Method for establishing an autoimmune hepatitis mouse model and its application

Abstract

The present invention discloses a method for creating an autoimmune hepatitis mouse model and its application, relating to the field of medical research. The method addresses the existing problems of low success rate and complex creation steps in creating autoimmune hepatitis mouse models. The key technical solution is: a method for creating an autoimmune hepatitis mouse model, comprising a single injection of an AAV8-CYP2D6 adeno-associated virus into a mouse. This method achieves simple operation, high infection efficiency, and a high modeling success rate.

Description

Technical Field

[0001] The present invention relates to the field of medical research, and more particularly to a method for creating an autoimmune hepatitis mouse model and its application. Background Art

[0002] Autoimmune hepatitis (AIH) is a chronic, progressive inflammatory liver disease mediated by an autoimmune response. It is more common in Europe and the United States. While there are no specific statistics on the incidence of AIH in my country, the number of reported cases has increased significantly in recent years. The pathogenesis of AIH remains incompletely elucidated, and the lack of an ideal animal model is a significant factor. In 1992, Tiegs et al. successfully established a T cell-dependent experimental liver injury model in mice using concanavalin A (ConA). However, this model was an acute liver injury model, and the mice failed to develop autoantibodies or liver fibrosis, thus failing to mimic the chronic course of AIH. Cytochrome P450 2D6 (CYP2D6) is a known autoantigen in human autoimmune hepatitis. Antibodies that recognize CYP2D6 are anti-LKM-1 antibodies, a hallmark antibody in AIH patients. In 2008, Holdener et al. used an adenovirus expression vector of human CYP2D6 (Ad-CYP2D6) to establish an AIH model in mice (J Exp Med. 2008; 205(6): 1409-1422). However, due to the instability of adenovirus and the off-target effects of a single injection, this model is not highly reproducible, lacks representativeness, and is still controversial (J Autoimmun. 2017 Mar; 78: 39-45). Currently, there is no ideal chronic AIH animal model at home and abroad. Patent 202111553602.6 discloses a method for creating an autoimmune hepatitis animal model, which uses multiple injections of CYP2D6 naked plasmids to induce autoimmune hepatitis in mice. However, this method requires multiple and frequent injections of plasmids, which can easily induce heart failure in mice and lead to model failure. In addition, CYP2D6 expression may be located anywhere in the mouse body, so there are many uncertainties in model creation. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for establishing an autoimmune hepatitis mouse model, in which mice are infected with the AAV8-CYP2D6 adeno-associated virus, and modeling can be achieved with a single injection.

[0004] The above technical objectives of the present invention are achieved through the following technical solutions: a method for creating an autoimmune hepatitis mouse model, comprising the step of injecting AAV8-CYP2D6 adeno-associated virus into the mouse; the number of injections is 1.

[0005] Furthermore, the AAV8-CYP2D6 adeno-associated virus gene sequence contains a liver-specific promoter.

[0006] Furthermore, the liver-specific promoter is ApoE / hAAT or TBG.

[0007] Furthermore, the injection method is tail vein injection.

[0008] Furthermore, the titer of the AAV8-CYP2D6 adeno-associated virus is not less than 1×10 11 vg / mL; injection dose is 100 μL per mouse.

[0009] Furthermore, the mice are 6 to 8 week old male C57 / BL6 mice.

[0010] The present invention also provides a use of the autoimmune hepatitis mouse model obtained by the creation method described above in screening, preventing or treating drugs for autoimmune hepatitis.

[0011] Beneficial effects:

[0012] 1. AAV8 is used as a viral vector, which targets liver transfection and has a high infection efficiency, which can increase the success rate of model construction.

[0013] 2. Only one injection is needed to establish a mouse model with a high success rate, eliminating the need for frequent injections. This reduces injection-related complications in mice, such as superimposed acute hepatitis or injection-induced heart failure and death, and reduces the workload of researchers.

[0014] 3. Adding AAV8-CYP2D6 adeno-associated virus to a liver-specific promoter can anchor CYP2D6 to the liver for sustained and stable expression, resulting in a high modeling success rate and low experimental difficulty. It can also prevent the interference of expression in other tissues on liver inflammation itself. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figures 1 to 13 This is the verification result of the occurrence of autoimmune hepatitis in mouse models.

