Construction method of double-person-derived hepatitis B mouse model characterized by T cell immune reconstruction

Abstract

The invention provides a construction method of a two-person-derived hepatitis B mouse model characterized by T cell immune reconstruction. The method is characterized by promoting the reconstruction of humanized T cells and comprises the steps of constructing a human liver chimeric immunodeficiency mouse model, infecting the human liver chimeric immunodeficiency mouse with HBV, implanting human immune cells and the like, and results show that hAlb, HBV DNA and HBsAg can be continuously detected in blood of the constructed mouse, HBsAg and HBcAg can be detected in the liver, and the human liver chimeric immunodeficiency mouse can be used for detecting HBcAg. Human-derived immune cell infiltration mainly comprising CD3 + T cells can be detected in organs such as blood and liver, so that a hepatitis B mouse model with double-derived liver and immune systems is formed. The technology for constructing the humanized mouse model can be used for researching the interaction mechanism of the HBV and the immune cells in the hepatitis B progress, and the technical scheme provides a good animal model for researching the hepatitis B progress, the immune cell depletion mechanism, clinical hepatitis B drug treatment and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of animal model construction, and specifically, is a method for constructing a hepatitis B mouse model with liver and immune system double humanized and characterized by T cell immune reconstruction. Technical Background

[0002] Hepatitis B virus (HBV) is one of the major challenges facing global public health, with more than 250 million people infected and about 1 million people dying each year from various liver diseases caused by HBV, such as liver failure, cirrhosis and hepatocellular carcinoma (HCC). HBV infection and development are usually closely related to the immune system, involving the interaction between innate and acquired immune responses. After HBV enters the body, it interacts with a variety of immune cells and immune factors, among which T cells, especially CD8+ cytotoxic T cells (CTL), play a core role in clearing HBV infection. These CTLs directly kill infected cells by recognizing viral antigens (such as HBV surface antigen HBsAg, core antigen HBcAg, etc.) presented on the surface of HBV-infected hepatocytes, thereby effectively inhibiting the spread of the virus, thereby affecting the body's immune status and treatment effect, and ultimately determining the patient's clinical outcome.

[0003] Since HBV mainly infects humans and has strong species specificity, only a few animals, such as chimpanzees, crab-eating macaques, and tree shrews, can be infected with HBV. However, these non-human primate models are restricted in research by ethical issues, high costs, and limitations of experimental operations. Although other hepatotropic DNA viruses, such as duck hepatitis virus (DHV) and woodchuck hepatitis virus (GBV-B), have been used to study their pathogenesis and immune response in the host, due to the significant differences between these viruses and HBV in host species and immune response mechanisms, their research results are difficult to fully reflect the infection process and immune response of HBV in humans.

[0004] As a common experimental animal, mice have the advantages of simple operation and low feeding cost, and are widely used in biomedical research. However, the mouse models currently used to study HBV infection, such as HBV transgenic mouse model and HBV transfected mouse model, have certain limitations. Although these models can simulate HBV infection to a certain extent, due to the significant differences between the mouse autoimmune system and the human immune system, especially in the immune response to HBV, the existing mouse models fail to effectively simulate the human immune response, and this difference makes these models have great limitations in studying human specific immune response and immune function. The human T cells reconstructed in the humanized mice constructed based on hematopoietic stem cells (HSC) are naive T cells, which hinders the study of human specific immune response and immune function caused by HBV infection. Therefore, this model constructed by the present invention will help to provide model selection for HBV biological pathogenesis and immune response, drug screening, and new therapy verification optimization. Summary of the invention

[0005] The purpose of the present invention is to address the defects of the existing HBV mouse infection model and propose a method for constructing a double humanized hepatitis B mouse model characterized by T cell immune reconstruction.

[0006] The technical solution adopted by the present invention comprises the following steps:

[0007] (1) Construction of human liver chimeric immunodeficient mouse model, including; Step 1-1, prepare commercially isolated or frozen human hepatocytes, and perform HLA-A, HLA-B, and HLA-C typing and identification; Step 1-2, select 6-7 week old male URG immunodeficient mice, weighing 23 ± 3 g; Step 1-3: After mice were injected intraperitoneally with 15 mg / kg of doxycycline to induce spontaneous liver injury for 1 week, human hepatocytes were transplanted by intrasplenic injection at a dose of 1×10 6 Each mouse; Steps 1-4: After human hepatocyte transplantation, doxycycline was added to the drinking water of mice. The concentration was started from 0.1 mg / ml, and increased by 0.1 mg / ml to 0.5 mg / ml every week and then maintained unchanged; Step 1-5: Detect human albumin hAlb in peripheral blood of mice 6 weeks after transplantation of human hepatocytes. 6 ng / ml, and immunodeficient mice with humanized liver system were successfully constructed and will be used for subsequent HBV infection and PBMC transplantation.

