Method for screening drugs for autoimmune liver diseases

Abstract

The present invention discloses a method for screening drugs for autoimmune liver diseases, comprising: providing a test group of animals and a reference group of animals, wherein both the test group of animals and the reference group of animals include an animal model of autoimmune liver disease, and the genotype of the animal model of autoimmune liver disease is a regulatory T cell conditional knockout of adenosine monophosphate-activated protein kinase / apolipoprotein genotype; administering a candidate drug for autoimmune liver disease to the test group of animals; monitoring the changing trend of the first biochemical index data of the test group of animals and the changing trend of the second biochemical index data of the reference group of animals; and determining a target drug from the candidate drugs by comparing the changing trend of the first biochemical index data with the changing trend of the second biochemical index data.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to a method for screening drugs for autoimmune liver diseases. Background Art

[0002] Autoimmune liver diseases are chronic liver diseases that potentially threaten life, including autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and the recently discovered IgG4-related sclerosing cholangitis. They are caused by the immune system attacking liver cells or bile ducts, with different mechanisms and clinical manifestations, but the exact pathogenesis is not clear.

[0003] Animal models are powerful tools for understanding the immune pathogenesis of such diseases. Successfully constructing animal models can achieve the purpose of discovering serological markers for early diagnosis and immune therapy targets. Currently, many animal models of autoimmune liver diseases have been developed, most of which are mouse models, including transgenic, chemically induced, and infection-induced models. However, most of these mouse models have certain limitations.

[0004] The first spontaneous lethal AIH mouse model is the NTxPD-1- / - mouse model, which is similar to acute-onset AIH. However, these mice start to die at two weeks of age and cannot be used for further mechanism research and drug treatment observation. In addition to the spontaneous model, there are also some alternative antigen-induced models and liver autoantigen-induced models for AIH. These alternative antigen-induced AIH models are cytokine-dependent and antigen-independent liver injury models, and are acute rather than chronic processes. The liver autoantigen-induced model can only be induced in the NOD mouse model. The animal models of PBC and PSC are also divided into spontaneous models and induced models. The disadvantage of the spontaneous model NOD.c3.c4 mouse model of PBC is that the liver injury symptoms shown are more extensive than those in humans, while most of the induced models cannot replicate the histological features of PBC. The limitation of the spontaneous model Mdr2- / - mouse model of PSC is that it does not have the key features of PSC, and the chemically induced model of PSC also lacks the key features of PSC and has a high mortality rate of mice due to the high toxicity of chemical drugs. When such animal models are applied to the screening of drugs for autoimmune liver diseases, the metabolic cycle of the administered drugs in mice is too short, which is not conducive to monitoring the disease development process of mice and the impact of drugs on the disease development process. Summary of the Invention

[0005] In view of this, the present invention provides a method for screening drugs for autoimmune liver diseases, including:

[0006] Provide test group animals and reference group animals, wherein both the test group animals and the reference group animals include animal models of autoimmune liver disease, and the genotype of the animal model of autoimmune liver disease is the genotype of conditional knockout of adenosine monophosphate-activated protein kinase / apolipoprotein in regulatory T cells;

[0007] Administer a candidate drug for autoimmune liver disease to the test group animals;

[0008] Monitor the changing trend of the first biochemical index data of the test group animals and the changing trend of the second biochemical index data of the reference group animals;

[0009] Determine the target drug from the candidate drugs by comparing the changing trend of the first biochemical index data and the changing trend of the second biochemical index data.

[0010] According to an embodiment of the present invention, the method for screening drugs for autoimmune liver disease further includes:

[0011] After a preset time period, sacrifice the test group animals and the reference group animals;

[0012] Perform pathological sectioning on the test group animals and the reference group animals respectively;

[0013] Determine the target drug from the candidate drugs by analyzing the pathological section results of the test group animals and the reference group animals.

[0014] According to an embodiment of the present invention, the method for establishing an animal model of autoimmune liver disease includes:

[0015] Mate apolipoprotein genotype mice with first genotype mice in a preset ratio to obtain first-generation double-gene heterozygous mice; wherein, the genotype of the first genotype mice is the genotype of conditional knockout of adenosine monophosphate-activated protein kinase in regulatory T cells.

