した背部皮膚を引き伸ばし、DSC（安久工機）のバックフレームをマウスに装着した。手前側の皮膚（表皮、真皮、皮下組織、筋肉層、結合組織）を切り取り（約7mm×7mm）、奥側の皮膚の筋肉層を露出させた。露出した筋肉層上にヒト腫瘍組織懸濁液を載置し、その上にカバーグラス付きのフロントフレームを置いて空気が入らないように固定した（図１参照）。その後、マウスがDSCを壊さないようにDSCの周囲にシールドを装着した。 (4) Transplantation of human tumor tissues into mice Three-type mixed anesthesia was intraperitoneally administered to the NOD / ShiJic-scid mice that had undergone hair removal on the previous day. The back frame of DSC (Ahiko) was attached to the mouse. The skin on the near side (epidermis, dermis, subcutaneous tissue, muscle layer, connective tissue) was cut out (about 7 mm × 7 mm) to expose the muscle layer of the back skin. A human tumor tissue suspension was placed on the exposed muscle layer, and a front frame with a cover glass was placed on the muscle layer and fixed so that air could not enter (see FIG. 1). Thereafter, a shield was attached around the DSC so that the mouse did not break the DSC.
（５）血管のイメージング
　ヒト腫瘍組織移植後27日目にAlexa Fluor 488抗ヒトCD31抗体（Biolegend）10μgをマウスに静脈内投与した。同時にマウス血管をイメージングするためにAlexa Fluor 647抗マウスCD31抗体（Biolegend）10μgをマウスに静脈内投与した。翌日、オールインワン小動物用麻酔器（室町機械）を用いて、マウスをイソフルラン麻酔（流量 1L/min、濃度1％）し、マルチフォトン顕微鏡 Leica TCS SP8 MP（Leica）で、DSC内を観察した。 (5) Blood vessel imaging Alexa Fluor 488 anti-human CD31 antibody (Biolegend) 10 μg was intravenously administered to mice on the 27th day after transplantation of human tumor tissue. At the same time, 10 μg of Alexa Fluor 647 anti-mouse CD31 antibody (Biolegend) was intravenously administered to mice in order to image mouse blood vessels. On the next day, the mice were anesthetized with isoflurane (flow rate 1 L / min, concentration 1%) using an all-in-one small animal anesthesia machine (Muromachi Kikai), and the inside of the DSC was observed with a multiphoton microscope Leica TCS SP8 MP (Leica).
＜結果＞
　結果を図２に示した。スケールバーは200μmを示す。DSC内にtdTomato陽性細胞が観察されたことから（右上）、移植したヒト腫瘍組織由来細胞の生着が示された。また、宿主マウスに静脈内投与した抗体によりヒトCD31陽性のヒト血管が観察できたことから（左上）、マウス血管とヒト血管が連結していることが確認できた。さらに、マウスCD31陽性のマウス血管の画像（左下）とヒト血管の画像（左上）とtdTomato陽性細胞の画像（右上）を重ねた合成画像（右下）では、ヒト血管とマウス血管は蛍光色の違いにより区別可能であり、ヒト血管とマウス血管が近接して存在していることが示された。 <Result>
The results are shown in FIG. The scale bar indicates 200 μm. Since tdTomato positive cells were observed in the DSC (upper right), engraftment of transplanted human tumor tissue-derived cells was shown. Further, since human CD31-positive human blood vessels could be observed with the antibody administered intravenously to the host mouse (upper left), it was confirmed that the mouse blood vessels and human blood vessels were connected. Furthermore, in the composite image (lower right) of the mouse CD31 positive mouse blood vessel image (lower left), human blood vessel image (upper left) and tdTomato positive cell image (upper right), the human blood vessels and mouse blood vessels are fluorescent. It was possible to distinguish by the difference, indicating that human blood vessels and mouse blood vessels existed in close proximity.
実施例２：tdTomato陽性細胞およびヒトCD31陽性が移植したヒト腫瘍組織由来であることの確認
＜実験方法＞
（１）ヒト腫瘍組織片移植マウスの作製
　実施例１（１）～（４）と同じ手順でヒト大腸腫瘍組織を8週齢の雌のNOD/ShiJic-scidマウスに移植した。移植後17日目に、実施例１（５）と同じ手順でAlexa Fluor 488抗ヒトCD31抗体をマウスに静脈内投与した。 Example 2: Confirmation that tdTomato positive cells and human CD31 positivity are derived from transplanted human tumor tissue <Experimental method>
(1) Preparation of human tumor tissue-grafted mouse A human colon tumor tissue was transplanted into an 8-week-old female NOD / ShiJic-scid mouse by the same procedure as in Examples 1 (1) to (4). On the 17th day after transplantation, Alexa Fluor 488 anti-human CD31 antibody was intravenously administered to mice in the same procedure as in Example 1 (5).
