Construction method and application of animal model

Abstract

The invention provides a construction method and application of an animal model, and the construction method of the animal model comprises the following steps: exposing animals of right age to E3Water containing 4% (w / v) fructose, excessively feeding high-fat feed, and changing liquid at regular time. According to the method, the zebra fish is excessively fed with high-glucose and high-fat feed, so that the non-alcoholic steatohepatitis zebra fish model is established, and the method is a method for quickly and economically establishing the non-alcoholic steatohepatitis zebra fish model with complete NASH pathological characteristics.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction, and particularly relates to a method for constructing an animal model and its application. Background Art

[0002] The gene homology between zebrafish and humans is higher than 85%. In addition, due to its advantages such as low breeding cost, short modeling cycle, and transparent body for easy observation compared with mice, zebrafish has become an ideal model organism for disease modeling. The liver structure and lipid metabolism functions of zebrafish larvae at 5 days post fertilization (5dpf) are similar to those of humans, so it has been tried as a model organism to study liver metabolism-related diseases, including non-alcoholic fatty liver disease (NAFLD).

[0003] NAFLD is a metabolic liver injury closely related to insulin resistance and genetic susceptibility. A clinical diagnosis can be made as long as fatty degeneration of more than 5% of hepatocytes caused by alcohol or other known liver injury factors is excluded. NAFLD includes non-progressive simple hepatic steatosis (NAFL) and progressive non-alcoholic steatohepatitis (NASH). If NASH is not effectively controlled, it can further develop into cirrhosis or even hepatocellular carcinoma. Therefore, it is crucial to clarify the pathogenesis of NASH and find effective intervention measures. Establishing a disease model that can simulate the pathological characteristics of human NASH (hepatocyte steatosis, hepatocyte ballooning, and lobular inflammation) and can balance economic and efficient requirements is an urgent problem for those skilled in the art.

[0004] In recent years, although some researchers have tried to establish zebrafish NAFL or models that can partially simulate the pathological characteristics of NASH by means of single high-fat diet, overfeeding, fructose or toxic drug exposure, etc. within a time range from 1 week to 3 months. However, the previous methods have problems such as inconsistent induction methods and treatment times, and the model liver does not fully conform to the pathological characteristics of NASH. Based on this, there is still a need to explore a method for constructing a standardized, rapid zebrafish model with complete NASH pathological characteristics.

[0005] Therefore, providing a method for establishing a zebrafish model of non-alcoholic steatohepatitis is an urgent problem for those skilled in the art. Summary of the Invention

[0006] In view of the defects of the prior art, the purpose of the present invention is to provide a method for constructing an animal model and its application. The animal model constructed by the method meets the complete pathological characteristics of NASH and is fast and economical.

[0007] The present invention first provides a method for constructing an animal model, which comprises the following steps: exposing animals of appropriate age to E3 Water containing 4% (w / v) fructose, overfeeding them with high-fat feed, and changing the liquid at regular intervals.

[0008] In the above construction method, preferably, the scheduled fluid change is every 24 hours, and the continuous fluid change time is 14-21 days, and more preferably, the continuous fluid change time is 21 days.

[0009] In the above construction method, preferably, the animal of appropriate age is a young zebrafish, and the zebrafish is an AB zebrafish or a macrophage transgenic (Tg(mpeg:EGFP)) zebrafish.

[0010] In the above construction method, preferably, the composition of the E3 Water is: 5mM NaCl, 0.17mM KCl, 0.33mM CaCl 2 , 0.33 mM MgSO 4 .

[0011] In the above construction method, preferably, the overfeeding of high-fat feed is feeding zebrafish twice a day at 10:00 and 13:30, with 18 mg of high-fat feed each time.

[0012] Preferably, the number of the zebrafish is 100-150.

[0013] The present invention also provides application of the above-mentioned method for constructing an animal model in constructing an animal model of non-alcoholic fatty liver disease.

[0014] In the above application, preferably, the application evaluates the deposition of fat in the liver of the animal model by staining with Oil Red O. Oil Red O is a fat-soluble azo dye that can specifically stain neutral triglycerides, lipids and lipoproteins in tissues and cells. When the tissue is immersed in the dye solution, the dye leaves the dye solution and dissolves in the lipids in the tissue, making the lipids in the tissue appear red.

[0015] In the above application, preferably, the application evaluates hepatocyte fatty degeneration, hepatocyte ballooning degeneration and lobular inflammation by H&E staining of pathological sections.