[0016] Mate the first-generation double-gene heterozygous mice with each other to obtain second genotype mice, wherein the genotype of the second genotype mice is the genotype of conditional knockout of adenosine monophosphate-activated protein kinase / apolipoprotein in regulatory T cells;

[0017] After feeding the second genotype mice for a preset time period, obtain an animal model of autoimmune liver disease.

[0018] According to an embodiment of the present invention, the method for constructing the model of the first genotype mice includes:

[0019] The zero-generation mice are mated with wild mice to obtain conditional knockout of adenosine monophosphate-activated protein kinase genotype heterozygous mice; among them, the zero-generation mice are obtained by artificially implanting target fertilized eggs into the body of female mice, and the target fertilized eggs include guide ribonucleic acid, targeting adenosine monophosphate-activated protein kinase, and endonuclease;

[0020] The conditional knockout of adenosine monophosphate-activated protein kinase genotype heterozygous mice are mated with each other to obtain conditional knockout of adenosine monophosphate-activated protein kinase genotype homozygous mice;

[0021] The conditional knockout of adenosine monophosphate-activated protein kinase genotype homozygous mice are mated with mice carrying regulatory T cell lineage-specific transcription factor to obtain regulatory T cell conditional knockout of adenosine monophosphate-activated protein kinase genotype heterozygous mice;

[0022] The regulatory T cell conditional knockout of adenosine monophosphate-activated protein kinase genotype heterozygous mice are mated with each other to obtain the first genotype mice.

[0023] According to an embodiment of the present invention, the targeting adenosine monophosphate-activated protein kinase is obtained by specifically labeling and modifying adenosine monophosphate-activated protein kinase.

[0024] According to an embodiment of the present invention, the specific labeling and modification of adenosine monophosphate-activated protein kinase includes:

[0025] By using a chromosomal site-specific recombinase system, a specific labeling sequence is placed at each end of adenosine monophosphate-activated protein kinase to obtain the targeting adenosine monophosphate-activated protein kinase.

[0026] According to an embodiment of the present invention, the method for constructing a model of apolipoprotein genotype mice includes:

[0027] Targetedly knockout the apolipoprotein gene in mouse embryonic stem cells to obtain targeted cells;

[0028] Inject the targeted cells into the blastocysts of tool mice to obtain chimeras;

[0029] The chimeras are bred according to a preset mating method to obtain apolipoprotein genotype mice.

[0030] According to an embodiment of the present invention, breeding the chimeras according to a preset mating method to obtain apolipoprotein genotype mice includes:

[0031] The chimeras are first backcrossed and then self-crossed to obtain homozygous mice of the intermediate generation;

[0032] The homozygous mice of the intermediate generation are backcrossed to obtain apolipoprotein genotype mice.

[0033] According to an embodiment of the present invention, an apolipoprotein genotype mouse is mated with a first genotype mouse in a preset ratio to obtain a first-generation double-gene heterozygous mouse, including:

[0034] The apolipoprotein genotype mouse and the first genotype mouse are placed in a laminar flow animal house in a preset ratio for mating to obtain a first-generation double-gene heterozygous mouse, wherein the temperature in the laminar flow animal house is 18-22 °C, and the humidity in the laminar flow animal house is 40%-60%.

[0035] According to an embodiment of the present invention, the preset ratio includes any one of the following:

[0036] The ratio of the number of male apolipoprotein genotype mice to the number of female first genotype mice is 1:2;

[0037] The ratio of the number of male first genotype mice to the number of female apolipoprotein genotype mice is 1:2.

[0038] According to an embodiment of the present invention, since the animal model provided by the embodiment of the present invention is a spontaneous autoimmune liver disease model, a chronic pathological change appears in the animal liver, which is beneficial to observing the change process of the disease course and the influence of drugs on the disease course, and improving the efficiency of drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematically shows the HE staining result of the liver section of AMPKα1 fl / fl / Foxp3 cre / ApoE - / - mouse.

[0040] Figure 2 Schematically shows the serum immunoglobulin G (IgG) level of AMPKα1 fl / fl / Foxp3 cre / ApoE - / - mouse.

[0041] Figure 3 Schematically shows the serum antinuclear antibody (ANA) level of AMPKα1 fl / fl / Foxp3 cre / ApoE - / - mouse.

