Construction and application of lipid metabolism related gene Nrf1-453 subtype specific overexpression mouse model

Abstract

The invention discloses construction and application of a lipid metabolism related gene Nrf1-453 specific overexpression mouse model, and relates to the technical field of animal models, in particular to the research field of transgenic functional mice and metabolism related fatty liver diseases. According to the animal model disclosed by the invention, a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) technology is applied to construct a mouse model with Nrf1-453 conditional gene overexpression; the method comprises the following steps of: establishing a liver cell Nrf1-453 specific overexpression mouse model (Nrf1-453 (L)-OE), exploring the effect of a 453 transcript in homeostasis imbalance of cholesterol metabolism through a high cholesterol feed (HCD), and comprehensively and carefully elaborating the effect and molecular mechanism of the subtype in the occurrence and development of MAFLD caused by the HCD. The metabolism-related fatty liver disease animal model prepared by adopting the construction method disclosed by the invention can be used for research on effects and molecular mechanisms in occurrence and development of related diseases and screening and / or evaluation of drugs for the related diseases.

Description

Technical Field

[0001] The present invention relates to the field of animal model technology, specifically to the field of transgenic functional mice and metabolism-related fatty liver disease research, and more specifically to the establishment of a lipid metabolism-related gene Nrf1 Construction and application of -453 isoform-specific overexpression mouse model. Background Art

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD) is considered to be one of the most common liver diseases in the world. Its disease spectrum includes simple steatosis, metabolic-associated fatty liver hepatitis, and end-stage liver fibrosis and cirrhosis. Hypercholesterolemia is highly correlated with the occurrence of MAFLD, and feeds rich in high-cholesterol diets are more likely to induce the occurrence of MAFLD. The transcription factor Nuclear factor erythroid-derived 2-related factor 1 (NF-2) is a transcription factor that is involved in the development of MAFLD. Nfe2l1 ) belongs to the CNC-bZIP transcription factor subfamily and plays a core role in transcriptional control. Its downstream genes are usually involved in antioxidant response, proteasome homeostasis, lipid metabolism, etc., referred to as Nrf1 .because Nrf1 The embryonic lethality of α-synuclein makes it impossible to obtain adult mice with systemic deletion, but the specific deletion in hepatocytes ( Nrf1 In the absence of other exposure factors, mice with (L)-KO spontaneously develop fatty liver and gradually develop terminal liver cancer. Based on the latest version of the Ensemble genomebrowser database, mice Nrf1 The gene (Gene ID: ENSMUSG00000038615) is located at the distal end of chromosome 11, with a total length of 12,554 bp and 10 exons. It can produce 12 transcripts through variable splicing and can synthesize at least 4 protein subtypes that may have transcriptional regulatory functions, containing 742, 741, 583, and 453 amino acids, respectively. According to the number of amino acids, it can be divided into: long-form NRF1, namely L-NRF1s, mainly including two subtypes of NRF1-741aa and NRF1-742aa, and short-form NRF1, namely S-NRF1s, refers to two subtypes of NRF1-583aa and NRF1-453aa. Cellular molecular biology studies have shown that NRF1-453aa negatively regulates the expression of genes related to antioxidant response, but there is no animal model to study the physiological and pathological functions of NRF1-453aa. Therefore, it is urgent to construct NRF1-453aa subtype-specific overexpression mice, based on which Nrf1 The role of -453(L)-OE mice in the occurrence and development of MAFLD also has important application value. Summary of the invention

[0003] In view of the problems existing in the prior art, the present invention aims to provide a lipid metabolism related gene Nrf1 -453 specific overexpression mouse model and its application. This model was constructed using CRISPR-Cas9 technology Nrf1 -453 conditional gene overexpression mouse model; then establish hepatocyte Nrf1 -453 specific overexpression mouse model, through HCD diet exposure, to explore the role of 453 transcripts in the imbalance of cholesterol metabolism homeostasis, and comprehensively and meticulously explain the role and molecular mechanism of this subtype in the occurrence and development of MAFLD caused by HCD.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0005] The present invention discloses an animal model of metabolic-related fatty liver disease, characterized in that the model is a liver cell Nrf1 -453-specific overexpression mouse model.

