CN113519458A - Method for constructing liver and immune double humanized large animal model by single stem cell transplantation - Google Patents
Method for constructing liver and immune double humanized large animal model by single stem cell transplantation Download PDFInfo
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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
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a method for constructing a large animal model with double humanization of liver and immunity by utilizing single stem cell transplantation, which comprises the steps of obtaining human stem cells, transplanting a single stem cell into a large animal with liver injury, embedding human liver cells into a humanized immune system and the like.
Description
Technical Field
The invention belongs to the fields of clinical medicine, experimental medicine, regenerative medicine and virology, and particularly relates to a method for constructing a large animal model with double humanization of liver and immunity by utilizing single stem cell transplantation.
Background
The experimental animal disease model is an indispensable research tool for researching etiology and pathogenesis of human diseases and developing prevention and treatment technologies and medicines. However, the species difference between humans and animals and the difference between the experimental results and clinical phenotype of disease models lead to serious consequences of drugs developed and researched in common animal models, such as a large amount of toxic and side effects, liver and kidney failure, and the like. If human cells and tissues are introduced into a common animal model body to establish a humanized animal model, an important tool is provided for rapidly converting the development of basic medical research and the achievement thereof into a new clinical treatment method or means.
Infectious diseases such as hepatitis, AIDS, novel coronavirus pneumonia, dengue fever, Ebola, Zika, malaria, sepsis and the like have wide epidemic range, the caused diseases have great harm to human, and the research on the diseases caused by the viruses does not have great breakthrough at present. The natural infection process and treatment of the viruses in human bodies are relegated to the outcome, and the viruses are urgently needed to be researched by a human animal model. Diseases of the immune system such as acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelocytic leukemia and chronic myelocytic leukemia require animal models with human immune cells to study the development and treatment of the diseases. Liver system diseases such as hepatotrophic hepatitis, fatty liver, liver cirrhosis caused by hepatitis, liver cancer and the like require animal models with human liver cells and immune cells to study development and treatment of the diseases. Animal models of liver and immune double humanization provide a good model basis for the study of these diseases.
The size, anatomical structure, physiological index and functional level of the liver of the pig and the liver of the human are similar, if the natural infection process of the human infection virus is simulated in the body of a large animal such as the pig, the better mechanism, progress and regression research of the human disease is realized, the optimization of the treatment scheme of the human disease is promoted to a great extent, and the optimized treatment scheme can be more quickly applied to clinical treatment. A double chimeric and double humanized model of two different cells of human liver cells and immune cells is established in a large animal body such as a pig and the like, and a natural infection process of human infection virus is simulated on the basis, so that the scientific significance and the application value of the model cannot be simulated by small animals such as a mouse and the like. Establishing a double humanized double chimeric model of large animals such as humanized pigs and the like provides a good research carrier for researching human diseases and treating and disease regression.
The stem cells (including human bone marrow mesenchymal stem cells, human embryonic stem cells, human adipose mesenchymal stem cells, induced pluripotent stem cells and the like) are CD34 and CD45 negative stem cells, have multidirectional differentiation potential and can differentiate to various types of cells. The previous researches show that the human mesenchymal stem cells (hBMSC) can be embedded into fulminant liver failure pigs, which lays a foundation for transplanting the stem cells into the liver of large animals such as liver-damaged pigs and the like and establishing a novel liver and immune double humanized large animal model. The double humanized model provides a model basis for infectious disease models, liver disease and drug metabolism models, tumor growth and cancer immune models, tumor-immune system interaction models, organ transplantation models and autoimmune disease models. The double humanized model provides an important tool for the development of basic medical research and the rapid conversion of the results thereof into a new clinical treatment method or means.
Disclosure of Invention
The invention provides a method for constructing a double-chimeric double-humanized animal model of two different cells of human liver cells and immune cells in a large animal body by transplanting a single human stem cell, and provides an important tool for rapidly converting the development of basic medical research and the achievement thereof into a new clinical treatment method or means. . The invention is realized by the following technical scheme:
the invention discloses a method for constructing a large animal model with double humanization of liver and immunity by utilizing single stem cell transplantation, which comprises the following steps:
1) obtaining human stem cells;
2) transplanting a single stem cell into a large animal with liver damage;
3) human hepatocyte chimerization: in the liver of a large animal, the transplanted human stem cells are differentiated to form human liver cells; humanized immune system: in a large animal body, the transplanted human stem cells are differentiated to form a human immune system;
as a further improvement, the invention can construct a double-embedded double-humanized model of two different systems of human liver cells and immune cells by transplanting one cell.