（２）ヒト腫瘍組織移植部位の皮膚の回収
　抗体投与の翌日、マウスをソムノペンチル（共立製薬）麻酔下、左心室よりPhosphate buffered saline （PBS）10mLを灌流した後、4％パラホルムアルデヒド-PBS（pH7.4）を10mL灌流して灌流固定した。DSCをマウスから切り離し、脂肪などの周辺組織を取り除いた皮膚を、4％パラホルムアルデヒド-PBS中で4℃1時間、振盪しながら固定した。組織を15％スクロース（和光純薬工業社）/PBS、30％スクロース/PBSの順にスクロース置換後、Surgipath FSC22包埋コンパウンド青色（Leica社）に包埋し、-80℃ディープフリーザーにて凍結した。 (2) Collection of skin at human tumor tissue transplantation site The day after antibody administration, mice were perfused with 10 mL of Phosphate buffered saline (PBS) from the left ventricle under anesthesia with somnopentyl (Kyoritsu Pharmaceutical), and then 4% paraformaldehyde-PBS (pH 7) .4) was perfused and fixed with 10 mL. The skin from which DSC was cut from the mouse and peripheral tissues such as fat were removed was fixed in 4% paraformaldehyde-PBS at 4 ° C. for 1 hour with shaking. Tissue was replaced with sucrose in the order of 15% sucrose (Wako Pure Chemical Industries) / PBS, 30% sucrose / PBS, then embedded in Surgipath FSC22 embedded compound blue (Leica), and frozen in a -80 ° C deep freezer .
（３）皮膚断面の免疫染色
　クライオスタット（Leica社）を用いて、包埋した組織から厚さ7μmの切片を作製した。0.1％TritonX-100を含むPBS（PBS-T）を用いて室温で10分間、2回洗浄することにより、包埋コンパウンドを洗い流した。ブロッキング溶液（5％ normal goat serum/1％BSA/2％ skim milk/PBS）を切片上に滴下し、湿潤箱内で室温1時間ブロッキングを行った。一次抗体にはAnti-Human Nuclei Antibody (clone 235-1) Biotin Conjugate（MERCK）およびAnti-RFP pAb（MBL）を用い、ブロッキング溶液で100倍希釈して切片上に滴下し、湿潤箱内で4℃一晩反応させた。PBS-Tで10分間の洗浄を3回行った。二次抗体には Streptavidin Alexa Fluor 647 Conjugate（Thermo Fisher Scientific）および Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 546（Thermo Fisher Scientific）を用い、ブロッキング溶液で200倍に希釈して切片上に滴下し、1.5時間室温遮光条件下にて湿潤箱内で反応させた。PBS-Tで10分間の洗浄を5回行った。ProLong Diamond Antifade Mountant（Thermo Fisher Scientific）を数滴滴下し、カバーガラスで封入した。共焦点レーザー顕微鏡 Leica TCS SP5（Leica）を用いて、作製した組織標本の観察および写真撮影を行った。 (3) Immunostaining of skin cross section Using a cryostat (Leica), a 7 μm thick section was prepared from the embedded tissue. The embedded compound was washed away by washing twice with PBS containing 0.1% TritonX-100 (PBS-T) at room temperature for 10 minutes. A blocking solution (5% normal goat serum / 1% BSA / 2% skim milk / PBS) was dropped on the section, and blocking was performed in a humid box at room temperature for 1 hour. Anti-Human Nuclei Antibody (clone 235-1) Biotin Conjugate (MERCK) and Anti-RFP pAb (MBL) were used as primary antibodies, diluted 100-fold with blocking solution, dropped onto the sections, and placed in a wet box. The reaction was allowed to proceed overnight at 0 ° C. Washing with PBS-T for 10 minutes was performed 3 times. Secondary antibodies are Streptavidin Alexa Fluor 647 Conjugate (Thermo Fisher Scientific) and Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 546 (Thermo Fisher Scientific), diluted 200-fold with blocking solution Then, the solution was dropped on the section and reacted in a wet box for 1.5 hours under light shielding conditions at room temperature. The plate was washed 5 times with PBS-T for 10 minutes. A few drops of ProLong Diamond Antifade Mountant (Thermo Fisher Scientific) were dropped and sealed with a cover glass. The prepared tissue specimen was observed and photographed using a confocal laser microscope Leica TCS SP5 (Leica).
＜結果＞
　結果を図３に示した。スケールバーは50μmを示す。上段の矢頭はMerge画像（右上）でtdTomato陽性細胞とヒト核特異的な抗体によって染色された細胞が重なった位置を示しており、矢頭で示されたTomato陽性細胞はヒト細胞であることが確認された。また、下段の矢印はMerge画像（右下）でヒトCD31陽性細胞とヒト核特異的な抗体によって染色された細胞が重なった位置を示しており、ヒトCD31陽性細胞はヒト細胞であることが確認された。この結果から、tdTomato陽性細胞およびヒトCD31陽性細胞は移植したヒト腫瘍組織由来の細胞であることが判明した。さらに、ヒト核特異的な抗体によって染色されるがヒトCD31陰性かつtdTomato陰性である細胞が、ヒト腫瘍組織内に存在することが確認された。 <Result>
The results are shown in FIG. The scale bar indicates 50 μm. The top arrow indicates the position where the tdTomato positive cells and cells stained with human nucleus-specific antibodies overlap in the Merge image (upper right), confirming that the Tomato positive cells indicated by the arrow are human cells It was done. In addition, the arrow at the bottom indicates the position where human CD31 positive cells and cells stained with human nucleus-specific antibodies overlap in the Merge image (lower right), confirming that human CD31 positive cells are human cells It was done. From these results, it was found that tdTomato positive cells and human CD31 positive cells were derived from transplanted human tumor tissue. Furthermore, it was confirmed that cells that are stained with a human nucleus-specific antibody but are negative for human CD31 and negative for tdTomato are present in human tumor tissue.
