Construction method of double humanized hepatitis b mouse model characterized by t cell immune reconstruction
By constructing a human liver chimeric immunodeficient mouse model and transplanting HLA-matched human hepatocytes and PBMCs, the problem that existing HBV mouse models cannot effectively simulate human immune responses has been solved. This has enabled a dual-humanized hepatitis B mouse model with T cell regeneration as the main component, supporting research on the biological pathogenesis and immune response of HBV.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
- Filing Date
- 2025-02-18
- Publication Date
- 2026-06-02
AI Technical Summary
Existing HBV mouse models have failed to effectively mimic human immune responses, particularly in T-cell immune responses, thus limiting research on human-specific immune responses and immune function induced by HBV infection.
By constructing a human liver chimeric immunodeficient mouse model, transplanting HLA-matched human hepatocytes and infecting them with HBV, and then transplanting PBMCs to reconstruct T cells, a humanized hepatitis B mouse model with functional T cells was formed.
It enables continuous detection of human CD45+ cells in a mouse model, reduces the time required to reconstruct the human immune system, detects human immune cells in the liver, and forms a dual humanized model of the liver and immune system with T cell reconstruction as the main component. Intrahepatic immunofluorescence shows co-staining of HBsAg, HBcAg and human CD3+ T cells.
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Figure CN119969344B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of animal model construction technology. Specifically, it is a method for constructing a mouse model of hepatitis B with dual humanization of the liver and immune system, characterized by T-cell immune reconstitution. Technical Background
[0002] Hepatitis B virus (HBV) is one of the major challenges facing global public health, infecting over 250 million people and causing approximately one million deaths annually from various liver diseases caused by HBV, such as liver failure, cirrhosis, and hepatocellular carcinoma (HCC). HBV infection and development are typically closely related to the immune system, involving the interaction between innate and adaptive immune responses. After entering the body, HBV interacts with various immune cells and factors, among which T cells, particularly CD8+ cytotoxic T cells (CTLs), play a central role in clearing HBV infection. These CTLs directly kill infected cells by recognizing viral antigens presented on the surface of HBV-infected hepatocytes (such as HBV surface antigen HBsAg and core antigen HBcAg), thereby effectively inhibiting viral spread. This, in turn, affects the body's immune status and treatment efficacy, ultimately determining the patient's clinical outcome.
[0003] Because HBV primarily infects humans and exhibits strong species specificity, only a few animals, such as chimpanzees, cynomolgus monkeys, and tree shrews, can be infected with HBV. However, these non-human primate models are constrained in research due to ethical concerns, high costs, and limitations in experimental procedures. Although other hepatotropic DNA viruses, such as duck hepatitis virus (DHV) and marmot hepatitis virus (GBV-B), have been used to study their pathogenesis and immune responses in hosts, the results cannot fully reflect the HBV infection process and immune response in humans due to significant differences between these viruses and HBV in host species and immune response mechanisms.
[0004] Mice, as commonly used laboratory animals, are widely used in biomedical research due to their ease of handling and low breeding costs. However, current mouse models used to study HBV infection, such as HBV transgenic mouse models and HBV-transfected mouse models, have certain limitations. Although these models can simulate HBV infection to some extent, the significant differences between the mouse and human immune systems, especially in the immune response to HBV, mean that existing mouse models fail to effectively mimic the human immune response. This difference significantly limits their ability to study human-specific immune responses and immune function. Furthermore, the human T cells reconstituted in humanized mice based on hematopoietic stem cells (HSCs) are naive T cells, which hinders research on human-specific immune responses and immune function induced by HBV infection. Therefore, the model constructed in this invention will help provide model selection for HBV biological pathogenesis and immune response, drug screening, and the validation and optimization of novel therapies. Summary of the Invention
[0005] The purpose of this invention is to address the shortcomings of existing HBV mouse infection models by proposing a method for constructing a dual-humanized hepatitis B mouse model characterized by T-cell immune reconstitution.
[0006] The technical solution adopted in this invention includes the following steps:
[0007] (1) Construction of a human liver chimeric immunodeficient mouse model, including;
[0008] Step 1-1: Prepare commercially available isolated or cryopreserved human hepatocytes for HLA-A, HLA-B, and HLA-C typing identification;
[0009] Steps 1-2: Select 6-7 week old male URG immunodeficient mice with a weight of 23±3g;
[0010] Steps 1-3: One week after mice were induced to suffer spontaneous liver injury by intraperitoneal injection of doxycycline at a dose of 15 mg / kg, human hepatocytes were transplanted via intrasplenic injection at a dose of 1 × 10⁻⁶. 6 Each mouse;
[0011] Steps 1-4: After human hepatocyte transplantation, doxycycline was added to the drinking water of mice, starting at a concentration of 0.1 mg / ml, increasing by 0.1 mg / ml weekly to 0.5 mg / ml, and then maintaining the concentration unchanged.