As a further improvement, the stem cells of the invention are isolated and cultured human stem cells, or commercial isolated or cryopreserved human stem cells or cell lines.
As a further improvement, the liver injury provided by the invention comprises any one of acute and chronic liver injury and acute, subacute and chronic liver failure, the liver injury mode is liver injury medicine or surgical partial hepatectomy, wherein the liver injury medicine comprises acetaminophen (APAP), D-galactose (D-Gal) and carbon tetrachloride (CCL4), and the liver injury medicine intake mode is abdominal cavity, muscle, peripheral intravenous injection, oral administration or intragastric administration.
As a further improvement, the experimental animal is a pig or a macaque or a rabbit or a dog which is similar to the size, the anatomical structure, the physiological index and the functional level of the liver organ of the human being.
As a further improvement, the step 2) of the present invention is to transplant 1X 10 by means of peripheral vein, portal vein, spleen or liver injection6-9Human stem cells.
As a further improvement, tissues such as whole mouse blood, liver, spleen, thymus, bone marrow and the like are collected at 1, 2, 4, 6, 8, 12, 16 and 20 weeks after cell transplantation and used for evaluating the chimeric rate of human liver cells and immune cells to confirm that the model is successfully established.
Compared with the prior art for constructing the humanized animal model, the invention has the following beneficial effects:
in order to rapidly convert the development of basic medical research and the achievement thereof into a new clinical treatment method or means, the invention carries out research on a plurality of aspects such as biochemical indexes, immunohistochemistry, gene expression level, proteomics and the like. The discovery shows that by inducing severe liver damage and transplanting human stem cells, human-derived hepatocytes in livers of large animals such as pigs have a high chimerism rate of 30-50%, and human-derived immune cells can be continuously separated from organs such as spleens, blood, livers, bone marrow of large animals such as pigs, so that large animal models such as double humanized pigs with livers and immune cells are formed. Besides being used for researching pathogenesis of human diseases, researching and developing and screening new clinical drugs, the method can also obtain infectious disease models, liver disease and drug metabolism models, tumor growth and cancer immune models, tumor-immune system interaction models, organ transplantation models, autoimmune disease models and the like which are more in line with the development history of human diseases.
The technology for constructing the double humanized pig model of the liver and the immune cells provides basic guarantee for infectious disease models, liver disease and drug metabolism models, tumor growth and cancer immune models, tumor-immune system interaction models, organ transplantation models and autoimmune disease models. The technical scheme provides a convenient, simple and easily-obtained humanized model with high similarity with a human body for clinical treatment and research of liver diseases.
Drawings
FIG. 1 shows 3 days after the injection of physiological saline without cells in the control group and 3X 10 days after the injection in the transplant group7Schematic representation of HE staining results of miniature pig liver 3 weeks after hbmscs; (A) as a control group, 3 days after the injection of physiological saline without cells; (B) for IPT group, hbmscs were transplanted 3 weeks later.
FIG. 2 is a graph showing the results of chimeric histochemistry of hepatocytes in humanized pig liver; after 5 weeks of hBMSC portal vein transplantation, alb (a) and hsa (b) positive cells were widely distributed in liver lobules.
Detailed Description
The invention discloses a method for constructing a large animal model with double liver and immune humanization by single stem cell transplantation, which comprises the following steps: firstly, obtaining and culturing human hepatocytes to ensure that the hepatocytes are cultured to be in sufficient quantity; secondly, before the human stem cell transplantation, liver injury treatment is carried out on large animals such as pigs; transplanting a sufficient number of human stem cells into a large animal body such as a pig with damaged liver by means of portal vein, peripheral vein, spleen or liver injection; and finally, collecting tissues such as whole blood, liver, spleen, thymus, bone marrow and the like of the mouse, and evaluating the chimeric rate of the human liver cells and the immune cells to confirm the successful establishment of the model.
1) As a further improvement, the stem cell of the invention is a separation cultured human stem cell, or a commercial separation or frozen human stem cell or cell line;
2) as a further improvement, the liver injury of the invention comprises any one of acute and chronic liver injury, acute, subacute and chronic liver failure. The liver damage mode is liver damage drug or surgical partial hepatectomy, wherein the liver damage drug comprises acetaminophen (APAP), D-galactose (D-Gal), carbon tetrachloride (CCL4) and the like; the liver damage drug intake mode is abdominal cavity, muscle, peripheral intravenous injection, oral administration or intragastric administration;
3) as a further improvement, the number of the human stem cells used for transplanting large animal models such as pigs is 1 multiplied by 106-9；
4) As a further improvement, the method for determining the liver cell chimerism rate of the humanized mouse is to detect the human ALB concentration in the serum of the mouse by ELISA; the quantity of human ALB and human Hepatocyte Specific Antigen (HSA) double positive cells in mouse liver tissue is analyzed by immunohistochemistry; quantitatively sorting Human Leukocyte Antigen (HLA) positive cells in mouse whole hepatocytes by using flow cytometry, and performing in-vitro culture and immunohistochemical identification on the amount of human ALB positive cells; quantitatively analyzing the ratio of the human mRNA in mouse mononuclear cells and liver tissues by using transcriptome sequencing; quantitatively analyzing the ratio of the humanized ALB peptide in the plasma and the liver tissue of the mouse by utilizing a proteomics technology; and (4) determining the liver cell chimerism rate of the humanized mouse by combining the results, and confirming that the establishment of the liver and immune double humanized large-scale animal model is successful.