[0016] Figure 14 This is the GV651 vector map. DETAILED DESCRIPTION

[0017] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0018] Example 1:

[0019] like Figure 1 As shown, this embodiment provides a method for creating an autoimmune hepatitis mouse model. Using adeno-associated virus (AAV) as a vector, the human cytochrome P4502D6 (CYP2D6) gene, embedded in the hepatocyte promoter sequence, was injected into the tail vein to achieve overexpression of the CYP2D6 gene in anchored hepatocytes, resulting in sustained overexpression in the liver. Serum transaminases, autoantibodies, and liver pathology in the mice were dynamically monitored. The results showed: 1. Elevated serum transaminases ALT and AST in the model mice; 2. Autoantibodies appeared in the serum of the model mice at 7 weeks; 3. HE staining of liver sections of the model mice at 7 weeks revealed characteristic pathological changes of AIH, such as chronic inflammation at the interface, rosettes, and lymphocyte infiltration; 4. Liver inflammation in the mice was chronic and gradually worsened over time; and significant liver fibrosis was observed at 5 and 8 weeks. These results indicate that this animal model can simulate the occurrence and development of AIH in vivo, with clear histological and serological characteristics, persistent inflammation, and progressive liver fibrosis. Therefore, it can simulate the occurrence and development of AIH in vivo and can serve as a new and effective research tool for AIH, solving the bottleneck problem of the lack of animal models in current AIH research.

[0020] The specific steps are as follows:

[0021] 1. Inject AAV8-CYP2D6 adeno-associated virus into the tail vein of 6-8 week old male C57 / BL6 mice. The virus titer is 1×10 11 vg / mL; the injection dose was 100 μL per mouse.

[0022] C57 / BL6 mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0023] AAV8-CYP2D6 adeno-associated virus was purchased from Shanghai GeneCare Gene Technology Co., Ltd. The AAV8-CYP2D6 adeno-associated virus gene sequence contains the ApoE / hAAT liver-specific promoter. In other possible embodiments, the liver-specific promoter contained in the gene sequence is TBG.

[0024] 2. Three weeks after the injection, the mice were sacrificed and the liver tissues were frozen and sectioned in the dark. The eGFP fluorescence brightness of the frozen tissue sections was observed under an immunofluorescence microscope. Figure 2 The results showed that CYP2D6 was successfully transferred into hepatocytes for expression.

[0025] 3. Three weeks after injection, mice were sacrificed and their liver tissues were ground and RNA and protein were extracted. PCR ( Figure 3 ) Verify the mRNA level, Western blot ( Figure 4 ) Verify transfection efficiency at the protein level. Transfection efficiency was good, with mRNA and protein levels 2-3 times higher than those in the normal control group.

[0026] 4. Starting from the day of injection, mice were sacrificed at 7, 8, and 9 weeks to obtain mouse serum, which was then used to immunize rat liver tissues to observe the production of autoantibodies. Autoantibodies appeared in the mouse serum at 7 weeks, and the expression of autoantibodies gradually increased with time. Figure 5 The indirect Elisa method was used to detect changes in autoantibody titers, which showed that autoantibody titers gradually increased over time ( Figure 6 ).

[0027] 5. Starting from the day of injection, mice were sacrificed at 7, 8, and 9 weeks, and serum was collected. The kit test showed that the serum transaminase AST ( Figure 7 ), ALT( Figure 8 ) is elevated, indicating liver cell damage and liver inflammation.

[0028] 6. Starting from the day of injection, the mice were sacrificed at 7, 8, and 9 weeks, and liver tissue sections were taken and embedded in paraffin. HE staining was used to identify the characteristic pathology of AIH (rosetted rings, lymphocyte invasion, and interface hepatitis). Figure 9 ) production; over time, liver lymphocyte infiltration gradually increases and liver inflammation gradually worsens ( Figure 10 、 Figure 11 ).

[0029] 7. Starting from the injection date, at 8 weeks, Sirius red staining of the liver of AIH mice showed obvious scattered red collagen fibers, indicating the occurrence of mild to moderate fibrosis; at 9 weeks, Sirius red staining of the liver of AIH mice showed significant red collagen fiber proliferation, indicating further progression of liver fibrosis ( Figure 12 、 Figure 13 ).

[0030] The method described in this application can successfully establish a stable and reproducible AIH animal model. This AIH mouse model, through a single tail vein injection of adeno-associated virus, can produce autoantibodies and has clear histological and serological characteristics, persistent inflammation, and progressive liver fibrosis. Therefore, it can simulate the development and progression of AIH in humans and can serve as an effective research tool for AIH. It can be said that it fundamentally solves the current problem of the lack of reliable chronic animal models in the field of AIH.

[0031] Choosing AAV as an overexpression vector has the following advantages:

[0032] It has high expression levels and is non-pathogenic, and has been approved by the EU and FDA for use as a vector for gene therapy drugs.

[0033] Suitable for the expression of this model, excluding the interference of the virus itself on liver pathogenicity.

[0034] AAV8 is a specific serotype for the liver, muscles, eyes, and nervous system, providing the possibility of achieving liver-specific overexpression of human CYP2D6 antigen while avoiding the influence of other organs and tissues.

[0035] ApoE / hAAT was selected as the liver-specific promoter and integrated into the constructed vector, achieving the purpose of anchoring hepatocytes to overexpress human CYP2D6 and avoiding the influence of other non-parenchymal cells in the liver. This constructed an autoimmune hepatitis model and truly achieved the overexpression of autoantigens by hepatocytes, prompting the immune system to launch a chain reaction against autoantigens.

