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WO2018161417A1 - 一种利用干细胞构建人源化慢性乙型肝炎鼠模型的方法 - Google Patents

一种利用干细胞构建人源化慢性乙型肝炎鼠模型的方法 Download PDF

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WO2018161417A1
WO2018161417A1 PCT/CN2017/081750 CN2017081750W WO2018161417A1 WO 2018161417 A1 WO2018161417 A1 WO 2018161417A1 CN 2017081750 W CN2017081750 W CN 2017081750W WO 2018161417 A1 WO2018161417 A1 WO 2018161417A1
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stem cells
constructing
mouse model
liver
humanized
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李君�
李江
孙苏婉
李兰娟
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Zhejiang University ZJU
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2207/00—Modified animals
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2207/00—Modified animals
    • A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
    • A—HUMAN NECESSITIES
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    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases

Definitions

  • the invention belongs to the fields of clinical medicine, experimental medicine, regenerative medicine and virology, and in particular, is a new method for constructing a humanized mouse model and for researching human diseases such as viral hepatitis.
  • Hepatotropic viruses have a wide range of epidemics, and millions of people die each year from liver failure caused by viral hepatitis, liver cirrhosis and hepatocellular carcinoma. Hepatotropic viruses can only cause disease in high-grade primates. Animal experimental models often require the use of simian animals. The simians have high cost, complicated operation and long experimental period. Therefore, it is possible to establish a virus that can infect hepatovirus and cause disease. Small animal models have great scientific significance and application value.
  • Stem cells including human embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells, etc.
  • functional cells such as bone marrow mesenchymal stem cells.
  • hBMSCs human bone marrow mesenchymal stem cells
  • the present invention is directed to the defects of the existing hepatovirus research model, and provides a technique for constructing a humanized mouse model using human stem cells.
  • the present invention is achieved by the following technical solutions:
  • the invention discloses a method for constructing a humanized mouse model by using stem cells, and the steps are as follows:
  • the stem cells of the present invention are isolated cultured human stem cells, or commercially available isolated or cryopreserved human stem cells or cell lines.
  • the steps of obtaining the isolated cultured human stem cells of the present invention are as follows:
  • the step g of the present invention further comprises the step h: fractionally administering the liver damage drug.
  • the experimental mouse of the present invention is a normal mouse or an immunodeficient mouse or a normal rat or an immunodeficient rat
  • the liver damage includes acute, chronic liver injury, acute, subacute, chronic liver failure. Any one.
  • the liver damage drug is administered by intraperitoneal, intramuscular, peripheral intravenous injection, oral or intragastric administration.
  • the step g of the present invention is to transplant 1 x 10 4-8 human stem cells by injection into the peripheral vein, portal vein, spleen or liver.
  • step 3) of the present invention is to inject each type of hepadnavirus into each mouse by peripheral vein, subcutaneous, intramuscular or abdominal cavity.
  • the step 3) of the present invention further comprises 4) detecting the viral load once within 3-30 days after the infection of the mouse, confirming that the model is successfully established; or detecting the virus within 3-30 days after the infection of the mouse After the load was once, the viral load was measured in stages to confirm that the model was successfully established.
  • the present invention studies various aspects such as biochemical indicators, immunohistochemistry, gene expression levels, and proteomics.
  • human liver cells derived from rat liver have a high chimeric rate of 50-95%, and human immune cells can be stably separated into organs such as spleen, blood, liver and bone marrow to form humanized liver.
  • a mouse model of immune cells Various types of hepadnaviruses were infected with the above-mentioned humanized mice to form a humanized mouse model of hepadnavirus infection.
  • the model studies the entire life cycle of the hepadnavirus and the immune response between the hepadnavirus infection and the humanized immune system formed by transdifferentiation. From 10 weeks to 50 weeks after humanized mice were infected with hepadnavirus, liver injury, chronic hepatitis B, liver fibrosis and cirrhosis occurred successively, and hepatocellular carcinoma gradually appeared. It is in line with the natural history of human infection with hepadnavirus and the development of viral hepatitis.
  • liver cirrhosis and hepatocellular carcinoma that are more in line with the history of human disease development.
  • the idea of the technical scheme can also be used to construct models of other humanized organs.
  • the technical solution provides a convenient, simple and easy to obtain humanized model for clinical treatment of liver diseases.
  • Figure 1 is a schematic diagram showing the construction of a bone marrow mesenchymal stem cell humanized FRGS mouse (hBMSC-FRGS mouse) model;
  • FIG. 2 is a schematic diagram showing the construction of an embryonic stem cell humanized uPAS mouse (ES-uPAS mouse) model
  • Figure 3 is a schematic diagram showing the construction of a pluripotent stem cell humanized galactosamine normal mouse (iPS-normal mouse) model
  • Figure 4 is a diagram showing the construction of a normal rat model of bone marrow mesenchymal stem cells
  • Figure 5 is a diagram showing the construction of a normal rat model of adipose-derived mesenchymal stem cells.
