CN114365716A

CN114365716A - Preparation method of non-alcoholic simple fatty liver golden hamster model

Info

Publication number: CN114365716A
Application number: CN202111475510.0A
Authority: CN
Inventors: 陈鑫; 王黎明; 应剑; 肖杰; 赵瑾凯
Original assignee: Cofco Corp; Cofco Nutrition and Health Research Institute Co Ltd
Current assignee: Cofco Corp; Cofco Nutrition and Health Research Institute Co Ltd
Priority date: 2021-12-06
Filing date: 2021-12-06
Publication date: 2022-04-19

Abstract

The invention provides a method for constructing a golden hamster model of non-alcoholic simple fatty liver, which comprises the following steps: randomly dividing golden hamsters into two groups, namely a model group of high-fat high-cholesterol feed and a control group of common feed, wherein the high-fat high-cholesterol feed is obtained by adding 1-10g of cholesterol and 10-200g of corn oil or olive oil to each kilogram of common feed; allowing golden hamster in the model group and the control group to freely take food and drink water; and feeding the mice to 8-12 weeks, and randomly extracting golden yellow hamster from each group for detection, thereby confirming the establishment of the non-alcoholic simple fatty liver golden yellow hamster animal model. In the invention, the non-alcoholic simple fatty liver model established by using the vegetable oil is more in line with the dietary structure of people at present, and the method for establishing the simple fatty liver animal model by using the golden hamster is safe and reliable, the required molding time is short, the molding success rate and the molding efficiency are high, and the method has good application prospect.

Description

Preparation method of non-alcoholic simple fatty liver golden hamster model

Technical Field

The invention relates to construction of an animal model, in particular to a method for constructing a golden hamster model of non-alcoholic simple fatty liver by feeding vegetable oily high-fat feed.

Background

Non-alcoholic fatty liver disease refers to a metabolic liver disease in which accumulation of lipid mainly composed of triglyceride in liver cells is a pathological change, which is caused by long-term drinking of large amounts of alcohol and other definite liver damage factors. Non-alcoholic fatty liver disease can be classified into: simple fatty liver, steatohepatitis and cirrhosis. The incidence of fatty liver rapidly rises in europe, the united states and China in recent years, and becomes the second largest liver disease after viral hepatitis. Epidemiological investigation shows that the incidence rate of the nonalcoholic fatty liver disease in China is about 15 percent, and the incidence rate of the nonalcoholic fatty liver disease in Europe and America is more than 20 percent. The average incidence rate of fatty liver in professional population such as white-collar workers and high-level knowledge molecules is about 25%; the incidence of fatty liver in obese people and type 2 diabetic patients is about 50%; the incidence of fatty liver in alcoholics and alcoholics is about 58%; the incidence of fatty liver in sub-healthy people with frequent insomnia, fatigue, poor appetite and gastrointestinal dysfunction is about 60%.

The establishment of an ideal fatty liver animal model has very important significance for the research of pathogenesis of fatty liver, drug screening, treatment strategy and the like. An ideal non-alcoholic simple fatty liver animal model should have the following characteristics: (1) the evolution process of the human non-alcoholic simple fatty liver is simulated, and the human non-alcoholic simple fatty liver has the characteristics of human fatty liver pathological changes; (2) the forming rate is high, the controllability is strong, the repeatability is good, and the death rate is low; and (3) the method is simple, convenient and easy to implement and low in cost.

Due to the complex liver function of human body, the metabolic mechanism of blood lipid of rat, mouse and human is very different, for example, the main carrier of cholesterol in rat serum is high density lipoprotein cholesterol (HDL-C), the transport of cholesterol in human body depends on low density lipoprotein cholesterol (LDL-C), and the cholesterol removal rate in rat body is obviously higher than that of human, therefore, the essence of simple fatty liver of human can not be completely reflected. In addition, researchers use techniques such as gene modification to modify the genes of rats and mice to construct fatty liver animal models, but the model has high technical requirements and long time-consuming period. The nonhuman primate such as a monkey with a close relationship with human, a monkey with a marmoset, a rhesus monkey, an African green monkey, a tree shrew and the like are suitable for being used as fatty liver model animals, but the non-human primate is rare and expensive, and the application and popularization difficulty is large. In order to better reflect the characteristics of the human fatty liver, it is important to find experimental animals which are closer to the synthesis, transportation and metabolism of the human lipid to establish a non-alcoholic simple fatty liver model. The golden hamster is closer to the human lipid metabolism, has the characteristics similar to the human fatty liver pathological changes, can reflect and research the nature of the human simple fatty liver, is economic and easy to obtain, and is convenient to popularize in practical application.