[0042] Figure 4 Schematically shows the anti-double-stranded DNA (anti-dsDNA) level of AMPKα1 fl / fl / Foxp3 cre / ApoE - / - mouse.

[0043] Figure 5a Schematically shows the flow cytometry analysis of AMPKα1 fl / fl / Foxp3 cre / ApoE - / - Expression of CD62L and CD44 in activated CD4 + T cells in the mesenteric lymph nodes of mice at 8 weeks;

[0044] Figure 5b Schematically shows the flow cytometry analysis of AMPKα1 fl / fl / Foxp3 cre / ApoE - / - Expression of CD62L and CD44 in unactivated CD4 + T cells in the mesenteric lymph nodes of mice at 8 weeks;

[0045] Figure 6 Schematically shows the flow cytometry analysis of AMPKα1 fl / fl / Foxp3 cre / ApoE - / - Expression of IL17 in CD4 + T cells in the mesenteric lymph nodes of mice at 8 weeks.

[0046] Figure 7 Schematically shows the gene determination results of the animal model of autoimmune liver disease in the present invention. Detailed implementation manners

[0047] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further describes the present invention in detail with reference to specific embodiments and the accompanying drawings.

[0048] According to an embodiment of the present invention, a method for establishing an animal model of autoimmune liver disease includes: mating apolipoprotein genotype mice with first genotype mice in a preset ratio to obtain first-generation double-gene heterozygous mice; wherein, the genotype of the first genotype mice is a genotype in which regulatory T cells conditionally knockout adenosine monophosphate-activated protein kinase.

[0049] Interbreeding the first-generation double-gene heterozygous mice to obtain second genotype mice, wherein the genotype of the second genotype mice is a genotype in which regulatory T cells conditionally knockout adenosine monophosphate-activated protein kinase / apolipoprotein genotype;

[0050] After feeding the second genotype mice for a preset duration, an animal model of autoimmune liver disease is obtained.

[0051] According to an embodiment of the present invention, the model construction method of the first genotype mice includes:

[0052] The zero-generation mice are mated with wild mice to obtain heterozygous mice with conditional knockout of adenosine monophosphate-activated protein kinase; among them, the zero-generation mice are obtained by artificially implanting target fertilized eggs into the body of a female mouse, and the target fertilized eggs include guide ribonucleic acid, adenosine monophosphate-activated protein kinase targeting, and endonuclease.

[0053] The heterozygous mice with conditional knockout of adenosine monophosphate-activated protein kinase are mated with each other to obtain homozygous mice with conditional knockout of adenosine monophosphate-activated protein kinase.

[0054] The homozygous mice with conditional knockout of adenosine monophosphate-activated protein kinase are mated with mice carrying the regulatory T cell lineage-specific transcription factor to obtain heterozygous mice with conditional knockout of adenosine monophosphate-activated protein kinase in regulatory T cells.

[0055] The heterozygous mice with conditional knockout of adenosine monophosphate-activated protein kinase in regulatory T cells are mated with each other to obtain the first genotype mice.

[0056] According to the embodiments of the present invention, the zero-generation mice can be obtained by co-injecting guide ribonucleic acid (sgRNA), adenosine monophosphate-activated protein kinase targeting (Donor DNA), and endonuclease (Cas9) into fertilized eggs, and then implanting the fertilized eggs into the uterus of pseudopregnant female mice to produce the F0 generation mice. Among them, Donor DNA is constructed by designing and in vitro transcribing sgRNA and simultaneously performing flox modification on the AMPKα1 gene.

[0057] According to the embodiments of the present invention, through the Cre / LoxP system, one loxP sequence can be placed at each end of a target DNA sequence to be knocked out to obtain flox (flanked by loxP) mice. The flox mice are mated with mice carrying cell-specific expression of Cre to obtain mice with specific knockout of the AMPKα1 gene in Treg cells.

[0058] According to the embodiments of the present invention, the method for constructing a model of apolipoprotein genotype mice includes:

[0059] Targetedly knocking out the apolipoprotein gene in mouse embryonic stem cells to obtain targeted cells;

[0060] Injecting the targeted cells into the blastocysts of tool mice to obtain chimeras;

[0061] Breeding the chimeras according to a preset mating method to obtain apolipoprotein genotype mice.