  • the invention discloses a method for constructing a humanized mouse model by using human stem cells and discloses a humanized mouse model for constructing research on viral hepatitis by using the method.
  • the technical scheme of the present invention is further described below:
  • mice included normal mice, immunodeficient mice, normal rats and immunodeficient rats.
  • liver damage drugs through the abdominal cavity, muscle, peripheral intravenous injection, oral or intragastric administration of liver damage drugs, or surgical partial hepatectomy, establish a rat model of liver injury.
  • Each type of hepadnavirus is injected into each mouse by peripheral vein, subcutaneous, intramuscular or abdominal cavity.
  • the technical scheme of the present invention is further illustrated by the specific examples and comparative examples according to the accompanying drawings.
  • the present invention is to study hepatotropic viruses by injecting different humanized mouse models into a rat model of liver injury by using different human stem cells. Infection mechanism and the occurrence, development mechanism, outcome and treatment of hepadnavirus infection.
  • FIG. 1 is a schematic diagram of the construction of humanized FRGS mouse model of bone marrow mesenchymal stem cells.
  • Human bone marrow mesenchymal stem cells hBMSCs
  • FRGS mice established humanized FRGS of bone marrow mesenchymal stem cells.
  • Mouse model Mouse model.
  • hBMSCs Human bone marrow mesenchymal stem cells
  • FRGS mice gradually reduced the drug 2-(2-nitro-4-trifluoromethylbenzyl)-cyclohexane-1,3-dione (NTBC) and injected 0.2mg/kg anti-Fas antibody. (JO2), a mouse model of liver failure was established.
  • Each mouse in the tail vein was injected with A, B, C, and D type 1*10 6 hepatitis B virus.
  • hepatitis B virus load and liver function status were detected every 1 week, 2 weeks, and 4 weeks after the injection of hepatitis B virus, and the model was established successfully.
  • Figure 1 Using FRGS mice, first use the chemical drug NTBC to establish fulminant hepatic failure, and then use human Bone marrow mesenchymal stem cells were transplanted, differentiated into hepatocytes, and finally injected with hepatitis B virus to construct a mouse model of humanized chronic hepatitis B.
  • FIG. 2 is a schematic diagram of the construction of a humanized uPA mouse model of embryonic stem cells.
  • the human embryonic stem cell line was transplanted into a homozygous uPA mouse to establish a humanized uPA mouse model of embryonic stem cells.
  • Human embryonic stem cells were cultured in DMEM medium containing 10% fetal bovine serum.
  • the viral load and liver function status were detected every 1 week, 2 weeks, and 4 weeks after the injection of hepatitis C virus, and the model was established successfully.
  • FIG. 2 Using uPA mice, uPA spontaneously forms liver damage, then uses human embryonic stem cell transplantation to differentiate into hepatocytes, and finally injects hepatitis C virus to construct a humanized chronic hepatitis C mouse model.
  • FIG. 3 is a schematic diagram showing the construction of a normal mouse model of humanized galactosamine induced by pluripotent stem cells.
  • Human induced pluripotent stem cells hiPSCs
  • hiPSCs Human induced pluripotent stem cells
  • transcription factors are introduced into animal or human somatic cells by gene transfection technology, so that the somatic cells directly reconstitute the pluripotent stem cells, and cultured in DMEM medium containing 10% fetal bovine serum.
  • mice were intraperitoneally injected with galactosamine 1.5g/kg to establish a mouse model of liver failure.
  • Figure 3 Using mice, first use the chemical drug galactosamine to establish fulminant hepatic failure, then use induced pluripotent stem cell transplantation to differentiate into hepatocytes, and finally inject hepatitis E virus to construct humanized chronic hepatitis E mice. model.
  • FIG. 4 is a schematic diagram showing the construction of a normal rat model of bone marrow mesenchymal stem cells.
  • Human bone marrow mesenchymal stem cells hBMSCs
  • hBMSCs Human bone marrow mesenchymal stem cells
  • Human bone marrow mononuclear cells were isolated and purified from normal human bone marrow by lymphocyte separation solution, and cultured in DMEM medium containing 10% fetal bovine serum to obtain human bone marrow mesenchymal stem cells hBMSCs.
  • A, B, C, and D type 1*10 6 hepatitis B virus were injected into each mouse through the tail vein.
  • Figure 4 uses normal mice, first to establish acute liver injury by surgical 50% liver resection, then transplanted with human bone marrow mesenchymal stem cells, differentiated into hepatocytes, and finally injected hepatitis B virus to construct a rat model of humanized chronic hepatitis B. .
  • FIG. 5 is a schematic diagram showing the construction of a humanized normal rat model of human adipose-derived mesenchymal stem cells.
  • Human adipose-derived mesenchymal stem cells hADSCs
  • hADSCs Human adipose-derived mesenchymal stem cells
  • Human adipose-derived mesenchymal stem cells were isolated and purified from normal human adipose tissue and cultured in DMEM medium containing 10% fetal bovine serum to obtain human adipose-derived mesenchymal stem cells (hBMSCs).
  • Rats were established by intraperitoneal injection of carbon tetrachloride 0.5ml/100g to establish a rat model of acute liver injury.
  • Each mouse was intraperitoneally injected with 1*10 6 hepatitis C virus.
  • the viral load and liver function status were detected every 1 week, 2 weeks, and 4 weeks after the injection of hepatitis C virus, and the model was established successfully.
  • Figure 5 Using normal mice, first use the chemical drug carbon tetrachloride to establish acute liver injury, then use human adipose-derived mesenchymal stem cells to differentiate, form hepatocytes, and finally inject hepatitis C virus to construct humanized chronic hepatitis C rats. model.