The large animals comprise pigs or macaques or rabbits or dogs, and the technical scheme of the invention is further described by taking the pigs as an example in combination with the attached drawings of the specification:
first, obtaining human stem cell
1. Isolated culture of human stem cells
1) Purified human stem cells were obtained.
2) And (4) culturing stem cells and carrying out passage.
3) Culturing at 20-40 deg.C and 2-10% CO2In an incubator.
2. Commercial isolated or cryopreserved human stem cells or cell lines are obtained.
Second, the stem cells are transplanted into the large animals such as the liver-injured pigs
1. Obtaining experimental pigs of different strains
2. The pig model with liver injury is established by taking a liver injury medicament or surgical partial hepatectomy in modes of intraperitoneal, intramuscular and peripheral intravenous injection, oral administration or intragastric administration.
3. Transplanting by portal vein, peripheral vein, spleen or liver injection6-9A stem cell.
Thirdly, confirming successful establishment of large animal models such as pigs
1. Cell transplantation tissues such as whole blood, liver, spleen, thymus, bone marrow of mice were collected at 1, 2, 4, 6, 8, 12, 16, and 20 weeks.
2. Detecting the human ALB concentration in the mouse serum by ELISA; analyzing the number of human ALB and human Hepatocyte Specific Antigen (HSA) double positive cells in mouse liver tissues by immunohistochemistry; quantitatively sorting Human Leukocyte Antigen (HLA) positive cells in mouse whole hepatocytes by using flow cytometry, and performing in-vitro culture and immunohistochemical identification on the amount of human ALB positive cells; quantitatively analyzing the ratio of the human mRNA in mouse mononuclear cells and liver tissues by using transcriptome sequencing; quantitatively analyzing the ratio of the humanized ALB peptide in the plasma and the liver tissue of the mouse by utilizing a proteomics technology; and (4) determining the liver cell chimerism rate of the humanized mouse by combining the results, and confirming that the establishment of the liver and immune double humanized large-scale animal model is successful.
The technical scheme of the invention is further explained by specific embodiments and comparative examples according to the attached drawings: the invention obtains different large animal models such as double humanized pigs and the like by injecting various different human source stem cells into the liver-injured pigs.
Example 1: the human bone marrow mesenchymal stem cells (hBMSCs) are transplanted into the fulminant hepatic failure miniature pig to establish a human bone marrow mesenchymal stem cell double humanized pig model.
1. Culturing hBMSCs (human mesenchymal stem cells) in a DMEM medium containing 10% fetal bovine serum.
2. 8-10kg of Chinese experimental piglets were fasted for 12 hours and water was excluded for 8 hours before jugular vein cannulation. The injection method comprises the steps of injecting diazepam, ketamine and atropine, disinfecting and paving a towel after animals are anesthetized, performing unilateral external jugular vein intubation, returning the animals to a feeding room after the animals are awakened, and generally performing molding 2 days after the intubation.
3. D-galactose (D-gal) was slowly injected into the animals at a dose of 1.5g/kg through the jugular vein. All animals received no treatment with any drug or the like throughout the experiment.
4. The groups were randomized into 2 groups of 15. Transplantation group: the group will be 1 × 107hbmscs were suspended in 10ml of saline and transplanted through the hepatic portal vein at the B-ultrasonic location. Control group: the group was injected with 10ml of cell-free saline at the B-ultrasonic location via the portal vein. All animals received no treatment with any drug or the like throughout the experiment.
5. Tissues such as whole blood, liver, spleen, thymus, bone marrow and the like of the mice are collected after 1, 2, 4, 6, 8, 12, 16 and 20 weeks of cell transplantation, and detection technologies such as ELISA, flow cytometry quantitative sorting, immunohistochemistry, transcriptome sequencing, proteomics and the like are utilized to confirm that the model is successfully established.