[0062] According to an embodiment of the present invention, for example, by targeting and knocking out the ApoE gene in E14TG2a ES cells and injecting the targeted cells into C57BL / 6J blastocysts, the gene knockout model is established. The obtained chimeras are backcrossed for 10 generations (N10), and homozygous mice are obtained by self-crossing. Then, they are backcrossed for one more generation (N11) to obtain apolipoprotein genotype mice.

[0063] According to an embodiment of the present invention, ApoE- / - mice and AMPKα1fl / fl / Foxp3cre mice can be placed in a clean laminar flow animal room, with the room temperature controlled at 18 - 22 °C and the humidity controlled at 40% - 60%. The mouse cages, bedding, and feed are all sterilized, and the drinking water is sterilized by high-temperature and high-pressure treatment. During the whole feeding process, the feed and water are replenished in a timely manner, and the bedding is changed every 3 days. Then, the ApoE- / - mice and AMPKα1fl / fl / Foxp3cre mice are mated in the way of one male mouse and two female mice in the same cage to obtain F1 generation double-gene heterozygous mice. Then, the F1 generation double-gene heterozygous mice are interbred to obtain F2 generation mice with the genotype of AMPKα1 fl / fl / Foxp3 cre / ApoE - / - mice. The AMPKα1 fl / fl / Foxp3 cre / ApoE - / - mice are fed a Western diet containing 0.21% cholesterol and 21% fat for 6 - 8 weeks, and the mice show histological features similar to those of human autoimmune liver disease.

[0064] To verify the histological features of the animal model provided by the present invention, the liver of one-year-old mice in the above autoimmune liver disease animal model is fixed and dehydrated and then made into paraffin sections, and the experimental results as shown in Figure 1 are obtained by HE staining.

[0065] Figure 1 Schematically shows the HE staining results of the liver sections of AMPKα1 fl / fl / Foxp3 cre / ApoE - / - mice.

[0066] As shown in Figure 1 shown, Figure 1 in the experimental group, the liver of the mice shows the same histological features as human autoimmune liver disease, including portal vein inflammation (inflammatory infiltration around the hepatic portal vein), interface hepatitis (portal infiltration extending to the lobule), and lobular inflammation (infiltrating the hepatic parenchyma). Figure 1 The control group in is wild-type mice, which do not show the pathological features of the liver of clinical patients.

[0067] According to an embodiment of the present invention, the animal model of the present invention can exhibit histological features similar to those of human autoimmune liver disease and can be used as an animal model for screening drugs for autoimmune liver disease. It can be applied to study the role of AMPK in Treg cells and its indirect effect on autoimmune liver disease, as well as the molecular mechanism of pharmacological activation of AMPK in the treatment of autoimmune diseases.

[0068] In order to verify whether the change characteristics of the biochemical indexes of the autoimmune liver disease animal model in the embodiment of the present invention conform to the change characteristics of the biochemical indexes of clinically diagnosed patients with autoimmune liver disease, the serum biomarker biochemical indexes of the animal model mice were measured by enzyme-linked immunosorbent assay, such as serum immunoglobulin G, serum antinuclear antibody, and anti-double-stranded DNA. Mice at 8 weeks of age were used for the experiment. The experimental group was the animal model in the embodiment of the present invention, and the control group was wild normal mice, that is, mice without artificial gene fragment knockout. The experimental results are as Figures 2 - 4 shown.

[0069] As Figure 2 shown, from the results of measuring the serum immunoglobulin G in the serum of the animal model mice, it can be seen that the serum immunoglobulin G of the animal model mice increased.

[0070] As Figure 3 shown, from the results of measuring the serum antinuclear antibody in the serum of the animal model mice, it can be seen that the serum antinuclear antibody of the animal model mice increased.

[0071] As Figure 4 shown, from the results of measuring the anti-double-stranded DNA in the serum of the animal model mice, it can be seen that the anti-double-stranded DNA of the animal model mice increased.

[0072] Based on Figures 2 - 4 the comprehensive experimental results shown, it can be seen that the change characteristics of the biomarker biochemical indexes in the serum of the animal model mice provided in the embodiment of the present invention conform to the change characteristics of the biochemical indexes of clinically diagnosed patients with autoimmune liver disease.