[0012] Steps 1-5: Six weeks after mouse transplantation of human hepatocytes, peripheral blood human albumin (hAlb) was measured. When the peripheral blood hAlb level was greater than 1 × 10⁻⁶, the hAlb level was considered normal. 6 A humanized immunodeficient mouse model of the liver system was successfully constructed at ng / ml and will be used for subsequent HBV infection and PBMC transplantation.
[0013] (2) Human liver chimeric immunodeficient mice infected with HBV, including;
[0014] Step 2-1: Human liver chimeric immunodeficient mice were infected with HBV via vertebral vein injection at a dose of 1×10⁻⁶. 6 Each mouse received copies of HBV.
[0015] Step 2-2: After HBV infection, blood was collected from the ocular venous plexus of mice weekly to detect peripheral blood hAlb and virological markers (HBV DNA, HBsAg). When the peripheral blood HBV DNA of the model mice was >10... 4 If the number of copies / ml and HBsAg > 0.05 OD are considered to indicate successful HBV infection in mice.
[0016] (3) Implantation of human immune cells to construct a dual humanized hepatitis B mouse model with T cell regeneration as the main component and a humanized immune system, including;
[0017] Step 3-1: Extract peripheral blood mononuclear cells (PBMCs) from healthy individuals;
[0018] Step 3-2: PBMCs were transplanted via intravenous injection into the tail vein of mice, with an injection dose of 2.5 × 10⁻⁶. 6 One PBMC per mouse, with transplanted PBMC cell viability >90%.
[0019] (4) Identification of a dual-humanized hepatitis B mouse model with T-cell immune reconstitution of the liver and immune system, including:
[0020] Step 4-1: Perform flow cytometry analysis and detect hAlb and virological markers (HBV DNA, HBsAg) levels in the peripheral blood of model mice;
[0021] Step 4-2: Perform flow cytometry analysis and immunofluorescence staining on the liver tissue of the model mice.
[0022] Further, 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.
[0023] Furthermore, the HBV injected in step (2) came from a patient with chronic hepatitis B, whose ALT, AST, and coagulation function levels were all normal.
[0024] Furthermore, the human peripheral blood mononuclear cells (PBMCs) in step (3) are from normal blood donors whose HLA matching with the human hepatocytes in step (1) is >3 / 6, and the blood donors have no history of hepatitis B virus infection.
[0025] Furthermore, human PBMCs were transplanted into mice 8 weeks after HBV infection. The proportion of human CD45+ and human CD3+ immune cells in the peripheral blood of mice was detected weekly by flow cytometry. When human CD45+ immune cells accounted for >10% of the total number of immune cells and human CD3+ T cells accounted for >80% of the total number of human immune cells in the peripheral blood of the model mice, the dual humanized mouse model was successfully constructed.
[0026] Another object of the present invention is the application of the construction method in the construction of a dual-humanized mouse model of hepatitis B characterized by T-cell immune reconstitution.
[0027] The beneficial effects of this invention are that, compared with existing dual-humanized chimeric mouse models:
[0028] 1. The mouse model of the present invention can continuously detect human CD45+ cells in peripheral blood after transplantation of HLA-matched PBMCs, with human CD3+ T cells as the main component, and the time required to rebuild the human immune system is shorter.
[0029] 2. Human immune cells, mainly CD3+CD8+ T cells, can be detected in the liver of the mouse model of the present invention, forming a dual humanized hepatitis B mouse model with T cell reconstitution as the main feature of the liver and immune system;
[0030] 3. In the mouse model of the present invention, intrahepatic immunofluorescence showed co-staining of HBsAg, HBcAg and human CD3+ T cells. Attached Figure Description
[0031] Figure 1 Changes in peripheral blood Alb in mice after HBV infection.
[0032] Figure 2 Changes in peripheral blood HBsAg in mice after HBV infection.
[0033] Figure 3 Changes in peripheral blood HBV DNA in mice after HBV infection.
[0034] Figure 4 The change in the proportion of human CD45+ cells in peripheral blood of mice after PBMC transplantation.
[0035] Figure 5 This describes the chimerism of human immune cells in peripheral blood of mice after transplantation of PBMCs.
[0036] Figure 6 This image shows the infiltration of human immune cells in the liver of mice after transplantation of PBMCs.
[0037] Figure 7 The results of immunofluorescence staining of the liver in mice after PBMC transplantation are shown. Figure 7A and 7B are the results of CD45 and HBsAg fluorescent co-staining. Figure 7 C and 7D are the results of CD3 and HBsAg fluorescent co-staining. Detailed Implementation
[0038] The present invention will be further described in conjunction with the accompanying drawings and embodiments. However, the present invention is not limited to the above embodiments, and within the scope of knowledge possessed by those skilled in the art, several improvements and additions can be made without departing from the spirit of the present invention.