[0008] (2) Human liver chimeric immunodeficient mice infected with HBV, including; Step 2-1: Human liver chimeric immunodeficient mice were infected with HBV via caudal intravenous injection at a dose of 1×10 6 copies HBV per mouse; Step 2-2, after HBV infection, blood was collected from the ophthalmic venous plexus of mice once a week to detect peripheral blood hAlb and virological indicators (HBV DNA, HBsAg). 4 copies / ml, HBsAg>0.05OD, the mice were successfully infected with HBV.

[0009] (3) Human immune cell implantation to construct a hepatitis B mouse model with dual humanization of liver and immune system with T cell reconstruction as the main feature, including; Step 3-1, extracting peripheral blood mononuclear cells (PBMC) from healthy subjects; Step 3-2: PBMCs were transplanted via caudal intravenous injection in mice at a dose of 2.5×10 6 PBMCs were transplanted per mouse, and the cell viability of transplanted PBMCs was >90%.

[0010] (4) Identification of a dual humanized hepatitis B mouse model with T cell immune reconstruction of the liver and immune system, including: Step 4-1, performing flow cytometry analysis and detecting the levels of hAlb and virological indicators (HBV DNA, HBsAg) in the peripheral blood of the model mice; Step 4-2, flow cytometry analysis and immunofluorescence staining were performed on the liver tissues of the model mice.

[0011] Furthermore, the HLA identification results of the human hepatocytes in step (1) are: HLA-A*11:01, HLA-A*24:02; HLA-B*07:02, HLA-B*18:01; HLA-C*07:01, HLA-C*07:02.

[0012] Furthermore, the HBV injected in step (2) comes from a chronic hepatitis B patient, and the patient's ALT, AST, and coagulation function levels are normal.

[0013] Furthermore, the human peripheral blood mononuclear cells (PBMC) in step (3) are from normal blood donors whose HLA matching with the human liver cells in step 1) satisfies >3 / 6, and the blood donors have no history of hepatitis B virus infection.

[0014] Furthermore, the mice were infected with HBV and then transplanted with human PBMCs 8 weeks later. The proportions of human CD45+ and human CD3+ immune cells in the peripheral blood of the mice were detected by flow cytometry every week after transplantation. When the proportion of human CD45+ immune cells in the peripheral blood of the model mice was >10% of the total number of immune cells and the proportion of human CD3+T cells was >80% of the total number of human immune cells, the double humanized mouse model was successfully constructed.

[0015] Another object of the present invention is to apply the construction method to construct a double humanized hepatitis B mouse model characterized by T cell immune reconstitution.

[0016] The beneficial effects of the present invention are as follows: 1. The mouse model of the present invention can continuously detect human CD45+ cells in the peripheral blood after transplantation of HLA-matched PBMC, mainly human CD3+T cells, and the time required to rebuild the human immune system is shorter; 2. Human immune cells can be detected in the liver of the mouse model of the present invention, mainly CD3+CD8+T cells, forming a hepatitis B mouse model with dual humanization of liver and immune system with T cell reconstruction as the main feature; 3. Intrahepatic immunofluorescence of the mouse model of the present invention shows co-staining of HBsAg, HBcAg and human CD3+T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure shows the changes of peripheral blood Alb in mice infected with HBV.

[0018] Figure 2 The figure shows the changes of HBsAg in peripheral blood of mice infected with HBV.

[0019] Figure 3 The figure shows the changes of HBV DNA in peripheral blood of mice infected with HBV.

[0020] Figure 4 The changes in the proportion of human CD45+ cells in the peripheral blood of mice after PBMC transplantation.

[0021] Figure 5 This is the chimerism of human immune cells in the peripheral blood of mice after PBMC transplantation.

[0022] Figure 6 This is the infiltration of human immune cells in the liver of mice after PBMC transplantation.

[0023] Figure 7 The results of immunofluorescence staining of the liver after PBMC transplantation in mice. Figure 7 A and 7B are the results of CD45 and HBsAg fluorescence co-staining. Figure 7C and 7D are the results of CD3 and HBsAg fluorescence co-staining. DETAILED DESCRIPTION

[0024] The present invention is further described in conjunction with the accompanying drawings and embodiments. However, the present invention is not limited to the above embodiments, and within the knowledge of ordinary technicians in the relevant technical field, several improvements and supplements can be made without departing from the purpose of the present invention.

[0025] Example 1 The present invention discloses a method for constructing a hepatitis B mouse model with dual humanization of liver and immune system based on T cell reconstruction, and a humanized hepatitis B mouse model with functional T cells is reconstructed by injecting HLA-matched human peripheral blood mononuclear cells (PBMC). The technical scheme of the present invention is further described below.