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Abstract

提供了一种利用干细胞构建人源化慢性乙型肝炎鼠模型的方法，该方法包括获取人干细胞，将干细胞移植入肝损伤的鼠，HBV感染人源化鼠的步骤。

Description

一种利用干细胞构建人源化慢性乙型肝炎鼠模型的方法 技术领域
本发明属于临床医学、实验医学、再生医学与病毒学领域，具体地说，是一种构建人源化鼠模型并用于病毒性肝炎等人类疾病研究的新方法。
背景技术
嗜肝病毒(甲型肝炎病毒、乙型肝炎病毒、丙型肝炎病毒等)，流行范围广，每年上百万人死于病毒性肝炎所致肝衰竭、肝硬化和肝细胞癌。嗜肝病毒只能在高级灵长类动物中致病，动物实验模型常需使用猿猴类动物，猿猴类实验成本高，操作复杂，实验周期长，因此，建立能感染嗜肝病毒并致病的小动物模型有很大的科学意义和应用价值。但由于小动物无法感染嗜肝病毒，因此嗜肝病毒小动物研究模型建立困难，严重阻碍了病毒性肝炎机制、进展、转归研究，并很大程度上限制了治疗方案的优化。建立人源化鼠模型将为研究病毒性肝炎及其治疗与疾病转归提供良好的研究载体。干细胞(包括人胚胎干细胞、间充质干细胞、诱导多功能干细胞等)具有潜在分化能力，可以分化成多种功能细胞，如骨髓间充质干细胞。我们之前的研究发现人骨髓间充质干细胞(hBMSC)能嵌合暴发性肝功能衰竭猪，这为我们将干细胞移植到肝损伤的鼠肝内，建立新型人源化鼠模型奠定了基础。此人源化鼠对阐明嗜肝性病毒生物学特性、肝炎发病机制、疾病转归、研发和筛选抗嗜肝性病毒新药等提供保障。
发明内容
本发明正是针对现有嗜肝性病毒研究模型的缺陷，提供了一种利用人干细胞构建人源化鼠模型的技术，本发明是通过以下技术方案来实现的：
本发明公开了一种利用干细胞构建人源化鼠模型的方法，步骤如下：
1)、获取人干细胞；
2)、将干细胞移植入肝损伤的鼠；
3)、HBV感染人源化鼠。
作为进一步地改进，本发明所述的干细胞是分离培养的人干细胞，或是商品化的分离或冻存的人干细胞或细胞系。
作为进一步地改进，本发明所述的分离培养的人干细胞的获取步骤如下：
a、获得纯化人干细胞；
b、干细胞培养，传代；
c、培养于20℃-40℃，2％-10％CO2的培养箱中。
作为进一步地改进，本发明所述的步骤2)的具体步骤如下：
e、获得不同品系的实验鼠；
f、通过予以肝损药物，或予以外科部分肝切除术，建立肝损伤鼠模型；
g、移植1×104-8人干细胞进入鼠模型。
作为进一步地改进，本发明所述的步骤g后还包括步骤h：分次予以肝损药物。
作为进一步地改进，本发明所述的实验鼠是正常小鼠或免疫缺陷小鼠或正常大鼠或免疫缺陷大鼠，肝损伤包括急性、慢性肝损伤，急性、亚急性、慢性肝衰竭中的任意一种。
作为进一步地改进，本发明所述的步骤f中是予以肝损药物是通过腹腔、肌肉、外周静脉注射、口服或灌胃的方式。
作为进一步地改进，本发明所述的步骤g是通过外周静脉、门静脉、脾脏或肝脏注射的方式移植1×104-8人干细胞。
作为进一步地改进，本发明所述的步骤3)是通过外周静脉、皮下、肌肉或腹腔对每只鼠注射各型嗜肝性病毒。
作为进一步地改进，本发明所述的步骤3)后还包括4)在感染鼠后的3-30天内检测病毒载量一次，确认模型建立成功；或在感染鼠后的3-30天内检测病毒载量一次后，再分次检测病毒载量，确认模型建立成功。
与现有构建人源化鼠模型技术相比，本发明的有益效果如下：