[0016] In the above application, preferably, the application evaluates the liver inflammatory response by observing the number of macrophages aggregated in the liver of transgenic zebrafish with macrophage transgenic (Tg(mpeg:EGFP)) labeled with green fluorescence.

[0017] The technical solution of the present invention has the following beneficial technical effects:

[0018] The present invention overfeeds zebrafish with high-sugar and high-fat feed, causing disorders in the glycolipid metabolism of zebrafish, exceeding the ability of hepatocytes to transport triglycerides, resulting in excessive deposition of triglycerides in hepatocytes, causing oxidative stress damage, and while damaging hepatocytes, causing liver inflammatory response, thereby establishing a zebrafish non-alcoholic fatty liver disease model.

[0019] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for establishing a zebrafish model of non-alcoholic fatty liver disease. The present invention draws on and integrates the previous methods for establishing zebrafish models of NAFL or NASH, and proposes a new combination method of overfeeding with high-fat feed plus exposure to a certain concentration of fructose, and through experiments, it is explored that a model conforming to the significant pathological characteristics of NASH can be established at a fixed time (21 days). Compared with the NASH modeling cycle based on mice (12-16 weeks of feeding with high-fat feed), the time is significantly shortened and more economical. That is, the present invention discloses a method for quickly and economically constructing a zebrafish model of non-alcoholic fatty liver disease with complete NASH pathological characteristics. Brief Description of the Drawings

[0020] Figure 1 It is an intuitive diagram of Oil Red O staining to detect liver fat deposition in zebrafish after 14 days of induction by overfeeding with high-fat feed and exposure to a certain concentration of fructose.

[0021] Figure 2 It is a statistical chart of liver fat deposition in zebrafish after 14 days of induction by overfeeding with high-fat feed and exposure to a certain concentration of fructose.

[0022] Figure 3 It is an intuitive diagram of Oil Red O staining to detect liver fat deposition in zebrafish after 21 days of induction by overfeeding with high-fat feed and exposure to a certain concentration of fructose.

[0023] Figure 4 It is a statistical chart of liver fat deposition in zebrafish after 21 days of induction by overfeeding with high-fat feed and exposure to a certain concentration of fructose.

[0024] Figure 5 It is a liver H&E staining diagram of paraffin sections of zebrafish after 14 days of induction by overfeeding with high-fat feed and exposure to a certain concentration of fructose.

[0025] Figure 6 This is a H&E staining image of the liver paraffin section of zebrafish after 21 days of induction by overfeeding with high-fat diet and exposure to a certain concentration of fructose.

[0026] Figure 7 This is a visual image of macrophage aggregation in the liver region of zebrafish after 14 days of induction by overfeeding with high-fat diet and exposure to a certain concentration of fructose.

[0027] Figure 8 This is a statistical chart of macrophage aggregation in the liver region of zebrafish after 14 days of induction by overfeeding with high-fat diet and exposure to a certain concentration of fructose.

[0028] Figure 9 This is a visual image of macrophage aggregation in the liver region of zebrafish after 21 days of induction by overfeeding with high-fat diet and exposure to a certain concentration of fructose.

[0029] Figure 10 This is a statistical chart of macrophage aggregation in the liver region of zebrafish after 21 days of induction by overfeeding with high-fat diet and exposure to a certain concentration of fructose.

[0030] Figure 11 This is the expression level of lipidogenesis-related genes in zebrafish after 21 days of induction by overfeeding with high-fat diet and exposure to 4% fructose. Detailed implementation manners

[0031] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention are described in detail below, but it should not be construed as a limitation on the implementable scope of the present invention.

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0033] The zebrafish used in the present invention are AB strain zebrafish, purchased from Nanjing Yishulihua Biotechnology Co., Ltd. and propagated in the laboratory of Guangdong Core Selection Inspection and Testing Co., Ltd. The reagents used are shown in Table 1.

[0034] Table 1

[0035]

[0036]

[0037] The main equipment used is shown in Table 2.

[0038] Table 2

[0039]

[0040] Example 1: Establishment of a zebrafish model of non-alcoholic steatohepatitis

[0041] I. Detection of liver fat deposition

[0042] (1) Selection of juvenile fish: Healthy AB-strain zebrafish at 5 dpf (days post fertilization) were selected and placed in a 90-mm culture dish to obtain zebrafish for model construction.