実施例３：移植したヒト腫瘍組織内のヒト血管がマウス血管と連結していることの確認
＜実験方法＞
（１）ヒト腫瘍組織片移植マウスの作製
　実施例１（１）～（４）と同じ手順でヒト大腸腫瘍組織を8週齢の雌のNOD/ShiJic-scidマウスに移植した。移植後20日目に、実施例１（５）と同じ手順でAlexa Fluor 488抗ヒトCD31抗体およびAlexa Fluor 647抗マウスCD31抗体をマウスに静脈内投与した。 Example 3: Confirmation that human blood vessel in transplanted human tumor tissue is connected to mouse blood vessel <Experimental method>
(1) Preparation of human tumor tissue-grafted mouse A human colon tumor tissue was transplanted into an 8-week-old female NOD / ShiJic-scid mouse by the same procedure as in Examples 1 (1) to (4). On the 20th day after transplantation, Alexa Fluor 488 anti-human CD31 antibody and Alexa Fluor 647 anti-mouse CD31 antibody were intravenously administered to mice in the same procedure as in Example 1 (5).
（２）ヒト腫瘍組織移植部位の皮膚の回収およびホールマウント免疫染色
　抗体投与の翌日、実施例２（２）と同じ手順でマウスを灌流固定した。DSCをマウスから切り離し、脂肪などの周辺組織を取り除いた皮膚を、4％パラホルムアルデヒド-PBS中で4℃1時間、振盪しながら固定した。PBSで10分間の洗浄を2回行った。組織を振盪しながら、PBSで希釈した25％メタノール（×2回）、50％メタノール（×2回）、75％メタノール（×2回）、100％メタノール（×2回）にこの順で、1回5分浸漬して組織の脱水処理を行った。続いて、メタノールで希釈した 50％benzylbenzoate/benzylalchol（BABB）（×2回）、100％BABB（×2回）の順に1回5分組織を浸漬して透明化処理を行った。BABBは、組成比（benzylalchol（和光純薬）およびbenzylbenzoate（和光純薬））＝1：2のものを用いた。BABBを用いてスライドガラス上にカバーガラスで組織を封入し、マルチフォトン顕微鏡Leica TCS SP8（Leica）および25倍水浸レンズ（Leica）を用いて、作製した組織標本の観察および写真撮影を行った。 (2) Collection of skin at human tumor tissue transplantation site and whole mount immunostaining On the day after antibody administration, mice were perfusion-fixed by the same procedure as in Example 2 (2). The skin from which DSC was cut from the mouse and peripheral tissues such as fat were removed was fixed in 4% paraformaldehyde-PBS at 4 ° C. for 1 hour with shaking. The plate was washed twice with PBS for 10 minutes. While shaking the tissue, in this order to 25% methanol diluted in PBS (x2), 50% methanol (x2), 75% methanol (x2), 100% methanol (x2), The tissue was dehydrated by immersing once for 5 minutes. Subsequently, the tissue was dipped for 5 minutes in the order of 50% benzylbenzoate / benzylalchol (BABB) (× 2 times) and 100% BABB (× 2 times) diluted with methanol, and a transparent treatment was performed. As the BABB, a composition ratio (benzylalchol (Wako Pure Chemical Industries) and benzylbenzoate (Wako Pure Chemical Industries)) = 1: 2 was used. The tissue was sealed with a cover glass on a slide glass using BABB, and the prepared tissue specimen was observed and photographed using a multiphoton microscope Leica TCS SP8 (Leica) and a 25 × water immersion lens (Leica). .
＜結果＞
　結果を図４に示した。スケールバーは100μmを示す。同一標本の異なる３視野を写真撮影し、それぞれ位置１、位置２、位置３とした。図４から明らかなように、ヒトCD31陽性のヒト血管内皮細胞による血管の管腔と、マウスCD31陽性のマウス血管内皮細胞による血管の管腔が連結していることが判明した。 <Result>
The results are shown in FIG. The scale bar indicates 100 μm. Three different fields of view of the same specimen were photographed as position 1, position 2, and position 3, respectively. As is apparent from FIG. 4, it was found that the blood vessel lumen formed by human CD31-positive human vascular endothelial cells and the blood vessel lumen formed by mouse CD31-positive mouse vascular endothelial cells were connected.