FIG. 1 shows 3 days after the injection of physiological saline without cells in the control group and 3X 10 days after the injection in the transplant group7Graph of HE staining results of miniature pig liver 3 weeks after hbmscs. The control HE results showed extensive hepatocellular necrosis, hemorrhage of whole liver lobules, few residual hepatocytes near the fibrous plate, severe swelling of hepatocyte cytoplasm, and no evidence of hepatocyte regeneration in all specimens. The results of the transplanted group HE show that after the human bone marrow mesenchymal stem cells are transplanted for 3 weeks, new small leaves of the liver regenerate, the phenomena of blood sinus microthrombus formation or other types of microvascular hepatocyte necrosis are not found in the liver, and the structure of the living animal liver is close to normal.
FIG. 2 shows immunohistochemistry results, in which arrows indicate positive cells for the human hepatocyte-specific markers ALB and HSA in pig liver tissue. ALB and HSA positive cells represent human liver cells transdifferentiated from human mesenchymal stem cells. The results show that after 5 weeks of transplantation of the human bone marrow mesenchymal stem cells, the ALB and HSA positive staining cells, namely the liver cells from the hBMSC are widely distributed in liver lobules, the proportion of the liver cells accounts for about 30-50% of the total number of the liver cells, and the cells are present in the liver lobules in a conglomerate manner, a cluster manner or a scattered single cell manner. Indicating that adult BMSCs can enter FHF liver to colonize and have the capacity of differentiating into liver cells.
Example 2: the human embryonic stem cell line is transplanted into the fulminant hepatic failure miniature pig to establish a human embryonic stem cell double humanized pig model.
1. Human embryonic stem cells were cultured in DMEM medium containing 10% fetal bovine serum.
2. 8-10kg of Chinese experimental piglets were fasted for 12 hours and water was excluded for 8 hours before jugular vein cannulation. The injection method comprises the steps of injecting diazepam, ketamine and atropine, disinfecting and paving a towel after animals are anesthetized, performing unilateral external jugular vein intubation, returning the animals to a feeding room after the animals are awakened, and generally performing molding 2 days after the intubation.
3. Paracetamol (APAP) at a dose of 750mg/kg was injected slowly into the animals via the jugular vein. All animals received no treatment with any drug or the like throughout the experiment.
4. Will be 1 × 107Human embryonic stem cells were suspended in 10ml of physiological saline and transplanted through the hepatic portal vein under B-ultrasonic positioning. All animals received no treatment with any drug or the like throughout the experiment.
5. Tissues such as whole blood, liver, spleen, thymus, bone marrow and the like of the mice are collected after 1, 2, 4, 6, 8, 12, 16 and 20 weeks of cell transplantation, and detection technologies such as ELISA, flow cytometry quantitative sorting, immunohistochemistry, transcriptome sequencing, proteomics and the like are utilized to confirm that the model is successfully established.
Example 3: the method comprises the steps of transplanting small-sized pigs with fulminant hepatic failure by human induced pluripotent stem cells (hiPSCs) to establish a human induced pluripotent stem cell double humanized pig model.
1. Certain transcription factors are introduced into body cells of animals or human by gene transfection technology, so that the body cells are directly reconstructed into multifunctional stem cells, and the multifunctional stem cells are cultured by a DMEM medium containing 10% fetal bovine serum.
2. 8-10kg of Chinese experimental piglets were fasted for 12 hours and water was excluded for 8 hours before jugular vein cannulation. The injection method comprises the steps of injecting diazepam, ketamine and atropine, disinfecting and paving a towel after animals are anesthetized, performing unilateral external jugular vein intubation, returning the animals to a feeding room after the animals are awakened, and generally performing molding 2 days after the intubation.
3. D-galactose (D-gal) was slowly injected into the animals at a dose of 1.5g/kg through the jugular vein. All animals received no treatment with any drug or the like throughout the experiment.
4. Will be 1 × 107The human induced pluripotent stem cells were suspended in 10ml of physiological saline and transplanted through the hepatic portal vein under B-ultrasonic positioning. All animals received no treatment with any drug or the like throughout the experiment.
5. Tissues such as whole blood, liver, spleen, thymus, bone marrow and the like of the mice are collected after 1, 2, 4, 6, 8, 12, 16 and 20 weeks of cell transplantation, and detection technologies such as ELISA, flow cytometry quantitative sorting, immunohistochemistry, transcriptome sequencing, proteomics and the like are utilized to confirm that the model is successfully established.
Example 4: the method comprises the steps of transplanting the small-sized pig with the fulminant hepatic failure by the human adipose-derived mesenchymal stem cells to establish a double humanized pig model of the human adipose-derived mesenchymal stem cells.