[0043] (2) Model construction: The experiment was set up with a normal group and a model group, each with 100 zebrafish for model construction. In the normal group, 50 mL of E3 Water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 ) was added, and the fish were fed with normal feed (fed 2 times a day, at 10:00 and 13:30 respectively; 6 mg each time); in the model group, 50 mL of E3 Water containing 4% fructose (w / v) was added, and the fish were overfed with high-fat feed (fed 2 times a day, at 10:00 and 13:30 respectively; 18 mg each time); the liquid was changed every 24 h; observations and comparisons were made on the 14th and 21st days respectively, and finally zebrafish after the intervention were obtained.

[0044] (3) Oil Red O staining: After the intervention, 15 zebrafish after the intervention were selected from each group, washed twice with E3 Water, fixed with 4% paraformaldehyde solution for 24 h, and dehydrated with 25%, 50%, 75%, and 100% 1,2-propanediol gradients for 25 min each. After dehydration, Oil Red O staining was performed for 48 h, then decolorized with 1,2-propanediol for 30 min, and observed and photographed under a stereomicroscope to record the liver fat accumulation in the zebrafish. The Image J software was used to analyze the Oil Red O staining of the liver in the zebrafish, and the corresponding gray value (S) was statistically analyzed. The formula for calculating the relative liver fat content of zebrafish is as follows:

[0045]

[0046] After 14 days of high-sugar and high-fat induction, the intuitive diagram of Oil Red O staining to detect liver fat deposition in zebrafish after 14 days of overfeeding with high-fat feed and exposure to a certain concentration of fructose is as Figure 1 shown, and the statistical chart of liver fat deposition in zebrafish after 14 days of overfeeding with high-fat feed and exposure to a certain concentration of fructose is as Figure 2 shown.

[0047] The Oil Red O staining results showed that compared with the normal group, there was a large amount of lipid deposition in the livers of zebrafish in the model group (induced by high sugar and high fat for 14 days) ( Figure 1 ). The relative liver fat content of zebrafish in the model group was 333.19 ± 41.76% (p < 0.001) ( Figure 2 ), indicating that after 14 days of high sugar and high fat feeding, fat deposition in the livers of zebrafish could be promoted.

[0048] After 21 days of high sugar and high fat induction, the intuitive diagram of liver fat deposition in zebrafish induced by excessive feeding of high-fat diet and exposure to a certain concentration of fructose for 21 days detected by Oil Red O staining was as Figure 3 shown, and the statistical chart of liver fat deposition in zebrafish induced by excessive feeding of high-fat diet and exposure to a certain concentration of fructose for 21 days was as Figure 4 shown.

[0049] The Oil Red O staining results showed that compared with the normal group, there was a large amount of lipid deposition in the livers of zebrafish in the model group (fed with high sugar and high fat for 21 days) ( Figure 3 ). At the same time, the relative liver fat content of zebrafish in the model group was 402.41 ± 36.15% (p < 0.001) ( Figure 4 ), indicating that after 21 days of high sugar and high fat feeding, fat deposition in the livers of zebrafish could be promoted.

[0050] II. Detection of hepatocyte fatty degeneration, ballooning degeneration and lobular inflammation

[0051] (1) Preparation of paraffin sections

[0052] ① Fixation and sampling: The livers of zebrafish in the control group and the model group were dissected respectively after the intervention ended, and then immediately immersed in 4% paraformaldehyde for more than 24 h. The livers of zebrafish in each model group after the intervention ended were taken out from the fixative, and the target tissue was trimmed flat with a scalpel in the fume hood. The trimmed liver tissue and the corresponding labels were placed in an embedding frame to obtain an embedding frame containing tissue samples.

[0053] ② Dehydration and wax infiltration: The dehydration box was placed in the dehydrator and dehydrated with gradient ethanol in turn, including the following steps: 75% ethanol for 4 h, 85% ethanol for 2 h, 90% ethanol for 2 h, 95% ethanol for 1 h, absolute ethanol I for 30 min, absolute ethanol II for 30 min, alcohol-benzene for 5 - 10 min, xylene I for 5 - 10 min, xylene II for 5 - 10 min, melted paraffin I at 65 °C for 1 h, melted paraffin II at 65 °C for 1 h, melted paraffin III at 65 °C for 1 h to obtain melted wax.

