HBV (Hepatitis B Virus) infected mouse model as well as construction method and application thereof

Abstract

The invention discloses an HBV infected mouse model and a construction method and application thereof, and the construction method comprises the following steps: step 1, taking a healthy female HBV-Tg mouse with a positive HBV marker as a liver donor, taking out the liver through an operation, and cutting the liver into small blocks; step 2, taking a healthy female NPG mouse with severe combined immunodeficiency as a receptor animal, pulling the maximum leaf of the liver to the outside of the abdominal cavity through an operation, placing a liver tissue block of the HBV-Tg mouse on the exposed liver parenchyma surface of the NPG mouse, performing auxiliary fixation, then returning the liver back to the abdominal cavity, and then suturing; 3, the copy number of HBV DNA in NPG mouse serum is measured, and if the two continuous detection values are remarkably higher than the background value and the index can be continuously maintained at a certain level in the subsequent observation period, the HBV infected mouse model is successfully constructed. According to the invention, the process from donor sampling to receptor transplantation can be completed within several hours, and the model can be verified through serological detection 1-2 weeks after operation, so that the modeling period is greatly shortened.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and laboratory animal science, and particularly relates to an HBV-infected mouse model, its construction method, and its application. Background Technology

[0002] Hepatitis B virus (HBV) infection is a major global public health problem that can lead to chronic hepatitis, cirrhosis, and even hepatocellular carcinoma. A deeper understanding of the HBV life cycle, host immune response, and pathogenic mechanisms relies on animal models that can accurately mimic the characteristics of human HBV infection.

[0003] Traditional HBV research models mainly suffer from the following technical shortcomings: 1. HBV transgenic mouse model: This model integrates the HBV genome into the mouse chromosome, enabling mice to continuously express HBV antigens and even replicate the virus. The drawback of this type of model is that viral expression usually originates from all hepatocytes or multiple tissues throughout the body, rather than a specific process under natural infection, and it often lacks a complete viral replication cycle and typical immunopathological features. More importantly, because viral antigens begin to be expressed during the embryonic period, mice develop immune tolerance to them, making it impossible to simulate the adaptive immune response and related liver inflammation and damage triggered by infection in adulthood.

[0004] 2. Immunodeficient mouse models based on human hepatocyte transplantation (e.g., uPA / SCID, FRG, NPG mice): These models first damage the hepatocytes of the host mouse (e.g., NPG mice, which lack T, B, and NK cells and have a high degree of immunodeficiency due to IL2rg gene deficiency) (e.g., through drugs or genetic engineering), and then transplant primary human or human-derived hepatocytes into them to construct humanized liver mice. Inoculating these mice with HBV establishes infection. While this model can simulate natural HBV infection well, the technical process is complex, requiring pre-treatment of the recipient mouse's liver (e.g., through drug injection or genetic tools) followed by transplantation of highly viable primary human hepatocytes. The modeling cycle typically lasts 2-4 months, and the scarcity of primary human hepatocytes, significant individual variability, and difficulties in in vitro culture, along with substantial batch-to-batch variations in cell viability and chimerism efficiency, result in high model construction costs, long cycles, and uncertainties regarding the consistency and reproducibility of experimental results, greatly limiting its widespread application.

[0005] In summary, existing technologies either fail to simulate the key immunopathological features of chronic HBV infection (such as transgenic models) or involve cumbersome, unstable, costly, and difficult-to-scale model establishment processes (such as human hepatocyte transplantation models). Therefore, there is an urgent need in this field for a novel mouse model construction method that is stable, reliable, economical, and can partially reflect the pathological features of natural HBV infection, in order to fill the aforementioned technological gaps and contribute to HBV-related research. Summary of the Invention

[0006] This invention aims to provide a mouse model of HBV infection, its construction method, and its applications. By transplanting liver tissue from HBV transgenic (HBV-Tg) donor mice into immunodeficient NPG mice, a chimeric mouse model is established that can stably carry and express hepatitis B virus (HBV) and can be used to simulate the progression of HBV-related liver diseases. This model has significant application value in researching the pathogenic mechanism of hepatitis B virus, screening and evaluating anti-HBV drugs, and preclinical evaluation of novel therapies (such as immunotherapy and gene therapy).