[0073] In order to verify whether the change characteristics of specific cells in the autoimmune liver disease animal model in the embodiment of the present invention conform to the change characteristics of specific cells in clinically diagnosed patients with autoimmune liver disease, flow cytometry was used to measure the changes in specific cells in the peripheral immune organ mesenteric lymph nodes of the animal model mice. The experimental results are as Figure 5a , Figure 5b and Figure 6 shown.

[0074] According to an embodiment of the present invention, the specific cell can be a specific subset of T cells. Classified by surface-expressed antigens, T cells can be divided into CD4+ T cell subsets and CD8+ T cell subsets. Classified by the antigen response situation, T cells can be divided into naive T cells, activated T cells, and memory T cells. Classified by function, T cells can be divided into helper T cells, inhibitory T cells, and cytotoxic T cells.

[0075] Figure 5a Schematically shows the expression of CD62L and CD44 in activated CD4+ T cells in the mesenteric lymph nodes of AMPKα1fl / fl / Foxp3cre / ApoE- / - mice at 8 weeks by flow cytometry analysis;

[0076] Figure 5b Schematically shows the expression of CD62L and CD44 in non-activated CD4+ T cells in the mesenteric lymph nodes of AMPKα1fl / fl / Foxp3cre / ApoE- / - mice at 8 weeks by flow cytometry analysis;

[0077] As Figures 5a - 5b shown, the knockout model mice of the embodiments of the present invention represented by the experimental group, and the control group represents wild-type, i.e., normal mice. It can be seen that the T cells of the model mice of the embodiments of the present invention are activated.

[0078] As Figure 6 shown, Figure 6 it shows that the CD4+ T cells producing IL-17 in this mouse model are significantly increased. Clinically, the CD4+ cells producing IL-17 in patients with autoimmune liver disease are significantly increased, which is consistent with the change trend shown by the animal model of the embodiments of the present invention.

[0079] Thus, it can be seen that the change characteristics of the specific cells of the animal model mice of the embodiments of the present invention are consistent with the change characteristics of the specific cells of clinically autoimmune liver disease patients.

[0080] During the establishment of the animal model, it is necessary to select mice with the target genotype from each generation of mice through genotype identification.

[0081] According to an embodiment of the present invention, the following steps can be used to identify the genotype of mice:

[0082] Mark the mice according to the Yale toe clipping method, obtain mouse tissues (tail, toe or ear, etc.) and put them into a 1.5 ml EP tube;

[0083] Add 100 μl of 50 mM NaOH, heat in a metal bath at 100 °C for 30 min; after cooling to room temperature, centrifuge at 3000 g for 30 s, and then add 8.3 μl of 1 M TrisHCl to each sample;

[0084] Vortex to mix well; after centrifuging at 12000 g for 5 min, pipette the supernatant into a new 1.5 ml EP tube, place it in a 4 °C refrigerator, and for long-term storage, it can be stored in a -20 °C refrigerator;

[0085] Prepare the premix (other components without the sample) according to a 15 μl PCR system, aliquot it into PCR tubes, and add it to the samples;

[0086] Place the PCR tubes in a PCR instrument, set the PCR cycle according to the mouse genotype identification protocol, and the PCR reaction conditions are shown in the following table:

[0087] Table of PCR reaction conditions

[0088]

[0089] Weigh the agarose according to the agarose gel concentration required in the mouse number and genotype identification protocol, place it in a conical flask, add a certain amount of TAE buffer, and boil it in a microwave oven at medium heat for 3 - 5 min until the liquid becomes transparent;

[0090] Let it stand for 2 min, add gelred according to a 1 / 10000 ratio, mix well and pour it into the electrophoresis tank, and let it stand for 20 min to solidify;

[0091] Take 7 μl of the PCR product and add it to the gel well, and add an equal amount of DNA marker to a separate well;

[0092] Set the electrophoresis instrument at 120 V for 40 min, and observe the band position and size in a gel imager. The experimental results are as Figure 7 shown. The PCR identification results of three genotypes of mice with conditional knockout of the AMPK α1 gene in Treg cells can be used to select the required genotype of mice according to the PCR identification results.