[0026] 1. Materials and Reagents 1.1 Animals: URG mice, male, 6-7 weeks old, weighing 23±3 g, were purchased from Beijing Weitongda Company and were adaptively fed in an SPF-level laboratory for one week; 1.2 Reagents: Commercially isolated or frozen human hepatocytes (LifeNet Health); doxycycline (Sangon Biotechnology); hAlb Elisa kit (Proteintech); human peripheral blood lymphocyte separation medium (Solarbio); trypan blue dye (Invitrogen). 1.3 Instruments: Low-temperature high-speed centrifuge (Eppendorf, Germany); disposable sterile syringes (Zhejiang Longde Pharmaceutical Co., Ltd.); disposable sterile insulin syringes (BD, USA); Fortessa flow analyzer (BD, USA); full-spectrum flow cytometer (Cytek, USA).

[0027] 2. Methods 2.1 Construction of human liver chimeric immunodeficient mouse model; 2.2 Human liver chimeric immunodeficient mice infected with HBV; 2.3 Human immune cell implantation to construct a hepatitis B mouse model with dual humanization of liver and immune system with T cell reconstruction as the main feature; 2.4 Identification of a dual humanized hepatitis B mouse model with liver and immune system reconstructed by T cell immunity.

[0028] Example 2 Preparation of human liver chimeric immunodeficient mouse model 1. Prepare commercial human hepatocytes and identify their HLA-A, HLA-B, and HLA-C typing. 2. URG immunodeficient mice were injected intraperitoneally with 15 mg / kg of doxycycline Dox to induce spontaneous liver injury. One week later, 1×10 6 After transplantation of human hepatocytes, doxycycline was continuously added to the drinking water of mice, starting from 0.1 mg / ml, and the concentration was increased by 0.1 mg / ml every week to 0.5 mg / ml and then maintained unchanged. The hAlb content of mice was detected 6 weeks after transplantation. The hAlb content of mice was greater than 1×10 6 ng / ml, indicating that human hepatocytes are colonized in mice.

[0029] Example 3 Human liver chimeric immunodeficient mice infected with HBV Liver chimeric immunodeficient mice were infected with HBV by intravenous injection at the caudal vertebrae with a dose of 1×10 6 After infection, 100ul of blood was collected from the mouse ophthalmic venous plexus every week to detect the concentration of hAlb and hepatitis B virus load (HBV DNA, HBsAg) in the mouse peripheral blood to confirm the infection effect.

[0030] The level of hAlb in peripheral blood of mice Figure 1 As shown in Figure 2, the level of hAlb in the peripheral blood of mice was stable after infection with HBV. Figure 2-3 As shown, Figure 2 is the change of HBV DNA level after infection, Figure 3 The changes in HBsAg levels after infection. After mice were infected with HBV, the levels of HBV DNA and HBsAg continued to increase. When peripheral blood HBV DNA>104copies / ml and HBsAg>0.05OD, the mice were considered to have been successfully infected.

[0031] Example 4 Human immune cell implantation to construct a hepatitis B mouse model with dual humanization of liver and immune system with T cell reconstruction as the main component

[0032] 1. Obtain anticoagulated whole blood from a healthy donor with an HLA match of >3 / 6 with the transplanted human hepatocytes, and dilute it with an equal volume of normal saline. Add 15 mL of separation solution to the lower layer of a 50 mL sterile centrifuge tube, and spread the diluted blood with a Pasteur pipette above the surface of the separation solution to form a clear stratified interface. Set the centrifuge speed to 1, the speed to 0, and centrifuge at 500g for 30 minutes at room temperature. After centrifugation, discard the upper plasma layer, carefully pipette the white film layer cells into a 15 mL sterile centrifuge tube, and wash the white film layer cells with 10 mL of PBS washing solution. Centrifuge again, centrifuge at 250g for 10 minutes at room temperature, and wash twice. After separating the PBMC, count the cells, and detect cell viability with trypan blue staining for later use or freeze in liquid nitrogen.

[0033] 2. Human liver chimeric immunodeficient mice successfully infected with HBV were transplanted with PBMCs via caudal intravenous injection at a dose of 2.5×10 6 PBMCs per mouse.

[0034] Example 5 Identification of a dual humanized hepatitis B mouse model with liver and immune system reconstituted by T cell immunity

[0035] After PBMC transplantation, 150ul of blood was collected from the mouse ophthalmic venous plexus every week, and the peripheral blood was analyzed by flow cytometry and the levels of hAlb and virological indicators (HBV DNA, HBsAg) were detected. Flow cytometry and immunofluorescence staining were performed on the mouse liver tissue 4 weeks after PBMC transplantation to confirm that the model was successfully constructed.