[0007] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides a method for constructing an HBV-infected mouse model, comprising the following steps: Step 1, Donor Liver Tissue Acquisition: Healthy, HBV-Tg mice with positive HBV markers were used as liver donors. The livers were surgically removed and cut into small pieces. The cut liver tissue pieces were transferred to a pre-cooled sterile PBS containing P / S double antibodies and stored at 0-4℃. Step 2, Recipient Liver Tissue Transplantation: Using healthy female NPG mice with severe combined immunodeficiency as recipient animals, the largest lobe of the liver was surgically pulled out of the abdominal cavity, and the liver tissue block of the HBV-Tg mouse was placed on the exposed liver parenchyma surface of the NPG mouse and assisted in fixation. Then the liver was returned to the abdominal cavity and sutured. Step 3, Model Qualification and Monitoring The copy number of HBV DNA in the serum of NPG mice was measured. If the value was significantly higher than the background value in two consecutive tests, and the index could be maintained at a certain level in the subsequent observation period, it would indicate that the liver tissue of HBV-Tg mice had survived and maintained function in NPG mice, and the HBV-infected mouse model was successfully established.

[0008] In the above technical solution, the specific method of step 1 is as follows: Step 11, Donor animal selection: Select healthy, HBV-Tg mice that are positive for HBV markers as liver donors; Step 12, Anesthesia and Disinfection: Anesthetize HBV-Tg mice, fix them in a supine position on a sterile surgical board after complete anesthesia, and disinfect the abdominal surgical area with 75% ethanol. Step 13, Liver Acquisition: Cut the skin and peritoneum along the midline of the abdomen to fully expose the liver; use sterile instruments to completely free each lobe of the liver, carefully separate the ligaments and blood vessels connected to the liver, quickly remove the whole liver, and immediately place it in a pre-cooled sterile PBS containing P / S double antibodies. Step 14, Liver tissue segmentation and preservation: Use sterile instruments to cut the liver tissue into uniform small pieces. Transfer the cut liver tissue pieces to a pre-cooled sterile PBS environment containing P / S double antibody and store at 0-4°C throughout the process.

[0009] In the above technical solution, the specific method of step 2 is as follows: Step 21, Recipient animal selection: Select healthy female NPG mice with severe combined immunodeficiency; Step 22, Anesthesia and preoperative treatment: Anesthetize NPG mice, fix them in a prone position after anesthesia, remove the hair in the area below the costal arch on the back, and disinfect the surgical area. Step 23, surgical exposure of the liver: Under aseptic conditions, make a longitudinal skin incision in the disinfected area, bluntly dissect the subcutaneous tissue, and then cut the abdominal wall muscles and peritoneum along the muscle fiber direction at the corresponding position to form a small incision; gently explore with sterile blunt forceps or cotton swabs, pull the largest lobe of the liver out of the abdominal cavity, and cover the surrounding tissue with sterile cotton pads soaked in physiological saline to keep it moist; Step 24, Liver tissue transplantation: Use sterile forceps to remove a prepared HBV-Tg mouse liver tissue block from the preservation solution, gently aspirate excess liquid, and place the tissue block on the exposed liver parenchyma surface of the NPG mouse. Step 25, graft fixation: Tissue adhesive is used for auxiliary fixation; Step 26, Closure of the abdomen and postoperative care: After confirming that the graft is firmly fixed, gently return the liver back into the abdominal cavity; use absorbable sutures to suture the peritoneum and muscle layer continuously or intermittently, and then close the skin incision with the same or finer sutures or skin suture clips.

[0010] In the above technical solution, the specific method of step 1 is as follows: the HBV markers are HBsAg and HBeAg.