为阐明嗜肝病毒生物学特性、病毒性发病的具体机制，本发明从生化指标、免疫组织化学，基因表达水平，蛋白组学等多个方面进行研究。发现通过诱导严重肝损 害并移植人干细胞，鼠肝内人来源肝细胞有50-95％的高嵌合率，鼠脾脏、血液、肝脏、骨髓等器官内可持续分离到人源性免疫细胞，形成人源化肝脏和免疫细胞的鼠模型。再将各类嗜肝病毒感染上述人源化鼠，形成嗜肝病毒感染的人源化鼠模型。该模型能研究嗜肝病毒的整个生命周期，及嗜肝病毒感染后与转分化形成的人源化免疫系统之间的免疫应答反应。在人源化鼠感染嗜肝病毒后10周至50周，先后出现肝损伤、慢性乙型肝炎、肝纤维化和肝硬化，最后逐渐出现肝细胞癌。符合人感染嗜肝病毒的自然史，及病毒性肝炎的发展转归。除能用于研究嗜肝病毒的生物学特性、病毒性肝炎发病机制、研发和筛选抗嗜肝病毒新药之外，还能获得更符合人类疾病发展史的肝硬化、肝细胞癌等模型。
该构建人源化鼠模型技术除了用于构建研究嗜肝病毒感染的模型外，该技术方案的思路还可用于构建其他人源化器官的模型。该技术方案为临床上肝疾病治疗研究提供了方便、简单、易得的人源化模型。
附图说明
图1为骨髓间充质干细胞人源化FRGS小鼠(hBMSC-FRGS小鼠)模型构建示意图；
图2为胚胎干细胞人源化uPAS小鼠(ES-uPAS小鼠)模型构建示意图；
图3为诱导多能干细胞人源化半乳糖胺正常小鼠(iPS-正常小鼠)模型构建示意图；
图4为骨髓间充质干细胞人源化正常大鼠模型构建图；
图5为脂肪间充质干细胞人源化正常大鼠模型构建图。
具体实施方式
本发明公开了一种利用人干细胞构建人源化鼠模型的方法并公开了一类利用该方法构建研究病毒性肝炎的人源化鼠模型，下面对本发明的技术方案作进一步地说明：
一、获取人干细胞
1、分离培养人干细胞
1)获得纯化人干细胞。
2)干细胞培养，传代。
3)培养于20℃-40℃，2％-10％CO2的培养箱中。
2、获取商品化的分离或冻存的人干细胞或细胞系。
二、干细胞移植入肝损伤的鼠
1、获得不同品系的实验鼠，实验鼠包括正常小鼠、免疫缺陷小鼠、正常大鼠和免疫缺陷大鼠。
2、通过腹腔、肌肉、外周静脉注射、口服或灌胃的方式予以肝损药物，或予以外科部分肝切除术，建立肝损伤鼠模型。
3、通过外周静脉、门静脉、脾脏或肝脏注射的方式移植1×104-8干细胞。
三、HBV感染人源化小鼠
通过外周静脉、皮下、肌肉或腹腔每只鼠注射各型嗜肝性病毒。
下面根据附图，通过具体实施例及对比例，对本发明的技术方案作进一步地说明：本发明通过多种不同人来源干细胞注入肝损伤鼠获得不同人源化鼠模型，来研究嗜肝性病毒感染机制及嗜肝性病毒感染的发生、发展机制、转归及治疗。
实施例1：图1为骨髓间充质干细胞人源化FRGS小鼠模型构建示意图，人骨髓间充质干细胞(hBMSCs)移植暴发性肝衰竭FRGS小鼠建立骨髓间充质干细胞人源化FRGS小鼠模型。
1、用含10％胎牛血清的DMEM培养基培养人骨髓间充质干细胞hBMSCs。
2、FRGS小鼠逐渐将药物2-(2-硝基-4-三氟甲基苄基)-环己烷-1，3-二酮(NTBC)减量，注射0.2mg/kg抗Fas抗体(JO2)，建立肝衰竭小鼠模型。
3、通过门静脉注射500ul 1×106hBMSCs。
4、移植后2、5、8天持续JO2注射。
5、通过尾静脉每只小鼠注射A、B、C、D型1*106乙肝病毒。
6、在注射乙肝病毒后的1周、2周、4周，后续每4周分别检测乙肝病毒载量和肝功能状态，确认模型建立成功。
图1运用FRGS小鼠，首先运用化学药物NTBC建立暴发性肝衰竭，再运用人 骨髓间充质干细胞移植，分化形成肝细胞，最后注射乙肝病毒，构建人源化慢性乙型肝炎小鼠模型。