実施例４：移植したヒト腫瘍組織内のヒト血管増生
＜実験方法＞
　実施例１（１）～（４）と同じ手順でヒト大腸腫瘍組織を8週齢の雌のNOD/ShiJic-scidマウスに移植した。移植後13日目と20日目に、実施例１（５）と同じ手順でAlexa Fluor 488抗ヒトCD31抗体をマウスに静脈内投与した。翌日（移植後14日目と21日目）、オールインワン小動物用麻酔器（室町機械）を用いて、マウスをイソフルラン麻酔（流量 1L/min、濃度1％）し、マルチフォトン顕微鏡 Leica TCS SP8 MP（Leica）で、DSC内を観察して写真撮影を行った。撮影した写真からAngioTool血管構造解析ソフトを用いて、ヒト血管の面積、ヒト血管の分岐数、ヒト血管の長さを測定した。 Example 4: Human vascular growth in transplanted human tumor tissue <Experimental method>
Human colon tumor tissue was transplanted into 8-week-old female NOD / ShiJic-scid mice in the same procedure as in Examples 1 (1) to (4). On days 13 and 20 after transplantation, Alexa Fluor 488 anti-human CD31 antibody was intravenously administered to mice in the same procedure as in Example 1 (5). The next day (14th day and 21st day after transplantation), using an all-in-one small animal anesthesia machine (Muromachi Kikai), mice were anesthetized with isoflurane (flow rate 1 L / min, concentration 1%) and a multiphoton microscope Leica TCS SP8 MP ( Leica) observed the DSC and took a photo. The area of the human blood vessel, the number of branches of the human blood vessel, and the length of the human blood vessel were measured from the photographed photos using AngioTool blood vessel structure analysis software.
＜結果＞
　結果を図５に示した。（Ａ）は移植後14日目（d14）と21日目（d21）のヒト血管（ヒトCD31陽性）の顕微鏡画像である。スケールバーは200μmを示す。（Ｂ）は血管面積の経時変化、（Ｃ）は血管の分岐数の経時変化、（Ｄ）は血管長の経時変化を示す図である。血管面積、分岐数および血管長はいずれも、14日目から 21日目までに増加していることが判明した。つまり、このモデルにおいては、移植したヒト組織に既に存在しているヒト血管を単に維持しているのではなく、移植したヒト組織に既に存在しているヒト血管がマウス血管と連結して還流できるようになるまで、ヒト組織内のヒト血管新生が旺盛になった。 <Result>
The results are shown in FIG. (A) is a microscopic image of human blood vessels (human CD31 positive) on day 14 (d14) and day 21 (d21) after transplantation. The scale bar indicates 200 μm. (B) is a time-dependent change of the vascular area, (C) is a time-dependent change of the number of branches of the blood vessel, and (D) is a view showing a time-dependent change of the blood vessel length. It was found that the vessel area, the number of branches, and the vessel length all increased from the 14th day to the 21st day. In other words, in this model, the human blood vessels already existing in the transplanted human tissue are not simply maintained, but the human blood vessels already present in the transplanted human tissue can be connected to the mouse blood vessels and refluxed. Until that time, human angiogenesis in human tissues became active.
実施例５：移植したヒト膵がん組織内のヒト血管増生
　ヒト大腸がん組織は元来血管が豊富ながんとして知られているが、乏血管がんとして知られている膵がんの場合、本発明の製造方法を用いてヒト血管を移植片の中で増生させることは、大腸がん組織の場合より困難であると考えられた。 Example 5: Human vascular augmentation in transplanted human pancreatic cancer tissue Human colon cancer tissue is originally known as a cancer rich in blood vessels, but it is a pancreatic cancer known as oligovascular cancer. In some cases, it was considered more difficult to grow human blood vessels in a graft using the production method of the present invention than in the case of colon cancer tissue.
＜実験方法＞
（１）移植前日のマウス処置
　実施例１（１）と同じ手順で移植前日のマウス処置を行った。 <Experiment method>
(1) Mouse treatment on the day before transplantation Mouse treatment on the day before transplantation was performed in the same procedure as in Example 1 (1).
（２）間葉系幹細胞スフェロイド（細胞塊）の作製
　間葉系幹細胞（Human Mesenchymal Stem Cells、Lonza、以下「huMSC」）をMSCGM SingleQuots添加因子セット（Mesenchymal Stem Cell Growth Medium SingleQuots Supplements and Growth Factors、Lonza）を加えたMSCBM基本培地（Mesenchymal Stem Cell Growth Medium、Lonza、以下「MSC培地」）で培養した。huMSCをPBSで洗浄し、トリプシン/EDTA（Lonza）を用いて剥離した細胞を6cm細胞培養ディッシュに播種し、コンフルエントになるまで培養した。培地をFAST-DiI培養液（0μg/mL FAST-DiI（invtorgen）6μLをMSC培地3mLで希釈したもの、DiI最終濃度は2μM）に交換し、37℃のCO2インキュベーター内で、小型振とう機（ワケンビーテック）を用いて2時間振盪した。2時間後、PBSで2度洗浄した後、トリプシン/EDTA（Lonza）を用いて細胞を培養ディッシュから剥離し、回収した。2×104個/30μL MSC培地の細胞懸濁液を調製し、ハンギングドロップ法を用いてスフェロイドを作製した。3日後にスフェロイドを回収した。 (2) Preparation of mesenchymal stem cell spheroids (cell mass) Mesenchymal stem cells (Human Mesenchymal Stem Cells, Lonza, hereinafter referred to as “huMSC”) are added to MSCGM SingleQuots additive factor set (Mesenchymal Stem Cell Growth Medium SingleQuots Supplements and Growth Factors, Lonza ) Was added to MSCBM basic medium (Mesenchymal Stem Cell Growth Medium, Lonza, hereinafter referred to as “MSC medium”). The huMSC was washed with PBS, and the detached cells were seeded on a 6 cm cell culture dish using trypsin / EDTA (Lonza) and cultured until confluent. Replace the medium with FAST-DiI medium (6 μL of 0 μg / mL FAST-DiI (invtorgen) diluted with 3 mL of MSC medium, the final concentration of DiI is 2 μM), and use a small shaker in a CO 2 incubator at 37 ° C. (Wakenbeetec) and shaken for 2 hours. Two hours later, after washing twice with PBS, the cells were detached from the culture dish using trypsin / EDTA (Lonza) and collected. A cell suspension of 2 × 10 4 cells / 30 μL MSC medium was prepared, and spheroids were prepared using the hanging drop method. Spheroids were collected after 3 days.