[0006] The present invention also discloses a method for constructing the above-mentioned animal model, characterized in that the method specifically comprises the following steps: (1) Construction Rosa26 Homologous recombination DNA vector; (2) Targeting through in vitro transcription Rosa 26 gRNA and Cas9 mRNA of the gene; (3) Injecting the homologous recombination DNA vector, Cas9 mRNA and gRNA into the fertilized eggs of mice to obtain chimeric mice F0; (4) F0 generation mice were mated with wild-type mice to obtain F1 generation heterozygous mice ( Nrf1 -453 fl / + ); (5) Self-fertilization of F1 heterozygous mice to obtain F2 homozygous mice ( Nrf1 -453 fl / fl ); (6) F2 homozygous mice were crossed with hepatocyte-specific mice ( Alb -Cre) hybridization to obtain hepatocyte-specific Nrf1 -453 overexpressing mice and their littermate control mice ( Nrf1 -453-KI; Nrf1 -453(L)-OE).

[0007] Furthermore, the specific process of step (1) is as follows: gRNA is located at Rosa26 On exon 1 of the gene, the homologous recombination DNA vector contains 2115 bp of 5' homology arm, 2153 bp of 3' homology arm, 1726 bp of enhancer, 3254 bp of "Stop" site, two 34 bp LoxP regions and 1368 bp of Nrf1 -453 coding region fragment.

[0008] Furthermore, the DNA sequence of the action site of gRNA is: 5'-CTCCAGTCTTTCTAGAAGATGGG-3'.

[0009] The present invention also discloses an application of the above-mentioned metabolic-related fatty liver disease animal model, characterized in that the application includes application in the study of the pathogenesis of metabolic-related fatty liver disease.

[0010] The present invention also discloses an application of the above-mentioned metabolic-related fatty liver disease animal model, characterized in that the application includes application in screening and / or evaluation of metabolic-related fatty liver disease drugs.

[0011] The present invention also discloses an application of a mouse model obtained by any of the above-mentioned methods for constructing an animal model in the study of the pathogenesis of metabolism-related fatty liver disease.

[0012] The present invention also discloses an application of a mouse model obtained by any of the animal model construction methods described above in drug screening and / or evaluation of metabolism-related fatty liver disease.

[0013] The principle of the present invention is: mice Rosa26 The gene is a non-coding gene located on chromosome 6 of the mouse. It has been proven to be expressed in most tissues and cells. So far, no functionally expressed protein has been found. Because its site is easy to insert and homologous recombination occurs easily, it is considered to be one of the safest fixed-site integration sites. The present invention constructs a homologous DNA vector in vitro, inserts it into a fertilized egg based on CRISPR-Cas9 technology, and then transfers it into the uterus of a pseudo-pregnant female mouse, and obtains F0 generation mice by natural delivery. In this model, the Cre-LoxP system is used to insert a "Stop" fragment containing LoxP fragments at both ends at the front end of the coding region of the target gene. The conditional overexpression mouse can only start the expression of the target gene by cutting the "Stop" site when it is hybridized with a mouse containing a specific Cre recombinase, thereby achieving the effect of overexpressing the target gene.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0015] This invention uses CRISPR-Cas9 technology to construct Nrf1 The mouse model of conditional gene overexpression of NRF1-453 can further clarify the role and mechanism of NRF1-453aa in the occurrence and development of MAFLD caused by a high cholesterol diet, and get rid of the problem of different transcript functions of all subtypes interfering with each other; it has been confirmed that HCD feeding will lead to the occurrence of MAFLD, which increases the feasibility of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 1A is a schematic diagram of the construction principle of transgenic mice; 1B is a diagram of the genotype identification results of transgenic mouse tails; 1C is the protein level of NRF1-453 subtypes in primary hepatocytes and its quantification.

[0017] Figure 2 The following are the basic indicators of the transgenic mice and their controls at 10 weeks of age under normal feeding conditions. 2A is the body weight parameter; 2B is the liver organ coefficient; 2C is the fasting blood glucose level; 2D is a representative liver macroscopic picture; 2E is a representative H&E staining representative picture.