[0093] As Figure 7 shown, fl / fl represents homozygous, fl / + represents heterozygous, + / + represents wild type, - / - represents homozygous, + / - represents heterozygous, + / + represents wild type. The mice we need are determined by looking at the combination of both genes, and both genes need to be homozygous. Usually, AMPKα1 fl / fl / Foxp3 - / - represents homozygous, and AMPKα1 fl / + / Foxp3 + / - represents heterozygous, and AMPKα1 + / + / Foxp3 + / + It represents the wild type. AMPKα1 and Prakaa1 in the figure have a corresponding relationship between the protein and the coding gene. That is, the Prakaa1 gene encodes the AMPKα1 protein.

[0094] Autoimmune liver disease (AILD) mainly refers to liver inflammatory lesions mediated by autoimmunity. Traditionally, it is clinically divided into three types: autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC). A large number of clinical studies have shown that prednisone is an immunosuppressant effective in treating AIH and can significantly improve the long-term survival rate of AIH patients. However, the condition of AIH is complex, and more drugs still need to be developed for refractory AIH patients. Currently, the most commonly used drug for PBC in clinical practice is ursodeoxycholic acid, but the therapeutic effect of ursodeoxycholic acid is still not obvious in some patients. For PSC, there is currently no drug that can control its basic pathological process. The main treatment goal is to relieve cholestasis and hepatitis-related symptoms, and ursodeoxycholic acid may have a certain therapeutic effect on it.

[0095] The characteristics of the animal model in the prior art are acute liver injury. This mouse gradually dies after ten days and has basically all died after 30 days. This kind of animal model cannot be used to study the therapeutic effect of ursodeoxycholic acid drugs.

[0096] In view of this, according to the embodiments of the present invention, an autoimmune liver disease animal model with a genotype of conditional knockout of adenosine monophosphate-activated protein kinase / apolipoprotein in regulatory T cells is used to screen candidate drugs.

[0097] Provide test group animals and reference group animals. Among them, both the test group animals and the reference group animals include an autoimmune liver disease animal model, and the genotype of the autoimmune liver disease animal model is conditional knockout of adenosine monophosphate-activated protein kinase / apolipoprotein in regulatory T cells;

[0098] Administer an autoimmune liver disease candidate drug to the test group animals;

[0099] Monitor the changing trend of the first biochemical index data of the test group animals and the changing trend of the second biochemical index data of the reference group animals;

[0100] Determine the target drug from the candidate drugs by comparing the changing trend of the first biochemical index data and the changing trend of the second biochemical index data.

[0101] According to an embodiment of the present invention, for example, the test group animals can be divided into 5 groups, and candidate drugs a1 to a5 are respectively administered. The reference group animals are not administered any drugs. The test group animals and the reference group animals are fed in the same environment, and the changing trend of the biochemical index data is monitored. The candidate drug corresponding to the group of test group animals whose changing trend of the biochemical index data is significantly decreased compared with that of the reference group animals can be used as the target drug.

[0102] According to an embodiment of the present invention, the above method for screening drugs further includes:

[0103] After a preset time period, the test group animals and the reference group animals are sacrificed;

[0104] Pathological sections are respectively made for the test group animals and the reference group animals;

[0105] By analyzing the pathological section results of the test group animals and the reference group animals, the target drug is determined from the candidate drugs.

[0106] According to an embodiment of the present invention, different preset time periods can be set, and by analyzing the pathological section results, the pathological change characteristics of chronic autoimmune liver disease and the influence of different candidate drugs on the pathological changes can be determined. While determining the effective drug, the side effect situation of the effective drug can be determined.

[0107] According to an embodiment of the present invention, since the animal model provided by the embodiment of the present invention is a spontaneous autoimmune liver disease model, a chronic pathological change appears in the animal liver, which is beneficial to observing the change process of the disease course and the influence of the drug on the disease course, and improving the efficiency of drug screening.