（３）移植用ヒト膵がん組織懸濁液の調製
　国立研究開発法人医薬基盤・健康・栄養研究所、泉南資源研究資源研究施設内より提供を受けたヒト膵がん組織を実験に用いた。患者から摘出した膵がん組織を、保存液（100μg/ml Kanamycin Sulfate（和光純薬工業）、0.5μg/ml Amphotericin B（Thermo Fisher Scientific）を希釈したHBSS（Thermo Fisher Scientific））で2度洗浄し、氷上の保存液に浸漬して、医療機関から発明者らの研究室に運搬した。 (3) Preparation of human pancreatic cancer tissue suspension for transplantation Human pancreatic cancer tissue provided by National Institute of Health Sciences, National Institute of Biomedical Innovation, Health and Nutrition, and Sennan Resources Research Resource Laboratory was used for the experiment. . Pancreatic cancer tissue removed from the patient was washed twice with preservation solution (100μg / ml Kanamycin Sulfate (Wako Pure Chemical Industries), 0.5μg / ml Amphotericin B (Thermo Fisher Scientific) diluted HBSS (Thermo Fisher Scientific)) The sample was immersed in a preservation solution on ice and transported from a medical institution to the inventors' laboratory.
　ヒト膵がん組織を大塚生食注（大塚製薬工場）で4回洗浄した（1回あたり50mL）。10％FBS（Sigma）、100units/mL penicillin/100μg streptomycin（Sigma）を含むDMEM（high glucose、no glutamine、no phenol red）（Thermo Fisher Scientific）を入れた10cmディシュ内で、ヒト膵がん組織を約1cm角の大きさに切断した。1.5 mLチューブにヒト膵がん組織を移し、100 units/mL penicillin/100μg/mL streptomycin（Sigma）を添加したHBSS（calcium/magnesium/no phenol red、以下「HBSS-p/s」）150μLを加え、ヒト膵がん組織をハサミで細切し、200μLワイドボアフィルターバリアピペットチップ（Axygen）で吸える大きさ（約1～2mm角）にした。HBSS-p/sでヒト膵がん組織を洗浄した後、huMSCスフェロイド10個とヒト膵がん組織30mgを50μLのHBSS-p/sに懸濁し、huMSCスフェロイドとヒト膵がん組織との混合懸濁液を調製した。 The human pancreatic cancer tissue was washed 4 times with Otsuka raw food injection (Otsuka Pharmaceutical Factory) (50 mL per time). Human pancreatic cancer tissue in a 10cm dish containing DMEM (high （glucose, no glutamine, no phenol red) (Thermo Fisher Scientific) containing 10% FBS (Sigma), 100units / mL penicillin / 100μg streptomycin (Sigma) It was cut into a size of about 1 cm square. Transfer human pancreatic cancer tissue to a 1.5 mL tube and add 150150L of HBSS (calcium / magnesium / no phenol red, hereinafter referred to as "HBSS-p / s") with 100 添加 units / mL penicillin / 100μg / mL streptomycin (Sigma) The human pancreatic cancer tissue was minced with scissors and sized to be sucked with a 200 μL wide bore filter barrier pipette tip (Axygen) (about 1-2 mm square). After washing human pancreatic cancer tissue with HBSS-p / s, 10 huMSC spheroids and 30 mg human pancreatic cancer tissue are suspended in 50 μL of HBSS-p / s and mixed with huMSC spheroids and human pancreatic cancer tissue A suspension was prepared.
（４）マウスへのヒト膵がん組織の移植
　上記（３）で調製したhuMSCスフェロイドとヒト膵がん組織との混合懸濁液を用いて、実施例１（４）と同じ手順でヒト膵がん組織をマウスに移植した。 (4) Transplantation of human pancreatic cancer tissue to mouse Using the mixed suspension of huMSC spheroid and human pancreatic cancer tissue prepared in (3) above, human pancreatic cancer was transplanted in the same procedure as in Example 1 (4). Cancer tissue was transplanted into mice.