[0054] ③ Paraffin embedding: Embed the liver tissues impregnated with wax in an embedding machine. First, put the melted wax into the embedding frame containing tissue samples. Before the wax solidifies, take out the tissues from the dehydration box, place them into the embedding frame according to the requirements of the embedding surface, and attach the corresponding labels. Cool on a -20°C freezing table. After the wax solidifies, take out the wax block from the embedding frame and trim the wax block to obtain a trimmed wax block.

[0055] ④ Paraffin sectioning: Place the trimmed wax block on a paraffin slicing machine to slice, with a slice thickness of 4 μm. Float the slices on warm water at 40°C on a spreading machine to flatten the tissues, pick up the tissues with glass slides, and bake the slides in an oven at 60°C. After the water is dried and the wax is melted, take them out and store at room temperature for standby to obtain sections.

[0056] (2) H&E staining steps

[0057] ① Dewaxing the paraffin sections to water: Put the sections into an environmentally friendly dewaxing solution Ⅰ for 20 min, then into an environmentally friendly dewaxing solution Ⅱ for 20 min, then into absolute ethanol Ⅰ for 5 min, then into absolute ethanol Ⅱ for 5 min, then into 75% alcohol for 5 min, and wash with tap water to obtain dewaxed sections.

[0058] ② Pretreatment: Immerse the dewaxed sections in a high-definition constant staining pretreatment solution for 1 min to obtain pretreated sections.

[0059] ③ Hematoxylin staining: Immerse the pretreated sections in hematoxylin stain for 3 - 5 min, wash with tap water, differentiate with a differentiating solution, wash with tap water, blue with a bluing solution, and rinse with running water to obtain hematoxylin-stained sections.

[0060] ④ Eosin staining: Immerse the hematoxylin-stained sections in 95% alcohol for dehydration for 1 min, and stain in eosin stain for 15 s to obtain eosin-stained sections.

[0061] ⑤ Dehydration and mounting: Immerse the eosin-stained sections successively in absolute ethanol I for 2 min - absolute ethanol II for 2 min - absolute ethanol III for 2 min - n-butanol I for 2 min - n-butanol II for 2 min - xylene I for 2 min - xylene II for 2 min for clearing, and mount with neutral balsam to obtain mounted sections.

[0062] ⑥ Imaging: Examine the relatively thick sections under a microscope and perform image acquisition and analysis.

[0063] Figure 5 It is the H&E staining map of the liver paraffin sections of zebrafish after 14 days of induction by overfeeding with a high-fat diet and exposure to a certain concentration of fructose. Among them, fatty degeneration - blue arrow, ballooning degeneration - green arrow, lobular inflammation - red arrow. From the pathological sections ( Figure 5) It can be seen that the hepatocytes of zebrafish in the normal group are arranged neatly, with clear cell boundaries. The nucleus is round and located in the central cell, and the cytoplasm is relatively abundant. In the model group (induced by high sugar and high fat for 14 days), some hepatocytes of zebrafish show a vacuolar shape, are arranged disorderly, and there is a small amount of microvesicular hepatocyte steatosis (blue arrow). In addition, it can be seen that some hepatocytes show an increase in volume, loose cytoplasm and cytoplasmic condensation, that is, ballooning degeneration (green arrow). There is a small amount of lymphocyte infiltration in some portal areas, that is, lobular inflammation (red arrow), indicating that after inducing zebrafish with high sugar and high fat for 14 days, the liver of zebrafish shows mild non-alcoholic steatohepatitis, but it is not obvious.

[0064] Figure 6 It is a hematoxylin and eosin staining diagram of the liver of zebrafish paraffin sections after 21 days of induction by overfeeding with high-fat feed and exposure to a certain concentration of fructose. Among them, steatosis - blue arrow, ballooning degeneration - green arrow, lobular inflammation - red arrow. From the pathological sections ( Figure 6 ) It can be seen that the hepatocytes of zebrafish in the normal group are arranged neatly, with clear cell boundaries. The nucleus is round and located in the central cell, and the cytoplasm is relatively abundant. In the model group (fed with high sugar and high fat for 21 days), the hepatocyte structure of zebrafish is severely damaged, with a large amount of macrovesicular steatosis (blue arrow), scattered hepatocyte volume increase, loose and transparent cytoplasm and cytoplasmic condensation, that is, ballooning degeneration (green arrow). There is lymphocyte infiltration in some portal areas, that is, lobular inflammation (red arrow), indicating that after inducing zebrafish with high sugar and high fat for 21 days, the liver of zebrafish shows obvious and complete pathological features of non-alcoholic steatohepatitis (hepatocyte steatosis, hepatocyte ballooning degeneration and lobular inflammation).