[0039] Example 1
[0040] This invention discloses a method for constructing a humanized hepatitis B mouse model primarily based on T cell regeneration, involving both the liver and immune system. The method involves injecting HLA-matched human peripheral blood mononuclear cells (PBMCs) to reconstruct a humanized hepatitis B mouse model with functional T cells. The technical solution of this invention is further described below.
[0041] 1. Materials and Reagents
[0042] 1.1 Animals: URG mice, male, 6-7 weeks old, weighing 23±3g, were purchased from Beijing Vitonda Company and acclimatized for one week in an SPF-grade laboratory.
[0043] 1.2 Reagents: Commercially available isolated or cryopreserved human hepatocytes (LifeNet Health); doxycycline (Sangon Biotech); hAlb ELISA kit (Proteintech); human peripheral blood lymphocyte separation medium (Solepro); trypan blue dye (Invitrogen).
[0044] 1.3 Instruments: Low-temperature high-speed centrifuge (Eppendorf, Germany); disposable sterile syringes (Zhejiang Longde Pharmaceutical Co., Ltd.); disposable sterile insulin syringes (BD Pharmaceuticals, USA); Fortessa flow cytometer (BD Pharmaceuticals, USA); full-spectrum flow cytometer (Cytek, USA).
[0045] 2. Methods
[0046] 2.1 Construction of a human liver chimeric immunodeficient mouse model;
[0047] 2.2 Human liver chimeric immunodeficient mice infected with HBV;
[0048] 2.3 Implantation of human immune cells to construct a dual-humanized mouse model of hepatitis B with T-cell regeneration as the main component of the liver and immune system;
[0049] 2.4 Identification of a dual-humanized mouse model of hepatitis B with reconstituted liver and immune system by T cell immune regeneration.
[0050] Example 2: Preparation of a human liver chimeric immunodeficient mouse model
[0051] 1. Prepare commercially available human hepatocytes for HLA-A, HLA-B, and HLA-C typing identification.
[0052] 2. In URG immunodeficient mice, spontaneous liver injury was induced by intraperitoneal injection of 15 mg / kg doxycycline (Dox), and one week later, 1 × 10⁻⁶ liver cells were transplanted via intrasplenic injection. 6 Personal hepatocytes were transplanted into mice. Doxycycline was continuously added to the drinking water, starting at 0.1 mg / ml and increasing weekly by 0.1 mg / ml to 0.5 mg / ml, then maintaining this concentration. Peripheral blood hAlb levels were measured 6 weeks after transplantation. HAlb levels in mice were greater than 1 × 10⁻⁶. 6 The concentration of ng / ml suggests that human hepatocytes have colonized in mice.
[0053] Example 3: Human liver chimeric immunodeficient mice infected with HBV
[0054] HBV was infected in chimeric liver immunodeficient mice via vertebral vein injection at a dose of 1×10⁻⁶. 6 Each mouse received copies of HBV. 100 μL of blood was collected weekly via ocular venous plexus after infection to measure peripheral blood hAlb concentration and hepatitis B virus load (HBV DNA, HBsAg) to confirm infection efficacy.
[0055] Mouse peripheral blood hAlb levels as Figure 1 As shown, the level of hAlb in the peripheral blood of mice remained stable after HBV infection. The viral load results in mice are as follows. Figure 2-3 As shown, Figure 2 Changes in HBV DNA levels after infection Figure 3 To measure the changes in HBsAg levels after infection, HBV DNA and HBsAg levels in mice remained elevated after HBV infection. When peripheral blood HBV DNA > 104 copies / ml and HBsAg > 0.05 OD, the mice were considered to have been successfully infected.
[0056] Example 4: Human immune cell implantation to construct a dual-humanized hepatitis B mouse model with T-cell regeneration as the main component of the liver and immune system.
[0057] 1. Obtain anticoagulated whole blood from healthy donors whose HLA typing is >3 / 6 compatible with transplanted human hepatocytes, and dilute it with an equal volume of physiological saline. Add 15 mL of separation buffer to the bottom layer of a 50 mL sterile centrifuge tube. Use a Pasteur pipette to spread the diluted blood evenly on top of the separation buffer to form a clear stratification interface. Set the centrifuge speed to 1 for the rise and 0 for the fall, and centrifuge at 500 g for 30 min at room temperature. After centrifugation, discard the upper plasma layer, carefully aspirate the white blood cell layer into a 15 mL sterile centrifuge tube, and wash the white blood cell layer with 10 mL of PBS washing buffer. Centrifuge again at 250 g for 10 min at room temperature, washing twice in total. After isolating PBMCs, count the cells, and perform trypan blue staining to detect cell viability before use or cryopreservation in liquid nitrogen.