1. Separating and purifying the human adipose-derived mesenchymal stem cells, and culturing the human adipose-derived mesenchymal stem cells by using a DMEM medium containing 10% fetal calf serum to obtain the human adipose-derived mesenchymal stem cells.
2. 8-10kg of Chinese experimental miniature pigs are fasted for 12 hours, and water is forbidden for 8 hours. Firstly, diazepam, ketamine and atropine are injected, 50% hepatectomy is performed after animals are anesthetized, and a miniature pig model with acute liver injury is established.
3. Will be 1 × 107The human induced pluripotent stem cells were suspended in 10ml of physiological saline and transplanted through the hepatic portal vein under B-ultrasonic positioning.
4. Tissues such as whole blood, liver, spleen, thymus, bone marrow and the like of the mice are collected after 1, 2, 4, 6, 8, 12, 16 and 20 weeks of cell transplantation, and detection technologies such as ELISA, flow cytometry quantitative sorting, immunohistochemistry, transcriptome sequencing, proteomics and the like are utilized to confirm that the model is successfully established.
The above examples are only preferred embodiments of the present invention, and the present invention is not limited to the above examples, and other modifications and variations directly derived or suggested by those skilled in the art without departing from the spirit and concept of the present invention should be considered as included in the protection scope of the present invention.
Claims (8)
1. A method for constructing a large animal model with double liver and immunity humanization by single stem cell transplantation is characterized by comprising the following steps:
1) obtaining human stem cells;
2) transplanting a single stem cell into a large animal with liver damage;
3) human hepatocyte chimerization: in the liver of a large animal, the transplanted human stem cells are differentiated to form human liver cells; humanized immune system: in a large animal body, the transplanted human stem cells are differentiated to form a human immune system;
4) and confirming successful establishment of large animal models such as pigs.
2. The method for constructing a large animal model for liver and immune double humanization by single stem cell transplantation according to claim 1, wherein a double chimeric double humanization model of two different systems of human liver cells and immune cells can be constructed by transplantation of a single cell.
3. The method of claim 1, wherein the stem cells are isolated human stem cells, or commercial isolated or cryopreserved human stem cells or cell lines.
4. The method of claim 1, wherein the liver injury comprises any one of acute and chronic liver injury, acute and subacute and chronic liver failure, and the liver injury is liver damage drug or surgical partial hepatectomy, wherein the liver damage drug comprises acetaminophen (APAP), D-galactose (D-Gal) and carbon tetrachloride (CCL4), and the liver damage drug is taken by abdominal cavity, muscle, peripheral vein injection, oral administration or intragastric administration.
5. The method for constructing a large animal model for liver and immunization double humanization according to claim 1, 2, 3 or 4, wherein the experimental animal is a pig, a macaque, a rabbit or a dog, which is similar to human in size, anatomical structure, physiological index and functional level of liver organ.
6. The method for constructing a large animal model with liver and immune double humanization according to claim 5, wherein the step 2) is performed by injecting the single stem cell into the liver by a peripheral vein, portal vein, spleen or liver injection method, and then transplanting the stem cell into the 1X 10 animal model6-9Human stem cells.
7. The method for constructing a large animal model with liver and immune double humanization according to claim 1, 2, 3, 4 or 6, wherein the step 4) of cell transplantation is performed by collecting tissues such as mouse whole blood, liver, spleen, thymus, bone marrow and the like at 1, 2, 4, 6, 8, 12, 16 and 20 weeks for evaluation of the chimeric ratio of human hepatocytes and immune cells, and confirming successful model establishment.
8. The method for constructing a large animal model for liver and immune double humanization using single stem cell transplantation according to claim 1, 2, 3, 4 or 6, wherein the step 4) comprises measuring the human ALB concentration in the serum of the mouse by ELISA; analyzing the number of human ALB and human Hepatocyte Specific Antigen (HSA) double positive cells in mouse liver tissues by immunohistochemistry; quantitatively sorting Human Leukocyte Antigen (HLA) positive cells in mouse whole hepatocytes by using flow cytometry, and performing in-vitro culture and immunohistochemical identification on the amount of human ALB positive cells; quantitatively analyzing the ratio of the human mRNA in mouse mononuclear cells and liver tissues by using transcriptome sequencing; quantitatively analyzing the ratio of the humanized ALB peptide in the plasma and the liver tissue of the mouse by utilizing a proteomics technology; and (4) determining the liver cell chimerism rate of the humanized mouse by combining the results, and confirming that the establishment of the liver and immune double humanized large-scale animal model is successful.
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