实施例2：图2为胚胎干细胞人源化uPA小鼠模型构建示意图，人胚胎干细胞系移植纯合uPA小鼠建立胚胎干细胞人源化uPA小鼠模型。
1、用含10％胎牛血清的DMEM培养基培养人胚胎干细胞。
2、获得纯合uPA/SCID小鼠模型。
3、出生8周时通过脾注射500ul 1×106人胚胎干细胞。
4、通过尾静脉每只小鼠注射1*107丙型肝炎病毒。
5、在注射丙肝病毒后的1周、2周、4周，后续每4周分别检测病毒载量和肝功能状态，确认模型建立成功。
图2运用uPA小鼠，uPA会自发形成肝损害，再运用人胚胎干细胞移植，分化形成肝细胞，最后注射丙肝病毒，构建人源化慢性丙型肝炎小鼠模型。
实施例3，图3为诱导多能干细胞人源化半乳糖胺正常小鼠模型构建示意图，人诱导多功能干细胞(hiPSCs)注射正常免疫的暴发性肝衰竭小鼠建立诱导多能干细胞人源化正常小鼠模型。
1、通过基因转染技术将某些转录因子导入动物或人的体细胞，使体细胞直接重构成多功能干细胞，用含10％胎牛血清的DMEM培养基培养。
2、每只小鼠腹腔注射半乳糖胺1.5g/kg，建立肝衰竭小鼠模型。
3、通过肝脏注射500ul 1×106人诱导多功能干细胞。
4、通过尾静脉每只小鼠注射1*105戊型肝炎病毒。
5、在注射戊肝病毒后的1周、2周、4周，后续每4周分别检测病毒载量和肝功能状态，确认模型建立成功。
图3运用小鼠，首先运用化学药物半乳糖胺建立暴发性肝衰竭，再运用诱导人多能干细胞移植，分化形成肝细胞，最后注射戊型肝炎病毒，构建人源化慢性戊型肝炎小鼠模型。
实施例4：图4为骨髓间充质干细胞人源化正常大鼠模型构建示意图，人骨髓间充质干细胞(hBMSCs)注射急性肝损伤正常大鼠建立骨髓间充质干细胞人源化正常大鼠模型。
1、正常人骨髓用淋巴细胞分离液分离纯化人骨髓单核细胞，用含10％胎牛血清的DMEM培养基培养，获得人骨髓间充质干细胞hBMSCs。
2、正常大鼠行50％肝切除术，建立急性肝损伤大鼠模型。
3、通过门静脉注射500ul 1×106hBMSCs。
4、通过尾静脉每只小鼠注射A、B、C、D型1*106乙肝病毒。
6、在注射乙肝病毒后的1周、2周、4周，后续每4周分别检测病毒载量和肝功能状态，确认模型建立成功。
图4运用正常小鼠，首先运用外科50％肝脏切除建立急性肝损伤，再运用人骨髓间充质干细胞移植，分化形成肝细胞，最后注射乙肝病毒，构建人源化慢性乙型肝炎大鼠模型。
实施例5：图5为人脂肪间充质干细胞人源化正常大鼠模型构建示意图，人脂肪间充质干细胞(hADSCs)注射肝损伤正常大鼠建立脂肪间充质干细胞人源化正常大鼠模型。
1、正常人脂肪组织分离纯化人脂肪间充质干细胞，用含10％胎牛血清的DMEM培养基培养，获得人脂肪间充质干细胞hBMSCs。
2、正常大鼠通过腹腔注射四氯化碳0.5ml/100g，建立急性肝损伤大鼠模型。
3、通过脾脏注射1000ul 5×106hADSCs。
4、每只小鼠腹腔注射1*106丙型肝炎病毒。
5、在注射丙型肝炎病毒后的1周、2周、4周，后续每4周分别检测病毒载量和肝功能状态，确认模型建立成功。
图5运用正常小鼠，首先运用化学药物四氯化碳建立急性肝损伤，再运用人脂肪间充质干细胞移植，分化形成肝细胞，最后注射丙肝病毒，构建人源化慢性丙型肝炎大鼠模型。
以上例举的仅是本发明的优选实施方式，本发明并不限于以上实施例，本领域技术人员在不脱离本发明的精神和构思的前提下直接导出或联想到的其他改进和变化，均应认为包含在本发明的保护范围内。