[0036] The changes in peripheral blood hAlb concentration and hepatitis B virus load (HBV DNA, HBsAg) in the mouse model are as follows Figure 1-3 As shown, after the injection of PBMCs at week 8, interaction between the reconstructed human immune system and human liver cells could be observed, and viral proliferation in mice was inhibited after immunization was reconstructed.

[0037] The flow cytometry analysis results are as follows Figure 4-7 As shown, Figure 4 The change in the proportion of human CD45+ cells in mouse peripheral blood. Figure 5 The data for the proportion of human cells infiltrating the peripheral blood of mice. Figure 6 The data show the proportion of human cells infiltrating mouse liver tissue. The proportion of human CD45+ immune cells in the peripheral blood of model mice continued to increase, and the human CD45+ cells infiltrating the mouse peripheral blood and liver were mainly CD3+C8+T cells (>85%). Figure 7 Immunofluorescence staining of mouse liver tissue showed that CD45+ and CD3+ cells were concentrated around infected (HBsAg+) hepatocyte clusters.

Claims

1. A method for constructing a double-humanized hepatitis B mouse model characterized by T cell immune reconstitution, characterized in that: This is achieved by following the steps below; (1) Construction of human liver chimeric immunodeficient mouse model: (a) Prepare commercially isolated or frozen human hepatocytes and perform HLA-A, HLA-B, and HLA-C typing and identification; (b) 6-7 week old male URG immunodeficient mice weighing 23 ± 3 g were selected; (c) One week after spontaneous liver injury was induced by intraperitoneal injection of 15 mg / kg doxycycline in mice, human hepatocytes were transplanted by intrasplenic injection at a dose of 1×10 6 Each mouse; (d) After human hepatocyte transplantation, doxycycline was added to the drinking water of mice. The concentration started from 0.1 mg / ml and was increased by 0.1 mg / ml to 0.5 mg / ml every week and then maintained unchanged; (e) Detection of human albumin hAlb in peripheral blood of mice 6 weeks after transplantation of human hepatocytes. When the level of human albumin in peripheral blood is greater than 1×10 6 ng / ml, immunodeficient mice with humanized liver system were successfully constructed and will be used for subsequent HBV infection and PBMC transplantation; (2) Human liver chimeric immunodeficient mice were infected with HBV; (a) Human liver chimeric immunodeficient mice were infected with HBV via caudal intravenous injection with a dose of 1×10 6 HBV copy number per mouse; (b) After HBV infection, blood was collected from the ophthalmic venous plexus of mice once a week to detect peripheral blood hAlb and virological indicators. 4 copies / ml, HBsAg>0.05OD, the mice were successfully infected with HBV; virological indicators were HBVDNA and HBsAg; (3) Human immune cells were implanted to construct a hepatitis B mouse model with dual humanization of liver and immune system with T cell reconstruction as the main feature; (a) extracting peripheral blood mononuclear cells from healthy subjects; (b) PBMCs were transplanted via caudal intravenous injection in mice at a dose of 2.5×10 6 Peripheral blood mononuclear cells per mouse, transplanted PBMC cell viability> 90%; (4) Identification of a dual humanized hepatitis B mouse model with liver and immune system reconstructed by T cell immunity: (a) The peripheral blood of model mice was analyzed by flow cytometry and the levels of hAlb and virological indicators were detected. The virological indicators were HBVDNA and HBsAg; (b) Flow cytometry and immunofluorescence staining of liver tissues of model mice.

2. The construction method according to claim 1, characterized in that: The HLA identification results of the human hepatocytes in step (1) are: HLA-A*11:01, HLA-A*24:02; HLA-B*07:02, HLA-B*18:01; HLA-C*07:01, HLA-C*07:

02.

3. The construction method according to claim 1, characterized in that: The HBV injected in step (2) comes from a chronic hepatitis B patient whose ALT, AST and coagulation function levels are normal.

4. The construction method according to claim 1, characterized in that: The human peripheral blood mononuclear cells in step (3) are from normal blood donors whose HLA matching with the human liver cells in step (1) satisfies >3 / 6, and the blood donors have no history of hepatitis B virus infection.

5. The construction method according to claim 1, characterized in that: Mice were infected with HBV and then transplanted with human PBMCs 8 weeks after transplantation. The proportion of human CD45+ and human CD3+ immune cells in the peripheral blood of the mice was detected by cell flow cytometry every week after transplantation. When the proportion of human CD45+ immune cells in the peripheral blood of the model mice was >10% of the total immune cells and the proportion of human CD3+T cells was >80% of the human immune cells, the double-humanized mouse model was successfully constructed.

6. The construction method of claim 1 is used to construct a double humanized hepatitis B mouse model characterized by T cell immune reconstitution.

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