[0011] In the above technical solution, in step 24, the tissue block is placed in the liver of the NPG mouse at the edge of the liver or in a region with rich blood supply.

[0012] In the above technical solution, the specific method of auxiliary fixation in step 25 is as follows: a small amount of medical-grade cyanoacrylate tissue glue is drawn with a sterile syringe and applied to the area around the placed donor liver tissue block and at the interface where it contacts the recipient liver.

[0013] In the above technical solutions, the HBV-Tg mice and NPG mice are 6-8 weeks old and weigh 20-25g.

[0014] In the above technical solution, in step 3, "two consecutive times" refers to the second and third weeks after surgery, and the subsequent observation period refers to the eighth to twelfth weeks after surgery.

[0015] Secondly, the present invention provides an HBV-infected mouse model, which is constructed using the above-described construction method.

[0016] Thirdly, the present invention provides applications of the above-mentioned HBV-infected mouse model in the following aspects: (1) Research on the pathogenic mechanism of HBV; (2) Screening and evaluation of anti-HBV drugs; (3) Preclinical evaluation of novel therapies.

[0017] The beneficial effects of this invention are as follows: (1) The model is stable and durable: the transplanted HBV-Tg liver tissue is not rejected in highly immunodeficient NPG mice, and can survive for a long time and continuously express HBV antigen and virus, providing a stable vector for the study of chronic HBV infection.

[0018] (2) The operation is relatively simple and the cycle is short: it avoids the complicated and unstable steps of primary human hepatocyte isolation, culture, and pre-transplantation treatment of recipient liver injury. The process from donor sampling to recipient transplantation can be completed within a few hours, and the model can be verified by serological testing 1-2 weeks after the operation, which greatly shortens the modeling cycle.

[0019] (3) Low cost and high reproducibility: Both HBV-Tg mice and NPG mice are commercial strains, which are easy to obtain and breed. The experimental procedures are highly standardized and less affected by individual differences, which is conducive to replication and promotion in different laboratories.

[0020] (4) It has a certain pathological research foundation: the transplanted liver tissue block may cause changes in the local microenvironment during the growth process. Combined with the continuous expression of HBV antigen, it provides a potential research platform for studying local immune cell infiltration (such as subsequent adoption of immune cells) and fibrosis initiation.

[0021] (5) Clear application prospects: This model is very suitable for evaluating the long-term inhibitory effect of anti-HBV drugs, evaluating the in vivo pharmacodynamics of novel therapies (such as siRNA, ASO, CAR-T cells, etc. that target HBV), and studying HBV-related host factors, providing a powerful tool for the development of new hepatitis B drugs. Attached Figure Description

[0022] Figure 1 : Schematic diagram of the modeling process.

[0023] Figure 2 HBV DNA test results data from 1-4 weeks. Detailed Implementation

[0024] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0025] This invention provides a method for constructing an HBV-infected mouse model, comprising the following steps (see schematic diagram). Figure 1 ): 1. Preparation of donor liver tissue (1) Selection of donor animals: Healthy, HBV-positive transgenic (HBV-Tg) female mice were selected as liver donors. The preferred age was 6-8 weeks and the weight was 20-25g.

[0026] Among them, a positive HBV marker refers to a positive test for a specific molecular / serological indicator related to the presence or replication of HBV. For example: HBsAg positive, HBeAg positive.

[0027] (2) Anesthesia and disinfection: HBV-Tg mice were anesthetized by inhalation (e.g., using isoflurane, induction concentration 3-4%, maintenance concentration 1.5-2.5%) or injection (e.g., intraperitoneal injection of sodium pentobarbital solution, at 40-50 mg / kg body weight). After the mice were completely anesthetized (e.g., no withdrawal response when the toes were pinched), they were fixed in a supine position on a sterile surgical board, and the abdominal surgical area was thoroughly disinfected with 75% ethanol.