[0036] Example 2

[0037] This embodiment provides a method for constructing an AAV8-CYP2D6 adeno-associated virus, comprising the following steps:

[0038] Step 1. Construction of vector

[0039] Obtain a linearized vector using restriction endonuclease digestion. Prepare the target gene fragment by PCR amplification. When designing the amplification primers used, a homologous recombination sequence must be added to their 5' end. Use these primers to amplify the target gene fragment, and the sequences at the 5' and 3' ends of the amplified product are completely consistent with the sequences at both ends of the linearized cloning vector. Prepare a reaction system with the linearized vector and the target gene amplification product, and perform a recombination reaction to achieve in vitro circularization of the linearized vector and the target gene fragment. Directly transform the recombinant product, pick a single clone on the plate for PCR identification, and sequence the positive clones and analyze the results. Expand the culture and extract the correct clone solution to obtain a high-purity plasmid.

[0040] In the above steps, the target gene is:

[0041] Gene name: CYP2D6

[0042] Gene ID: NM_000106

[0043] Genetic species: Human

[0044] The tool carrier is: GV651; its carrier map is as follows Figure 12 shown.

[0045] Carrier number: GV651

[0046] Element sequence: pAAV-ApoE / hAATp-MCS-EGFP-3Flag-SV40 PolyA

[0047] Fluorescent marker: EGFP

[0048] Cloning site: NcoI / NcoI

[0049] The vector was purchased from Shanghai Genetech Technology Co., Ltd.

[0050] The vector carries an ApoE / hAATp liver-specific promoter, which can anchor the target gene for expression in the liver.

[0051] In the above steps, the amplification primers are shown in SEQ ID: 1 and SEQ ID: 2; and the restriction enzyme cleavage site is "CCATG".

[0052] The gene sequence of the recombinant plasmid is shown in SEQ ID: 3. The sequencing results are completely consistent with the target sequence.

[0053] Step 2: AAV packaging and assay

[0054] The AAV adeno-associated virus was purchased from Shanghai GeneTech Co., Ltd.

[0055] Step 1: Co-transfect the recombinant expression plasmid from Step 1 with pHelper (carrying adenoviral genes) and pAAV-RC (carrying AAV replication and capsid genes) into AAV-293 cells (providing the trans-acting factors required for AAV replication and packaging). Recombinant AAV is assembled in the packaging cells 2 to 3 days after transfection.

[0056] Step 2: Collect AAV viral particles from infected AAV-293 cells. AAV particles are typically concentrated in the packaging cells, so harvesting the cells and then lysing them to release the AAV particles into the supernatant allows for the recovery of the majority of the AAV particles. The viral supernatant obtained in this step can then be used to infect various mammalian cell lines. The virus in the supernatant can also be concentrated and retained.

[0057] Step 3: Concentrate and purify the viral supernatant from the third step. The original supernatant contains many cellular protein molecules and fragments. Two CsCl density gradient centrifugations and one ultrafiltration can remove most of the cellular proteins and residual CsCl ions. Animal experiments require purified viruses; otherwise, the required dose will not be achieved and side effects may occur. After infecting host cells, the single-stranded virus must be transfected into a double-stranded virus before gene expression. This transfection is the limiting step in recombinant gene expression and can be accelerated by superinfection with adenovirus or etoposide (camptothecin or sodium butyrate). However, reagents that accelerate gene expression are toxic to the target cells and can kill them if they remain on the cells. Therefore, etoposide can only be used short-term or to increase viral titer.

[0058] Step 4: Determine the titer of the resulting virus using quantitative PCR. This method provides a physical titer of the AAV genome packaged into the particles. AAV infectious titers vary significantly depending on the cells infected, the AAV capsid protein, and the test conditions. Furthermore, in vitro data may not reflect in vivo infection. Therefore, when comparing AAVs, the physical titer obtained by quantitative PCR is a more objective value.

[0059] This specific embodiment is only an explanation of the present invention, and it is not a limitation of the present invention. After reading this specification, those skilled in the art can make non-creative modifications to this embodiment as needed, but as long as it is within the scope of the claims of the present invention, it is protected by patent law.

Claims

1. A method for establishing an autoimmune hepatitis mouse model, characterized by: The method comprises the steps of injecting AAV8-CYP2D6 adeno-associated virus into mice; the injection frequency is 1; the injection method is tail vein injection; the titer of AAV8-CYP2D6 adeno-associated virus is not less than 1×10 11 vg / ml; injection dose, 100 μL per mouse; The AAV8-CYP2D6 adeno-associated virus gene sequence contains a liver-specific promoter; the liver-specific promoter is ApoE / hAAT or TBG.

2. The method for creating an autoimmune hepatitis mouse model according to claim 1, wherein: The mice were 6 to 8 week old male C57 / BL6 mice.

3. Use of the autoimmune hepatitis mouse model obtained by the creation method according to any one of claims 1 to 2 in screening, preventing or treating drugs for autoimmune hepatitis.

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