（５）血管のイメージング
　huMSCスフェロイドとヒト膵がん組織を移植後5日目に、Alexa Fluor 488抗ヒトCD31抗体（Biolegend）10μgをマウスに静脈内投与した。移植後6日目と12日目に、オールインワン小動物用麻酔器（室町機械）を用いて、マウスをイソフルラン麻酔（流量 1L/min、濃度1％）し、マルチフォトン顕微鏡 Leica TCS SP8 MP（Leica）で、DSC内を観察した。 (5) Blood vessel imaging Alexa Fluor 488 anti-human CD31 antibody (Biolegend) 10 μg was intravenously administered to mice 5 days after transplantation of huMSC spheroids and human pancreatic cancer tissue. On the 6th and 12th day after transplantation, the mice were anesthetized with isoflurane (flow rate 1L / min, concentration 1%) using an all-in-one small animal anesthesia machine (Muromachi Kikai), and the multiphoton microscope Leica TCS SP8 MP (Leica) Then, the inside of the DSC was observed.
＜結果＞
　結果を図６に示した。スケールバーは250μmを示し、huMSCスフェロイドを点線で囲った。移植後6日目から12日目にかけて、間葉系幹細胞の周囲にヒト腫瘍血管の伸長が見られた。この結果から、ヒト間葉系幹細胞が膵がん患者由来腫瘍血管の伸長を促進していることが判明した。 <Result>
The results are shown in FIG. The scale bar indicated 250 μm and the huMSC spheroids were surrounded by a dotted line. From the 6th day to the 12th day after transplantation, the growth of human tumor blood vessels was seen around the mesenchymal stem cells. From these results, it was found that human mesenchymal stem cells promote the growth of tumor blood vessels derived from pancreatic cancer patients.
　以上のことから、血管が元来豊富ながん患者のがん組織を用いても、血管の少ないがん組織を用いても、がん組織の血管を誘導することができることが判明し、本発明の製造方法を使用すれば、がん種によらずヒト血管を製造できることが示された。また、間葉系幹細胞を同時に移植することで、血管形成が促進されたことから、血管形成を促進する薬剤や細胞を添加したヒト組織を移植して、ヒト血管に対する作用を観察することが可能であることも証明された。 From the above, it has been found that blood vessels in cancer tissues can be induced using cancer tissues of cancer patients with abundant blood vessels or cancer tissues with few blood vessels. It was shown that human blood vessels can be produced regardless of the type of cancer if the production method of the invention is used. In addition, since angiogenesis was promoted by transplanting mesenchymal stem cells at the same time, it is possible to transplant human tissues to which drugs or cells that promote angiogenesis are added and observe the effects on human blood vessels It was also proved that.
実施例６：移植したヒト正常組織内のヒト血管増生
＜実験方法＞
（１）移植前日のマウス処置
　実施例１（１）と同じ手順で移植前日のマウス処置を行った。 Example 6: Human vascular growth in transplanted human normal tissue <Experimental method>
(1) Mouse treatment on the day before transplantation Mouse treatment on the day before transplantation was performed in the same procedure as in Example 1 (1).
（２）移植用ヒト正常組織懸濁液の調製
　国立研究開発法人医薬基盤・健康・栄養研究所、泉南資源研究資源研究施設内より提供を受けたヒト大腸正常組織を実験に用いた。患者から摘出した大腸正常組織を、保存液（100μg/ml Kanamycin Sulfate（和光純薬工業）、0.5μg/ml Amphotericin B（Thermo Fisher Scientific）を希釈したHBSS（Thermo Fisher Scientific））で2度洗浄し、氷上の保存液に浸漬して、医療機関から発明者らの研究室に運搬した。 (2) Preparation of normal human tissue suspension for transplantation Normal human large intestine tissue provided by the National Institute of Biomedical Innovation, Health and Nutrition Research Institute, and Sennan Resources Research Resource Research Facility was used for experiments. Normal colon tissue removed from the patient was washed twice with preservation solution (100 μg / ml Kanamycin Sulfate (Wako Pure Chemical Industries), 0.5 μg / ml Amphotericin B (Thermo Fisher Scientific) diluted HBSS (Thermo Fisher Scientific)) The sample was immersed in a storage solution on ice and transported from a medical institution to the inventors' laboratory.
　ヒト大腸正常組織を大塚生食注（大塚製薬工場）で4回洗浄した（1回あたり50mL）。10％FBS（Sigma）、100units/mL penicillin/100μg streptomycin（Sigma）を含むDMEM（high glucose、no glutamine、no phenol red）（Thermo Fisher Scientific）を入れた10cmディシュ内で、ヒト大腸正常組織を約1cm角の大きさに切断した。1.5 mLチューブにヒト大腸正常組織を移し、100 units/mL penicillin/100μg/mL streptomycin（Sigma）を添加したHBSS-p/s 150μLを加え、ヒト大腸正常組織をハサミで細切し、200μLワイドボアフィルターバリアピペットチップ（Axygen）で吸える大きさ（約1～2mm角）にした。HBSS-p/s-VF（100ng/mL VEGF、100ng/mL bFGFを含有するHBSS-p/s）でヒト大腸正常組織を洗浄した後、40mgのヒト大腸正常組織に50μLのHBSS-p/s-VFを添加してヒト大腸正常組織懸濁液を調製した。 The normal human large intestine tissue was washed 4 times with Otsuka raw food injection (Otsuka Pharmaceutical Factory) (50 mL per time). Approximately 10% FBS (Sigma), 100units / mL penicillin / 100μg streptomycin (Sigma) in DMEM (high glucose, no glutamine, no phenol red) (Thermo Fisher Scientific) in a 10cm dish Cut into 1 cm square size. Transfer normal human colon tissue to a 1.5-mL tube, add HBSS-p / s-150 μL with 100 μunits / mL-penicillin / 100 μg / mL-streptomycin (Sigma), chop the normal human colon tissue with scissors, and 200 μL wide bore The filter barrier pipette tip (Axygen) can be sucked (about 1-2 mm square). After washing human normal colon tissue with HBSS-p / s-VF (HBSS-p / s containing 100ng / mL VEGF, 100ng / mL bFGF), 50μL HBSS-p / s was added to 40mg human colon normal tissue. -VF was added to prepare a human large intestine normal tissue suspension.