[0018] Figure 3 These are the basic indicators and pathological indicators after one week of high cholesterol diet modeling in the embodiment of the present invention. 3A is the body weight parameter; 3B is the liver organ coefficient; 3C is the fasting blood glucose level; 3D is a representative liver macroscopic picture, H&E staining picture, oil red staining picture, immunohistochemical F4 / 80 staining picture; 3E is the plasma and liver lipid levels; 3F is the liver damage index. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0020] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0021] A mouse model with increased susceptibility to metabolic-related fatty liver disease induced by high cholesterol diet is established, comprising the following steps: 1. Construction and identification of transgenic mice; 2. Construction of experimental animal models; 3. Phenotypic monitoring of experimental animals.

[0022] The above contents are explained below in combination with specific verification experiments.

[0023] according to Figure 1 The principle of A Nrf1 -453-KI transgenic mice, through Figure 1 The PCR results of B preliminarily confirmed that the mouse was successfully constructed. Figure 1 C Demonstrates effective overexpression of mouse 453 isoform at the translation (protein) level.

[0024] The transgenic mice were fed with conventional diets, and their body weights were measured at 10 weeks. Figure 2 A) Liver organ coefficient ( Figure 2 B) and measure fasting blood sugar levels ( Figure 2 C), take a gross picture of the liver ( Figure 2 D) and pathological staining of the liver's underlying structures ( Figure 2 E), compared with the KI group, Nrf1 There were no significant differences in the various indicators of mice in the -453(L)-OE group, indicating that under basic conditions Nrf1 Specific overexpression of the -453 isoform in hepatocytes did not affect the liver structure and metabolism of experimental mice.

[0025] The experimental mice were grouped according to genotype and fed with HCD for one week to construct the MAFLD model. Figure 3 As shown in A, compared with KI, Nrf1 The body weight of mice in the -453(L)-OE+HCD group did not differ, but the liver was enlarged and the organ coefficient was statistically different ( Figure 3 B), no significant changes in fasting blood sugar ( Figure 3 C).

[0026] Pathological staining of liver tissue revealed that Figure 3 As shown in D, compared with KI, Nrf1 In the -453(L)-OE+HCD group, obvious lipid droplets were observed (red arrows), the number of cells positive for oil red staining increased (yellow arrows), and more inflammatory foci appeared (blue arrows).

[0027] The results of lipid determination showed that Nrf1 -453(L)-OE+HCD group had elevated plasma triglyceride (TG) levels ( Figure 3 E), there was no significant difference in total cholesterol (T-CHO) levels, but there was no significant difference in liver TG levels, and T-CHO increased significantly, which was consistent with the results of pathological staining, proving Nrf1 -453(L)-OE increases the accumulation of cholesterol in the liver, accelerates lipid metabolism disorders in the liver of experimental mice under the same modeling cycle, and increases the susceptibility to MAFLD.

[0028] AST and ALT test results showed that Nrf1-453(L)-OE+HCD group had a higher ALT level and a statistically significant difference ( Figure 3 F), indicating that the liver damage is more serious. * P <0.05 vs the same treatment group Nrf1 -453-KI, # P <0.05 vs Nrf1 -453-OE control treatment group.

[0029] In summary, Nrf1 Overexpression of the -453 isoform specifically in hepatocytes accelerates the development of HCD diet-induced MAFLD.

[0030] Some of the experimental steps required for use in the above examples are shown below.

[0031] 1. Construction and identification of transgenic mice: Transgenic mice were transformed into Nrf1 The -453 coding region was integrated into the mouse genome, and its expression was restricted by the "Stop" site, which interacted with the hepatocyte-specific Alb -Cre mice were crossed to obtain control mice and hepatocyte-specific overexpression mice ( Nrf1 -453-KI, Nrf1 -453(L)-OE). The genotype of transgenic mice was identified by PCR amplification of mouse tail DNA, and primary mouse hepatocytes were obtained by in situ collagen perfusion. Total protein was extracted after adherent culture, and its overexpression effect was identified from the translation dimension.

[0032] The specific steps are as follows.

[0033] 1.1 Rat tail DNA identification.