[0028] (3) Liver Acquisition: The procedure was performed in a sterile laminar flow hood. The skin and peritoneum were incised along the midline of the abdomen to fully expose the liver. Using sterile ophthalmic scissors and forceps, the lobes of the liver were completely freed, and the ligaments and blood vessels connected to the liver were carefully separated. The intact liver was quickly removed and immediately placed in pre-chilled sterile phosphate-buffered saline (PBS, pH 7.2-7.4, containing 100 U / mL penicillin and 100 μg / mL streptomycin) on ice.

[0029] (4) Liver tissue segmentation and preservation: In a culture dish containing cold sterile PBS on ice, the liver tissue was segmented into pieces with a volume of approximately 9 mm using a sharp sterile scalpel. 3 Cut the liver tissue into uniform small pieces (3*3*3 mm). Avoid excessive compression of the tissue during the cutting process. Transfer the cut liver tissue pieces to new sterile centrifuge tubes containing cold sterile PBS (with P / S double antibodies) and store them in an ice-water mixture (0-4℃) throughout the process. This process should be completed within 1 hour to maximize the preservation of hepatocyte viability.

[0030] 2. Preparation of recipient mice and transplantation surgery (1) Selection of recipient animals: Healthy female NPG mice (NOD-Prkdcscid Il2rgem1) with severe combined immunodeficiency were selected. The preferred age was 6-8 weeks and the weight was 20-25g.

[0031] Among them, NPG mice (NOD-Prkdcscid Il2rgem1) are female individuals obtained by knocking out the Prkdc and Il2rg genes in a NOD background to establish a stable genetic strain, followed by targeted breeding and genotyping identification.

[0032] (2) Anesthesia and preoperative management: The NPG mice were anesthetized using the same or similar anesthesia protocol as the donor mice to ensure appropriate depth of anesthesia. After anesthesia, the mice were fixed in a prone position. Using an electric shaver, the hair in the area below the costal arch on the back (corresponding to the largest lobe of the liver—the left lateral or middle lobe) was carefully removed, covering an area of ​​approximately 2*2 cm. The surgical area was then disinfected with povidone-iodine and 75% ethanol in sequence, and covered with a sterile drape.

[0033] (3) Surgical exposure of the liver: Under aseptic conditions, make a longitudinal skin incision of about 1.0-1.5 cm in length in the disinfected area. Bluntly dissect the subcutaneous tissue, and then cut the abdominal wall muscles and peritoneum along the muscle fiber direction at the corresponding position to form a small incision. Gently explore with sterile blunt forceps or cotton swabs, carefully pull the largest lobe of the liver (usually the left lateral lobe) out of the abdominal cavity, and cover the surrounding tissue with sterile cotton swabs soaked in physiological saline to keep it moist.

[0034] (4) Liver tissue transplantation: A prepared HBV-Tg mouse liver tissue block (approximately 1 mm) was removed from the preservation solution using sterile fine forceps. 3 Gently aspirate any excess fluid. Place the tissue block on the exposed liver parenchyma surface of the NPG mouse. Preferred locations are the liver margins or areas with rich blood supply.

[0035] (5) Graft Fixation: To promote angiogenesis and integration of the graft with the recipient's liver tissue and prevent detachment or displacement, tissue adhesive is used for auxiliary fixation. Specifically, a small amount of medical-grade cyanoacrylate tissue adhesive (such as 3M™ Vetbond™ tissue adhesive or an equivalent product) is drawn into a sterile syringe and applied to the area around the placed donor liver tissue block and at the interface where it contacts the recipient liver. Care should be taken to avoid the adhesive covering the entire tissue block or flowing into the peritoneal cavity. Wait approximately 30-60 seconds for the adhesive to fully polymerize and harden, then firmly adhere the graft to the surface of the recipient liver.