（３）マウスへのヒト大腸正常組織の移植
　上記（２）で調製したヒト大腸正常組織懸濁液を用いて、実施例１（４）と同じ手順でヒト大腸正常組織をマウスに移植した。 (3) Transplantation of normal human large intestine tissue into mice Using the human large intestine normal tissue suspension prepared in (2) above, normal human large intestine tissue was transplanted into mice in the same procedure as in Example 1 (4).
（４）血管のイメージング
　ヒト大腸正常組織移植後19日目に、Alexa Fluor 488抗ヒトCD31抗体（Biolegend）10μgをマウスに静脈内投与した。翌日、オールインワン小動物用麻酔器（室町機械）を用いて、マウスをイソフルラン麻酔（流量 1L/min、濃度1％）し、マルチフォトン顕微鏡 Leica TCS SP8 MP（Leica）で、DSC内を観察した。 (4) Blood vessel imaging Alexa Fluor 488 anti-human CD31 antibody (Biolegend) 10 μg was intravenously administered to mice 19 days after transplantation of normal human large intestine tissue. On the next day, the mice were anesthetized with isoflurane (flow rate 1 L / min, concentration 1%) using an all-in-one small animal anesthesia machine (Muromachi Kikai), and the inside of the DSC was observed with a multiphoton microscope Leica TCS SP8 MP (Leica).
＜結果＞
　結果を図７に示した。スケールバーは500μmを示す。宿主マウスに静脈内投与した抗体によりヒトCD31陽性のヒト血管が観察できた。つまり、マウスの血管とヒトの正常組織の血管が連結して、マウスの静脈からの血流を介してヒトの血管が染色されていることが確認された。この結果から、本発明の製造方法を使用すれば、がん組織のみならず、正常組織のヒト血管を製造できることが示された。 <Result>
The results are shown in FIG. The scale bar indicates 500 μm. Human CD31-positive human blood vessels could be observed with the antibody administered intravenously to the host mouse. In other words, it was confirmed that the blood vessels of the mouse were connected to the blood vessels of normal human tissue, and the human blood vessels were stained via the blood flow from the veins of the mouse. From this result, it was shown that the use of the production method of the present invention can produce not only cancer tissues but also human blood vessels of normal tissues.
　なお本発明は上述した各実施形態および実施例に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考として援用される。 The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

Claims (9)

  1. 　ヒト血管の形成を促進もしくは抑制する物質、または、ヒト血管の構造もしくは機能を正常化する物質をスクリーニングする方法であって、以下の工程（１）～（７）を含むスクリーニング方法：
    （１）非ヒト免疫不全動物の皮膚の一部を切開し皮下組織または筋肉層を露出させる工程、
    （２）露出した皮下組織または筋肉層にヒト組織を載置する工程、
    （３）載置したヒト組織が空気と接触しないように、載置したヒト組織を封じる工程、
    （４）ヒト組織を非ヒト免疫不全動物に生着させる工程、
    （５）工程（１）～（４）により得られた、ヒト組織が生着した非ヒト免疫不全動物に被験物質を投与する工程、
    （６）生着したヒト組織内のヒト血管の形状もしくは構造を観察、および／または、ヒト血管の機能を評価する工程、および
    （７）被験物質を投与していない前記ヒト組織が生着した非ヒト免疫不全動物のヒト血管と比較して、ヒト血管の形成を促進もしくは抑制する被験物質、または、ヒト血管の構造もしくは機能を正常化する被験物質を選択する工程。     A screening method for a substance that promotes or suppresses the formation of human blood vessels, or a substance that normalizes the structure or function of human blood vessels, comprising the following steps (1) to (7):
    (1) a step of incising a part of the skin of a non-human immunodeficient animal to expose a subcutaneous tissue or a muscle layer;
    (2) placing a human tissue on the exposed subcutaneous tissue or muscle layer;
    (3) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air;
    (4) engrafting human tissue in a non-human immunodeficient animal;
    (5) A step of administering a test substance to a non-human immunodeficient animal engrafted with human tissue obtained by steps (1) to (4),
    (6) observing the shape or structure of a human blood vessel in the engrafted human tissue and / or evaluating the function of the human blood vessel, and (7) engrafting the human tissue not administered with the test substance. A step of selecting a test substance that promotes or suppresses the formation of human blood vessels, or a test substance that normalizes the structure or function of human blood vessels, compared to human blood vessels of non-human immunodeficient animals.