[0034] 1.1.1 Extraction of mouse tail DNA: Newborn mice were weaned 21 days after birth, and the mice were numbered by marking their toes with tattoo liquid. The scissors and the surface of the mouse tail were disinfected with 75% alcohol, and 1 mm of the mouse tail tip was cut. 100 μL of effective alkaline tissue lysis solution was added and heated at 95 °C in a dry-wet thermostat for 30 min (no more than 1 h). The digestion product was cooled to room temperature, and 100 μL of acidic neutralization solution was added to mix thoroughly. It can be used immediately (or frozen in a -20 °C refrigerator to avoid repeated freezing and thawing).

[0035] 1.1.2 PCR amplification.

[0036] ① Primer sequence and configuration.

[0037] According to the primer sequences, we commissioned Sangon Biotechnology (Shanghai) Co., Ltd. to synthesize the primers, see Table 1 below.

[0038] Table 1 Primer sequences .

[0039] Prepare 10 μM primer stock solution with the above primers according to the following ratios: Nrf1 -453-KI primer stock: 10 μL Nrf1 -453-KI-F+ 10 μL Nrf1 -453-KI-R + 10μL Nrf1 -453-WT-F+10 μL Nrf1 -453-WT-R+60 μL ddH 2 O; Alb -Cre primer stock: 10 μL Alb -Cre-F+10 μL Alb -Cre-R+10 μL 324Internal-F+10 μL 324 Internal-R+60 μL ddH 2 O.

[0040] ②PCR amplification system.

[0041] The PCR amplification system is shown in Table 2.

[0042] Table 2 PCR system .

[0043] ③PCR amplification conditions: Nrf1 -453-KI a. Top cover temperature 105 ℃; b. 94 ℃ 5 min; c. 94 ℃ 30 sec; d. 65 ℃ 30 sec; e. 72 ℃ 35 sec; ce repeated 35 cycles; f. 72 ℃ 20 min; g. 4 ℃ permanent storage; Alb -Cre a. Top cover temperature 105 ℃; b. 95 ℃ 5 min; c. 94 ℃ 30 sec; d. 55 ℃ 30 sec; e. 72 ℃ 35 sec; ce repeated 35 cycles; f. 72 ℃ 20 min; g. Permanent storage at 4℃.

[0044] 1.1.3 Agarose gel electrophoresis and imaging.

[0045] After the PCR reaction is completed, take 10 μL of the product, use agarose and 1 x TAE to prepare 2% agarose gel, run electrophoresis at 100 V for 30-40 min, and then develop it in a gel imaging system: Nrf1 -453-KI, 212bp; HET, 212+453bp; WT, 453 bp; Alb -Cre: 200 bp; Internal Control: 324 bp, to determine the mouse genotype.

[0046] 1.2. Identification of overexpression effects in primary hepatocytes.

[0047] 1.2.1 Extraction of primary hepatocytes.

[0048] Place the mouse in an isoflurane anesthesia machine and anesthetize the mouse with a steady slow flow for 2 min. Use a needle to fix the mouse's limbs on a mouse board, spray 75% alcohol on the abdomen for disinfection, cut the belly along the midline of the abdomen to expose the liver, use a cotton swab to move the liver to the right to fully expose the inferior vena cava, insert the perfusion bag needle into the descending vein and turn on the switch. If reflux occurs, it proves that the needle has entered the blood vessel. At this time, use a 37 ℃ preheated Wash bufffer to start perfusion, cut the lower side of the portal vein, and perfuse all the circulating blood until the liver turns white. After that, replace the collagen buffer at the interface of the perfusion bag, and perfuse again until obvious cords appear on the surface of the liver, indicating that the connection between the hepatocytes has been digested by collagenase, and the perfusion can be stopped. After clamping the liver connective tissue with tweezers, use scissors to separate the liver from the abdominal cavity into precooled ordinary culture medium, and use tweezers to gently scratch the surface membrane of the liver to prepare a hepatocyte suspension. Rinse the hepatocyte suspension twice with pre-cooled ordinary culture medium, centrifuge at low speed (30 g, 3 min) and discard the supernatant (if the supernatant is turbid after rinsing, increase the number of rinsing), add 37 ℃ pre-heated inoculation culture medium to resuspend and plate, keep the number of plates at 10 6 Live cells / mL culture medium were added according to the bottom area of ​​the culture dish. In this experiment, 6 cm culture dishes were used. After 2 h of attachment, they were washed three times with preheated PBS. The adherent cells in good condition were retained and replaced with ordinary culture medium. The medium was changed every 24 h and the cells were harvested after 48 h of attachment.