[0065] In addition, from Figure 5 and Figure 6 it can be seen that compared with zebrafish induced by high sugar and high fat for 14 days, the hepatocyte steatosis (blue arrow) and ballooning degeneration (green arrow) of zebrafish induced by high sugar and high fat for 21 days are significantly increased, and the pathological features of lobular inflammation (red arrow) in the portal area are also more obvious.

[0066] In the present invention, whether the hematoxylin and eosin staining results of zebrafish liver paraffin sections conform to the pathological features of NASH is used as a standard to judge whether the non-alcoholic steatohepatitis animal model is successfully established. Among them, about 10% of the sample size of the model is randomly selected for detection, and the success rate of model establishment reaches more than 70%.

[0067] III. Detection of liver macrophage recruitment

[0068] (1) Selection of juvenile fish: Select healthy Tg(mpeg:EGFP) line zebrafish (purchased from Nanjing Yishu Lihua Biotechnology Co., Ltd.) that have developed to 5 dpf and place them in a 90 mm culture dish to obtain zebrafish for constructing the model.

[0069] (2) Model construction: The experimental setup included a normal group and a model group, with 100 zebrafish in each group for model construction. In the normal group, 50 mL of E3 Water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 ) was added, and the zebrafish were fed with regular feed (twice a day, at 10:00 and 13:30 respectively; 6 mg each time); in the model group, 50 mL of E3 Water containing 4% fructose (w / v) was added, and the zebrafish were overfed with high-fat feed (twice a day, at 10:00 and 13:30 respectively; 18 mg each time); the liquid was changed every 24 h; this was continued for 14 days and 21 days respectively, and finally the zebrafish after the intervention ended were obtained.

[0070] (3) Microscopic imaging recording: On the 14th and 21st days of high-sugar and high-fat feeding, 20 zebrafish were selected from each group and placed under an electric inverted fluorescence microscope to observe and photograph the distribution of macrophages in the liver of the zebrafish after the intervention ended.

[0071] (4) Data statistics: The number of macrophages in the liver of the zebrafish after the intervention ended was counted. GraphPad Prism 6.0 software was used for statistical processing of the data. The experimental data were all expressed as mean ± SEM, and unpaired t-tests were used for analysis. Compared with the normal group: * p < 0.05, ** p < 0.01, *** p < 0.001.

[0072] Figure 7 This is an intuitive diagram of macrophage aggregation in the liver region of zebrafish induced by overfeeding with high-fat feed and exposure to a certain concentration of fructose for 14 days. Figure 8 This is a statistical chart of macrophage aggregation in the liver region of zebrafish induced by overfeeding with high-fat feed and exposure to a certain concentration of fructose for 14 days. As can be seen from Figure 7 and Figure 8 , after feeding zebrafish with regular feed (normal group) for 14 days, there was a small amount of macrophage aggregation (6.83 ± 0.74) in the liver of the zebrafish, while after inducing zebrafish with high sugar and high fat (model group) for 14 days, there was an increase in macrophage aggregation (12.18 ± 0.65) in the liver region of the zebrafish. The difference in the number of macrophages between the model group and the normal group (6.83 ± 0.74) was statistically significant (p < 0.01)( Figure 8 ), indicating that high sugar and high fat induced for 14 days can induce the infiltration of inflammation-related cells (macrophages) in the liver of zebrafish, suggesting a liver inflammatory response.

[0073] Figure 9An intuitive diagram of macrophage aggregation in the liver region of zebrafish after 21 days of induction by overfeeding with a high-fat diet and exposure to a certain concentration of fructose. Figure 10 A statistical chart of macrophage aggregation in the liver region of zebrafish after 21 days of induction by overfeeding with a high-fat diet and exposure to a certain concentration of fructose. It can be seen from Figure 9 and Figure 10 that after feeding zebrafish with a normal diet (normal group) for 21 days, there was a small amount of macrophage aggregation (8.80 ± 0.80) in the liver region of zebrafish, while after inducing zebrafish with a high-sugar and high-fat diet (model group) for 21 days, there was a large amount of macrophage aggregation (19.40 ± 2.26) in the liver region of zebrafish. The difference in the number of macrophages between the model group and the normal group (8.80 ± 0.80) was statistically significant (p < 0.01) ( Figure 10 ), indicating that after 14 days of induction with a high-sugar and high-fat diet, the liver inflammatory response was not obvious, while after 21 days, it could induce obvious liver inflammatory response in zebrafish.