  2. 　ヒト血管の形成を促進もしくは抑制する物質、または、ヒト血管の構造もしくは機能を正常化する物質をスクリーニングする方法であって、以下の工程（Ｉ）～（ＶＩ）を含むスクリーニング方法：
    （Ｉ）非ヒト免疫不全動物の皮膚の一部を切開し皮下組織または筋肉層を露出させる工程、
    （ＩＩ）露出した皮下組織または筋肉層に、ヒト組織と被験物質の混合物を載置する工程、
    （ＩＩＩ）載置したヒト組織が空気と接触しないように、載置したヒト組織を封じる工程、
    （ＩＶ）ヒト組織を非ヒト免疫不全動物に生着させる工程、
    （Ｖ）生着したヒト組織内のヒト血管の形状もしくは構造を観察、および／または、ヒト血管の機能を評価する工程、および
    （ＶＩ）被験物質と接触していない前記ヒト組織が生着した非ヒト免疫不全動物のヒト血管と比較して、ヒト血管の形成を促進もしくは抑制する被験物質、または、ヒト血管の構造もしくは機能を正常化する被験物質を選択する工程。     A method of screening for a substance that promotes or suppresses the formation of human blood vessels, or a substance that normalizes the structure or function of human blood vessels, comprising the following steps (I) to (VI):
    (I) a step of incising a part of the skin of a non-human immunodeficient animal to expose the subcutaneous tissue or muscle layer,
    (II) placing a mixture of human tissue and a test substance on the exposed subcutaneous tissue or muscle layer,
    (III) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air;
    (IV) engrafting human tissue in a non-human immunodeficient animal;
    (V) observing the shape or structure of a human blood vessel in the engrafted human tissue and / or evaluating the function of the human blood vessel, and (VI) engrafting the human tissue not in contact with the test substance A step of selecting a test substance that promotes or suppresses the formation of human blood vessels, or a test substance that normalizes the structure or function of human blood vessels, compared to human blood vessels of non-human immunodeficient animals.
  3. 　前記工程（２）または（ＩＩ）において、ヒト組織が血管形成を促進する薬剤または細胞を添加したヒト組織である請求項１または２に記載のスクリーニング方法。 The screening method according to claim 1 or 2, wherein in the step (2) or (II), the human tissue is a human tissue to which a drug or cell that promotes angiogenesis is added.
  4. 　非ヒト免疫不全動物に生着したヒト組織内のヒト血管が宿主動物の血管と連結している、請求項１～３のいずれかに記載のスクリーニング方法。 The screening method according to any one of claims 1 to 3, wherein the human blood vessel in the human tissue engrafted in the non-human immunodeficient animal is connected to the blood vessel of the host animal.
  5. 　前記工程（３）は、ドーサルスキンフォールドチャンバーを用いて載置したヒト組織を封じることを含む、請求項１～４のいずれかに記載のスクリーニング方法。 The screening method according to any one of claims 1 to 4, wherein the step (3) includes sealing a human tissue placed using a dorsal skin fold chamber.
  6. 　非ヒト免疫不全動物を用いるヒト血管の製造方法であって、以下の工程（Ａ）～（Ｅ）を含む製造方法：
    （Ａ）非ヒト免疫不全動物の皮膚の一部を切開し皮下組織または筋肉層を露出させる工程、
    （Ｂ）露出した皮下組織または筋肉層にヒト組織を載置する工程、
    （Ｃ）載置したヒト組織が空気と接触しないように、載置したヒト組織を封じる工程、
    （Ｄ）ヒト組織を非ヒト免疫不全動物に生着させ、ヒト血管を増生させる工程、および
    （Ｅ）生着したヒト組織を採取する工程。     A method for producing a human blood vessel using a non-human immunodeficient animal, comprising the following steps (A) to (E):
    (A) incising a part of the skin of a non-human immunodeficient animal to expose the subcutaneous tissue or muscle layer,
    (B) placing human tissue on exposed subcutaneous tissue or muscle layer;
    (C) a step of sealing the placed human tissue so that the placed human tissue does not come into contact with air;
    (D) engrafting human tissue in a non-human immunodeficient animal to proliferate human blood vessels, and (E) collecting engrafted human tissue.
  7. 　前記工程（Ｂ）において、ヒト組織が血管形成を促進する薬剤または細胞を添加したヒト組織である請求項６に記載の製造方法。 The manufacturing method according to claim 6, wherein in the step (B), the human tissue is a human tissue to which a drug or cell that promotes angiogenesis is added.
  8. 　前記工程（Ｃ）は、ドーサルスキンフォールドチャンバーを用いて載置したヒト組織を封じることを含む、請求項６または７に記載の製造方法。 The method according to claim 6 or 7, wherein the step (C) includes sealing the human tissue placed using a dorsal skin fold chamber.
  9. 　移植用ヒト血管の製造方法である請求項６～８のいずれかに記載の製造方法。 The production method according to any one of claims 6 to 8, which is a method for producing a human blood vessel for transplantation.
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