[0108] The above specific embodiments have further elaborated the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above is only the specific embodiments of the present invention and is not used to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for screening drugs for autoimmune liver diseases, comprising: Providing a test group of animals and a reference group of animals, wherein both the test group of animals and the reference group of animals include animal models of autoimmune liver diseases, and wherein the genotype of the animal model of autoimmune liver diseases is a regulatory T cell conditional knockout of adenosine monophosphate-activated protein kinase / apolipoprotein genotype; Administering a candidate drug for autoimmune liver diseases to the test group of animals; Monitoring the changing trend of the first biochemical index data of the test group of animals and the changing trend of the second biochemical index data of the reference group of animals; Determining a target drug from the candidate drugs by comparing the changing trend of the first biochemical index data and the changing trend of the second biochemical index data; Wherein the method for establishing the animal model of autoimmune liver diseases includes: Mating apolipoprotein genotype mice with first genotype mice in a preset ratio to obtain first-generation double-gene heterozygous mice; wherein the genotype of the first genotype mice is a regulatory T cell conditional knockout of adenosine monophosphate-activated protein kinase genotype; Intercrossing the first-generation double-gene heterozygous mice to obtain second genotype mice, wherein the genotype of the second genotype mice is a regulatory T cell conditional knockout of adenosine monophosphate-activated protein kinase / apolipoprotein genotype; After feeding the second genotype mice for a preset duration, obtaining the animal model of autoimmune liver diseases; The method for constructing the model of apolipoprotein genotype mice includes: Targetedly knocking out the apolipoprotein gene in mouse embryonic stem cells to obtain target cells; Injecting the target cells into the blastocysts of tool mice to obtain chimeras; Breeding the chimeras in a preset mating manner to obtain the apolipoprotein genotype mice.

2. The method according to claim 1, further comprising: After a preset duration, sacrificing the test group of animals and the reference group of animals; Performing pathological sectioning on the test group of animals and the reference group of animals respectively; Determining the target drug from the candidate drugs by analyzing the pathological section results of the test group of animals and the reference group of animals.

3. The method according to claim 1, wherein The method for constructing the model of the first genotype mice includes: Mating zero-generation mice with wild mice to obtain heterozygous mice with a conditional knockout of adenosine monophosphate-activated protein kinase genotype; wherein the zero-generation mice are obtained by artificially implanting target fertilized eggs into the body of a female mouse, and the target fertilized eggs include guide ribonucleic acid, targeting adenosine monophosphate-activated protein kinase, and endonuclease; Intercrossing the heterozygous mice with a conditional knockout of adenosine monophosphate-activated protein kinase genotype to obtain homozygous mice with a conditional knockout of adenosine monophosphate-activated protein kinase genotype; Mating the homozygous mice with a conditional knockout of adenosine monophosphate-activated protein kinase genotype with mice carrying a regulatory T cell lineage-specific transcription factor to obtain heterozygous mice with a regulatory T cell conditional knockout of adenosine monophosphate-activated protein kinase genotype; The heterozygous mice with conditional knockout of adenosine monophosphate-activated protein kinase in regulatory T cells were crossbred to obtain the first genotype mice.

4. The method according to claim 3, wherein The targeting adenosine monophosphate-activated protein kinase is obtained by specific labeling modification of adenosine monophosphate-activated protein kinase.

5. The method according to claim 4, wherein The specific labeling modification of adenosine monophosphate-activated protein kinase includes: By using the chromosomal site-specific recombinase system, a specific labeling sequence is placed at each end of the adenosine monophosphate-activated protein kinase to obtain the targeting adenosine monophosphate-activated protein kinase.

6. The method according to claim 1, wherein The step of breeding the chimeras according to a preset mating method to obtain the apolipoprotein genotype mice includes: The chimeras are first backcrossed and then self-crossed to obtain homozygous mice of the intermediate generation; The homozygous mice of the intermediate generation are backcrossed to obtain the apolipoprotein genotype mice.

7. The method according to claim 1, wherein The step of mating the apolipoprotein genotype mice and the first genotype mice in a preset ratio to obtain the first-generation double-gene heterozygous mice includes: The apolipoprotein genotype mice and the first genotype mice are placed in a laminar flow animal house and mated in a preset ratio to obtain the first-generation double-gene heterozygous mice. Among them, the temperature in the laminar flow animal house is 18-22 °C, and the humidity in the laminar flow animal house is 40%-60%.

8. The method according to claim 1, wherein The preset ratio includes any one of the following: The ratio of the number of male apolipoprotein genotype mice to the number of female first genotype mice is 1:2; The ratio of the number of male first genotype mice to the number of female apolipoprotein genotype mice is 1:2.

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