[0036] (6) Abdominal Closure and Postoperative Care: After confirming that the graft is securely fixed, gently return the liver (the entire liver, including the largest lobe and the liver tissue block previously pulled out of the abdominal cavity) back into the abdominal cavity. Use absorbable sutures to suture the peritoneum and muscle layer continuously or intermittently. Then close the skin incision with the same or finer sutures or skin suture clips. Immediately after the operation, transfer the mouse to a clean, warming pad preheated to 37°C and closely observe its recovery. Once it is fully awake, able to move and drink water independently, return it to a separate, ventilated cage. Antibiotics (such as sulfamethoxazole / trimethoprim) can be added to the drinking water for 3 consecutive days after the operation to prevent infection. Administer analgesics (such as ibuprofen or carboprofen) subcutaneously or via drinking water immediately after the operation and for at least 24-48 hours after the operation to relieve postoperative pain.

[0037] 3. Model identification and monitoring (1) Postoperative general observation: The mental state, activity level, diet and water intake, weight changes and wound healing of the recipient mice were observed at least once a day, and the survival rate was recorded.

[0038] (2) Blood sample collection and HBV marker detection: Starting one week after transplantation, mouse blood samples (approximately 50-100 μL) were collected weekly or bi-weekly via tail vein sampling or retro-orbital venous plexus sampling. After the blood samples were allowed to stand and coagulate, the serum was separated by centrifugation.

[0039] (3) Quantitative determination of HBV-related indicators: The following indicators were quantitatively detected in serum using commercially available high-sensitivity kits: HBV DNA: The copy number of HBV DNA in serum was measured using real-time quantitative PCR (qPCR) to directly reflect the viral replication level.

[0040] (4) Model success criteria: HBV DNA was detected in the serum of NPG mice twice consecutively (e.g., in the second and third weeks after surgery) at a significantly higher level than the background value, and the index was maintained at a certain level during the subsequent observation period (e.g., 8-12 weeks). This indicates that the liver tissue of HBV-Tg mice has survived and maintained function in NPG mice, and the model has been successfully constructed.

[0041] (5) Optional model success criteria: HBsAg or HBeAg is detected in NPG mouse serum at significantly higher levels than the background value twice consecutively (e.g., in the second and third weeks after surgery), and these indicators can be maintained at a certain level in the subsequent observation period (e.g., 8-12 weeks), which indicates that the liver tissue of HBV-Tg mice has survived and maintained function in NPG mice, and the model has been successfully constructed.

[0042] HBsAg (Hepatitis B surface antigen): The concentration of HBsAg in serum is quantitatively detected using chemiluminescent immunoassay (CLIA) or enzyme-linked immunosorbent assay (ELISA). HBeAg (Hepatitis B e antigen): The concentration of HBeAg in serum is quantitatively detected using CLIA or ELISA.

[0043] (6) Optional histological verification: At the end of the experiment, mice can be sacrificed and livers can be removed. Samples are taken from the transplantation site, fixed in formalin, embedded in paraffin, sectioned, and then hematoxylin and eosin (H&E) staining is performed to observe the tissue structure. Immunohistochemical staining (IHC) targeting HBV antigens (such as HBsAg and HBcAg) can be used to visually confirm that hepatocytes expressing HBV antigens are alive in the donor liver tissue block.

[0044] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a mouse model of HBV infection, characterized by: Comprising the following steps: Step 1, donor liver tissue acquisition: healthy, HBV marker positive female HBV-Tg mice are used as liver donors, and the liver is removed by surgery and cut into small pieces, and the cut liver tissue pieces are transferred to a pre-cooled sterile P / S double-antibiotic PBS environment and stored at 0-4°C; Step 2, liver tissue transplantation in recipient: healthy, severely combined immunodeficient female NPG mice are used as recipient animals, and the largest lobe of the liver is pulled out of the abdominal cavity by surgery, and the HBV-Tg mouse liver tissue pieces are placed on the exposed liver parenchymal surface of the NPG mouse, and auxiliary fixation is performed, then the liver is returned to the abdominal cavity, and sutured; Step 3, model identification and monitoring The copy number of HBV DNA in the serum of NPG mice is determined, and if the detection value is significantly higher than the background value for two consecutive times, and the index can be maintained at a certain level during the subsequent observation period, it indicates that the HBV-Tg mouse liver tissue has survived in the NPG mouse and maintained function, and the HBV infected mouse model is successfully constructed.