[0049] 1.2.2 Primary cell protein extraction and Western blot.

[0050] ①Protein extraction.

[0051] a. After the primary hepatocytes adhered to the wall for 48 h, the culture medium was discarded and the surface was rinsed with pre-cooled PBS. This was repeated three times and the water was fully absorbed.

[0052] b. Add Lysis buffer (containing 1 mM PMSF), scrape off all adherent cells with a cell scraper and transfer to a 1.5 mL ep tube.

[0053] c. Ultrasonic disruption and lysis of cells: ice bath, 25 Hz, 5 s, 3 times.

[0054] d. High-speed centrifugation: 4 ℃, 12000 g, 15 min.

[0055] e. Pipette the supernatant into a 1.5 mL EP tube; this is the protein stock solution.

[0056] f. Determine the protein concentration using the BCA method and then heat-deform (95°C, 10 mins) for later use.

[0057] ②Western blot.

[0058] a. Prepare electrophoresis gel and determine the loading volume according to the sample protein concentration and target protein abundance (the loading volume in this experiment is 40-60 μg).

[0059] b. Use Running buffer as the buffer system, concentrate the protein at 80 V for 30 min, then adjust to 100 V. Determine the electrophoresis time based on the relative position of the target band and the protein marker.

[0060] c. After electrophoresis, wet transfer was performed using Transfer buffer as the buffer system and 0.45 μm PVDF membrane as the carrier. The electrotransfer time was determined according to the molecular weight of the target protein (the electrotransfer time in this experiment was 100 min).

[0061] d. After electroporation, wash the front of the PVDF membrane with TBST (80 rpm, 5 min, 3 times), absorb the liquid thoroughly and add 3% BSA to block (40 rpm, 1 h), discard the blocking solution and wash with TBST (80 rpm, 5 min, 3 times) and then add the primary antibody (4 ℃, 30 rpm, overnight).

[0062] e. Recover the antibody, wash with TBST (80 rpm, 5 min, 3 times), and then add the secondary antibody (80 rpm, 1 h).

[0063] f. Discard the secondary antibody, wash with TBST (80 rpm, 5 min, 3 times) and then luminesce.

[0064] g. Data analysis.

[0065] 2. Customization of high cholesterol feed: high cholesterol feed (1.25% cholesterol, Fat 39.9 kcal%, XT109C, Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.).

[0066] 3. MAFLD diet-induced model: The experimental animals were divided into two groups according to their genotypes and given a high-cholesterol diet. In order to improve the adaptability of the experimental animals to the special diet, all mice were fasted for 24 h before changing the feed, and their body weight was monitored during feeding. After one week of feeding, the mice were collected after fasting for 16 h. In order to avoid errors in the experimental results caused by mice gnawing on the bedding, wood shavings were used as bedding during the operation.

[0067] 4. Phenotypic monitoring of experimental animals.

[0068] 4.1 Basic index monitoring: The mice were weighed before and after modeling, and the liver weight was weighed after collection to calculate the organ coefficient.

[0069] 4.2 Blood lipid determination: After fasting for 16 h, mice were anesthetized with carbon dioxide and their unilateral eyeballs were removed. Whole blood was squeezed into heparin anticoagulant tubes and centrifuged (4°C, 5000 rcf, 5 min). The supernatant was aspirated as plasma and tested using the Nanjing Jiancheng lipid kit for statistical analysis.

[0070] 4.3 Pathological staining: Fresh liver tissue was immersed in 4% paraformaldehyde for more than 48 h, and sliced ​​with a blade to a thickness of about 2 mm for gradient dehydration. After being transparentized with xylene, it was immersed in a wax bath for 2 h, embedded, and sliced ​​with 3 μm thickness for H&E staining and sealed with neutral resin. Paraffin sections were baked at 60 °C for 2 h before staining and then dewaxed to water. After antigen repair, they were blocked with goat serum, and then the primary antibody was added at 4 °C overnight, and the secondary antibody was added. After color development with DAB, the sections were sealed with neutral resin. After the above two staining seals were placed in a fume hood to air dry, they were scanned with a high-resolution panoramic imaging system for data analysis. After being soaked in paraformaldehyde, the tissue was cut with a thickness of about 1 mm and embedded in OTC. The slice thickness was 6 μm, and after rewarming, it was stained with oil red (ORO). After sealing with glycerol gelatin, it was immediately scanned with a high-resolution panoramic imaging system for data analysis.