[0058] 2. Human liver chimeric immunodeficient mice successfully infected with HBV underwent PBMC transplantation via intravenous injection into the tail vein, with an injection dose of 2.5 × 10⁻⁶. 6 One PBMC per mouse.
[0059] Example 5: Identification of a dual-humanized hepatitis B mouse model with T-cell immune reconstitution of the liver and immune system.
[0060] Following PBMC transplantation, 150 μL of blood was collected weekly from the ocular venous plexus of mice. Flow cytometry analysis and detection of hAlb and virological markers (HBV DNA, HBsAg) levels were performed on the peripheral blood. Four weeks after PBMC transplantation, flow cytometry analysis and immunofluorescence staining were performed on mouse liver tissue to confirm successful model establishment.
[0061] The results of changes in peripheral blood hAlb concentration and hepatitis B virus load (HBV DNA, HBsAg) in mouse models are as follows: Figure 1-3 As shown, after injection of PBMCs in week 8, an interaction between the reconstructed human immune system and human hepatocytes could be observed, and viral proliferation in mice was inhibited after immune reconstruction.
[0062] Flow cytometry results as follows Figure 4-7 As shown, Figure 4 The change in the proportion of human CD45+ cells in mouse peripheral blood. Figure 5 Data on the proportion of human cells infiltrating mouse peripheral blood. 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. The human CD45+ cells infiltrating the peripheral blood and liver of mice were mainly CD3+C8+ T cells (>85%). Figure 7 Immunofluorescence staining of mouse liver tissue showed that CD45+ and CD3+ cells were concentrated around the clusters of infected (HBsAg+) hepatocytes.
Claims
1. A method for constructing a dual-humanized mouse model of hepatitis B characterized by T-cell immune reconstitution, characterized in that, This can be achieved through the following steps; (1) Construction of a human liver chimeric immunodeficient mouse model: (a) Preparation of commercially isolated or cryopreserved human hepatocytes for HLA-A, HLA-B, and HLA-C typing identification; (b) Select 6-7 week old male URG immunodeficient mice with a weight of 23±3g; (c) One week after mice were induced with spontaneous liver injury by intraperitoneal injection of doxycycline at a dose of 15 mg / kg, human hepatocytes were transplanted via intrasplenic injection at a dose of 1 × 10⁻⁶. 6 Each mouse; (d) After human hepatocyte transplantation, doxycycline was added to the drinking water of mice. The concentration was increased by 0.1 mg / ml each week to 0.5 mg / ml and then maintained at the same concentration. (e) Six weeks after mouse transplantation of human hepatocytes, peripheral blood human albumin (hAlb) was measured. When the peripheral blood hAlb level was greater than 1 × 10⁻⁶, the hAlb level was considered normal. 6 A humanized immunodeficient mouse with liver system concentration of ng / ml was 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 vertebral vein injection at a dose of 1×10⁻⁶. 6 HBV copy number per mouse; (b) After HBV infection, blood was collected from the ocular venous plexus of mice weekly to detect peripheral blood hAlb and virological markers. When the peripheral blood HBV DNA of the model mice was >10... 4 If the number of copies / ml and HBsAg > 0.05 OD are considered as successful HBV infection in mice; the virological markers are HBV DNA and HBsAg. (3) Human immune cell implantation to construct a dual humanized hepatitis B mouse model with T cell regeneration as the main component of the liver and immune system; (a) Extraction of peripheral blood mononuclear cells from healthy individuals; (b) PBMCs were transplanted via intravenous injection into the tail vein of mice at a dose of 2.5 × 10⁻⁶. 6 Each mouse had one peripheral blood mononuclear cell, and the viability of the transplanted PBMC cells was >90%. (4) Identification of a dual-humanized mouse model of hepatitis B with T-cell immune reconstitution in the liver and immune system: (a) Flow cytometry analysis and detection of hAlb and virological marker levels in peripheral blood of model mice were performed. The virological markers were HBV DNA and HBsAg. (b) Flow cytometry analysis and immunofluorescence staining were performed on liver tissue from model mice.
2. The construction method according to claim 1, characterized in that, The HLA identification results of 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) came from a patient with chronic hepatitis B, whose ALT, AST and coagulation function were all 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 hepatocytes in step (1) is >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, Eight weeks after HBV infection, mice were transplanted with human PBMCs. The proportion of human CD45+ and human CD3+ immune cells in the peripheral blood of mice was detected weekly by flow cytometry. When human CD45+ immune cells accounted for more than 10% of the total number of immune cells in the peripheral blood of the model mice and human CD3+ T cells accounted for more than 80% of the total number of human immune cells, the dual humanized mouse model was successfully constructed.
6. The construction method described in claim 1 is applied to the construction of a dual-humanized mouse model of hepatitis B characterized by T-cell immune reconstitution.