2. The method of claim 1, wherein: The specific method of step 1 is: Step 11, donor animal selection: healthy, HBV marker positive female HBV-Tg mice are selected as liver donors; Step 12, anesthesia and disinfection: the HBV-Tg mouse is anesthetized, and after complete anesthesia, it is fixed in a supine position on a sterile surgical board, and the abdominal surgical area is disinfected with 75% ethanol; Step 13, liver acquisition: cut the skin and peritoneum along the midline of the abdomen, fully expose the liver; use sterile instruments to completely free the lobes of the liver, carefully separate the ligaments and blood vessels connected to the liver, quickly remove the complete liver, and immediately place it in a pre-cooled sterile P / S double-antibiotic PBS environment; Step 14, liver tissue segmentation and preservation: use sterile instruments to cut the liver tissue into uniform small pieces, and transfer the cut liver tissue pieces to a pre-cooled sterile P / S double-antibiotic PBS environment, and store them at 0-4°C throughout the process.

3. The method of claim 1, wherein: The specific method of step 2 is: Step 21, recipient animal selection: healthy, severely combined immunodeficient female NPG mice are selected; Step 22, anesthesia and preoperative treatment: the NPG mouse is anesthetized, and after anesthesia, it is fixed in a prone position, the hair in the area under the dorsal rib arch is removed, and the surgical area is disinfected; Step 23, surgical exposure of the liver: under sterile operating conditions, make a longitudinal skin incision in the disinfected area, bluntly separate the subcutaneous tissue, then cut the abdominal wall muscle and peritoneum along the muscle fiber direction at the corresponding position to form a small incision; gently probe with a sterile blunt forceps or cotton swab, pull the largest lobe of the liver out of the abdominal cavity, and cover the surrounding tissue with a sterile cotton swab soaked in normal saline to keep it moist; Step 24, liver tissue transplantation: use a sterile fine forceps to remove a prepared HBV-Tg mouse liver tissue piece from the storage solution, gently suck off excess liquid, and place the tissue piece on the exposed liver parenchymal surface of the NPG mouse; Step 25, graft fixation: auxiliary fixation is performed using tissue adhesive; Step 26, closing the abdomen and postoperative care: After confirming that the graft is fixed firmly, the liver is gently returned to the abdominal cavity; the peritoneum and muscle layer are sutured continuously or discontinuously using absorbable sutures, and the skin incision is closed using the same or thinner sutures or skin suture clips.

4. The method of claim 1, wherein: The specific method of step 1 is that the HBV marker is HBsAg or HBeAg.

5. The method of claim 1, wherein: In step 24, the position of the tissue block on the liver of the NPG mouse is the edge of the liver or an area rich in blood supply.

6. The method of construction of claim 1, wherein: In step 25, the specific method of auxiliary fixation is to use a sterile syringe to suck a small amount of medical-grade cyanoacrylate tissue glue, and drop the tissue glue around the placed donor liver tissue block and at the interface where it contacts the recipient liver.

7. The method of construction of claim 1, wherein: The HBV-Tg mice and NPG mice are 6-8 weeks old and weigh 20-25 g.

8. The method of construction of claim 1, wherein: In step 3, the two consecutive times refer to the 2nd and 3rd weeks after surgery, and the subsequent observation period refers to the 8th-12th week after surgery.

9. A mouse model of HBV infection, characterized in that: It is constructed by the construction method of any one of claims 1-8.

10. The use of the HBV-infected mouse model of claim 9 in the following aspects: (1) HBV pathogenesis research; (2) Anti-HBV drug screening and evaluation; (3) Preclinical evaluation of new therapies.

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