[0071] 4.4 Extraction and determination of liver fat: 30 mg of frozen liver was cut and added to 0.3 mL Lysis buffer (containing 1 mM PMSF) for homogenization. The supernatant was pipetted into a new 1.5 mL EP tube for ultrasonic disruption. After high-speed centrifugation, the supernatant was collected and 0.15 mL of each sample was dispensed into a new EP tube. 0.8 mL of chloroform-methanol mixture (chloroform: methanol = 2:1) was added in sequence. After high-speed vortexing and centrifugation (2000 rcf, 5 min), the lower layer of liquid was aspirated, 0.7 mL of chloroform-methanol mixture was added in sequence, centrifuged (2000 rcf, 5 min), and the lid was opened and placed in a fume hood to evaporate the liquid. 0.5 mL of 2% Triton-x-100 (solvent: chloroform) was added in sequence and vortexed to mix. The tube was placed in a fume hood to evaporate again. 0.25 mL of sterile PBS was added in sequence and vortexed to mix. Liver fat was determined using a lipid kit after vortexing. The protein stock solution was calibrated for lipid level after the concentration was determined using the BCA method.

[0072] 4.5 Detection of liver damage indicators: Fasting plasma was tested for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) using Nanjing Jiancheng kit to determine the degree of liver damage.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. 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 metabolic-related fatty liver disease animal model, characterized in that: The model is a hepatocyte NRF1-453 subtype-specific overexpression mouse model.

2. A method for constructing an animal model according to claim 1, characterized in that The method specifically comprises the following steps: (1) Construction Rosa26 Homologous recombination DNA vector; (2) Targeting through in vitro transcription Rosa 26 gRNA and Cas9 mRNA of the gene; (3) Injecting the homologous recombination DNA vector, Cas9 mRNA and gRNA into the fertilized eggs of mice to obtain chimeric mice F0; (4) F0 generation mice were mated with wild-type mice to obtain F1 generation heterozygous mice ( Nrf1 -453 fl / + ); (5) Self-fertilization of F1 heterozygous mice to obtain F2 homozygous mice ( Nrf1 -453 fl / fl ); (6) F2 homozygous mice were crossed with hepatocyte-specific mice ( Alb -Cre) hybridization to obtain hepatocyte-specific Nrf1 -453 overexpressing mice and their littermate control mice ( Nrf1 -453 fl / fl;Alb-Cre+ / - ; Nrf1 -453 fl / fl;Alb-Cre- / - ).

3. The construction method according to claim 2, characterized in that: The specific process of step (1) is as follows: gRNA is located in Rosa26 On exon 1 of the gene, the homologous recombination DNA vector contains 2115 bp of 5' homology arm, 2153 bp of 3' homology arm, 1726 bp of enhancer, 3254 bp of "Stop" site, two 34 bp LoxP regions and 1368 bp of Nrf1 -453 coding region fragment.

4. The construction method according to claim 3, characterized in that: The DNA sequence of the gRNA action site is: 5'-CTCCAGTCTTTCTAGAAGATGGG-3'.

5. A use of the metabolic-related fatty liver disease animal model according to claim 1, characterized in that: The application includes application in the study of the pathogenesis of metabolism-related fatty liver disease.

6. A use of the metabolic-related fatty liver disease animal model according to claim 1, characterized in that: The application includes application in drug screening and / or evaluation of metabolism-related fatty liver disease.

7. Use of a mouse model obtained by the method for constructing an animal model according to claims 2 to 4 in the study of the pathogenesis of metabolism-related fatty liver disease.

8. Use of a mouse model obtained by the method for constructing an animal model according to claims 2 to 4 in drug screening and / or evaluation for metabolism-related fatty liver disease.

Images