[0074] IV. Detection of the expression levels of lipid-generating genes sterol-regulatory element-binding proteins 1 (SREBP1), peroxisome proliferator-activated receptor γ (PPAR-γ), and fatty acid synthase (FASN)

[0075] (1) Selection of juvenile fish: Healthy AB-line zebrafish at 5 dpf were selected and placed in a 90-mm culture dish to obtain zebrafish for model construction.

[0076] (2) Model construction: The experiment was set up with a normal group and a model group, with 100 zebrafish for model construction in each group. In the normal group, 50 mL of E3 Water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 ) was added, and normal feed was fed (twice a day, at 10:00 and 13:30 respectively; 6 mg each time); in the model group, 50 mL of E3 Water containing 4% fructose (w / v) was added, and high-fat feed was fed (twice a day, at 10:00 and 13:30 respectively; 18 mg each time); the liquid was changed every 24 h; on the 21st day, the expression of lipid-generating related genes SREBP1, PPAR-γ, and FASN was detected.

[0077] Thirty zebrafish were selected from each group for total RNA extraction. The expression levels of SREBP1, PPAR-γ, and FASN mRNA were quantified by qRT-PCR, and the relative quantity of cDNA for each sample was calculated using the ΔΔCt method with β-actin as the calibration gene. The primer sequences for each gene are shown in Table 3.

[0078] Table 3

[0079]

[0080] (3) Data statistics: The data were statistically processed using GraphPad Prism 6.0 software. The experimental data were expressed as mean ± SEM and analyzed by unpaired t-test. Compared with the normal group: * p < 0.05, ** p < 0.01, *** p < 0.001.

[0081] Figure 11 It is the relative mRNA expression of lipidogenesis-related genes in zebrafish after 21 days of induction by overfeeding with high-fat diet and exposure to 4% fructose. As Figure 11 can be seen, compared with the normal group (SREBP1: 1.00 ± 0.02, PPAR-γ: 1.00 ± 0.07, FASN: 1.00 ± 0.01), the lipidogenesis genes in the model group (SREBP1: 2.80 ± 0.08, PPAR-γ: 17.40 ± 1.15, FASN: 2.93 ± 0.35) were significantly increased (p < 0.01), indicating that high-sugar and high-fat feeding for 21 days can promote the expression of lipidogenesis genes SREBP1, PPAR-γ, and FASN in zebrafish.

[0082] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing an animal model, comprising the following steps: Age-appropriate animals were exposed to E3 Water containing 4% (w / v) fructose, overfed with high-fat diet, and the fluid was changed regularly.

2. The construction method according to claim 1, wherein: The scheduled fluid replacement is performed every 24 hours, and the continuous fluid replacement time is 14-21 days. Preferably, the continuous fluid replacement time is 21 days.

3. The construction method according to claim 1, wherein: The age-appropriate animal is a young zebrafish, and the zebrafish is an AB zebrafish or a macrophage transgenic (Tg(mpeg:EGFP)) zebrafish.

4. The construction method according to claim 1, wherein: The composition of the E3 Water is: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4.

5. The construction method according to claim 1, wherein: The overfeeding of high-fat diet is feeding zebrafish twice a day at 10:00 and 13:30, with 18 mg of high-fat diet being fed each time.

6. The construction method according to claim 5, wherein: The number of the zebrafish is 100-150.

7. Use of the method for constructing an animal model according to any one of claims 1 to 6 in constructing an animal model of non-alcoholic fatty liver disease.

8. The use according to claim 7, wherein: The application evaluates fat deposition in the liver of an animal model by Oil Red O staining.

9. The use according to claim 7, wherein: The application evaluates hepatocyte fatty degeneration, hepatocyte ballooning and lobular inflammation by H&E staining of pathological sections.

10. The use according to claim 7, wherein: The application evaluates liver inflammatory response by observing the number of macrophages accumulated in the liver of the macrophage transgenic (Tg(mpeg:EGFP)) zebrafish with green fluorescence labeling.

Images