Claims (10)

  1. 一种利用干细胞构建人源化鼠模型的方法，其特征在于，所述方法的步骤如下：
    1)、获取人干细胞；
    2)、将干细胞移植入肝损伤的鼠；
    3)、HBV感染人源化鼠。
  2. 根据权利要求1所述的利用干细胞构建人源化鼠模型的方法，其特征在于，所述的干细胞是分离培养的人干细胞，或是商品化的分离或冻存的人干细胞或细胞系。
  3. 根据权利要求2所述的利用干细胞构建人源化鼠模型的方法，其特征在于，所述的分离培养的人干细胞的获取步骤如下：
    a、获得纯化人干细胞；
    b、干细胞培养，传代；
    c、培养于20℃-40℃，2％-10％CO2的培养箱中。
  4. 根据权利要求1或2或3所述的利用干细胞构建人源化鼠模型的方法，其特征在于，所述的步骤2)的具体步骤如下：
    e、获得不同品系的实验鼠；
    f、通过予以肝损药物，或予以外科部分肝切除术，建立肝损伤鼠模型；
    g、移植1×104-8人干细胞进入鼠模型。
  5. 根据权利要求4所述的用干细胞构建人源化鼠模型的方法，其特征在于，所述的步骤g后还包括步骤h：分次予以肝损药物。
  6. 根据权利要求4所述的利用干细胞构建人源化鼠模型的方法，其特征在于，所述的实验鼠是正常小鼠或免疫缺陷小鼠或正常大鼠或免疫缺陷大鼠，所述的肝损伤包括急性、慢性肝损伤，急性、亚急性、慢性肝衰竭中的任意一种。
  7. 根据权利要求4所述的利用干细胞构建人源化鼠模型的方法，其特征在于，所述的步骤f中是予以肝损药物是通过腹腔、肌肉、外周静脉注射、口服或灌胃的方式。
  8. 根据权利要求5或6或7所述的利用干细胞构建人源化鼠模型的方法，其特征在于，所述的步骤g是通过外周静脉、门静脉、脾脏或肝脏注射的方式移植1×104-8人干细胞。
  9. 根据权利要求1或2或3或5或6或7所述的利用干细胞构建人源化鼠模型的方法，其特征在于，所述的步骤3)是通过外周静脉、皮下、肌肉或腹腔对每只鼠注射各型嗜肝病毒。
  10. 根据权利要求1或2或3或5或6或7所述的利用干细胞构建人源化鼠模型的方法，其特征在于，所述的步骤3)后还包括4)在感染鼠后的3-30天内至少检测病毒载量一次，确认模型建立成功；在感染鼠后的3-30天内检测病毒载量一次后，每四周再分次检测病毒载量，确认模型建立成功。
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CN120616838A (zh) * 2025-08-08 2025-09-12 中国医学科学院基础医学研究所 一种诱导急性炎症性贫血的小鼠模型构建方法

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