Animal fat, such as lard, is usually selected for the construction of non-alcoholic simple fatty liver models by prior researchers. However, according to the results of national nutrition survey, the edible amount of animal fat and oil of residents in China is reduced year by year since 2002, and the edible amount of animal fat and oil is reduced by 44.8% from 8.7 g/person per day in 2002 to 4.8 g/person per day in 2012. Since 1982, the edible amount of vegetable oil and fat of our country increased year by year, and from 12.9 g/person/day in 1982 to 37.3 g/person/day in 2012, the edible amount of vegetable oil and fat increased 189%. Therefore, the establishment of the non-alcoholic simple fatty liver model by using the vegetable oil better conforms to the dietary structure of people at present, and has important significance for researching the pathogenesis of the fatty liver, drug screening and subsequent clinical application and popularization.

Therefore, the inventor selects high-fat feed made of vegetable oil (such as corn oil, olive oil and the like), and feeds golden yellow hamster close to human lipid metabolism to construct a non-alcoholic simple fatty liver animal model.

Disclosure of Invention

The invention aims to provide a preparation method of a non-alcoholic simple fatty liver animal model, which is carried out by utilizing an animal golden hamster which is closer to the synthesis, transportation and metabolism of human fat, thereby unexpectedly finding that the method can shorten the molding time and has high molding success rate and molding efficiency.

The invention provides a preparation method of a nonalcoholic simple fatty liver golden hamster animal model, which comprises the following steps:

step 1): randomly dividing golden hamsters into two groups, namely a model group of high-fat high-cholesterol feed and a control group of common feed, wherein the high-fat high-cholesterol feed is obtained by adding 1-10g of cholesterol and 10-200g of corn oil and/or olive oil to each kilogram of common feed;

step 2): allowing golden hamster in the model group and the control group to freely take food and drink water; and

step 3): raising to 8-12 weeks, preferably 9-10 weeks, and randomly extracting golden yellow hamster from each group for detection, thereby confirming establishment of non-alcoholic simple fatty liver golden yellow hamster animal model.

In the present invention, the golden hamster used is preferably an adult male golden hamster, i.e., 6 to 8 weeks old. In a further preferred embodiment, the golden hamster preferably has a body weight of 110g to 130 g.

Adult golden hamster obtained by purchase can also be acclimatized for about one week before step 1). The conditions for adaptive feeding may be: a clean animal house is provided with the temperature of 24 +/-2 ℃, the relative humidity of 40-60%, the artificial illumination/dark cycle for 12 hours and the ventilation frequency of 15 times/hour, and is free to eat and drink water, and the test is carried out after the animal house is adapted for one week. In a preferred embodiment, during the adaptive feeding process, the control group is fed with the common feed, and the model group is gradually added with the high-fat high-cholesterol feed on the basis of the common feed. For example, for the model group, starting with a common feed, 30 wt% of high-fat high-cholesterol feed is added every two days, the proportion of the high-fat high-cholesterol feed is gradually increased, and 100% of the high-fat high-cholesterol feed is fed before a formal experiment, so that the gradual adaptation of the hamster to the survival environment and diet is ensured. The feeding amount of golden hamster is 15g-20 g/day/mouse.

In the step 1), the golden hamster contained in each group can be determined according to actual requirements. For example, the number of golden hamster in the model group and the control group may be the same or substantially the same, or may be different.

In the present invention, the common feed for golden hamster may be a feed normally used for feeding golden hamster, which is commercially available or may be prepared by itself as required. For example, the main nutritional ingredients of the golden hamster common feed can be (in weight percent): about 30-60% carbohydrate, about 20-50% crude protein, about 2-20% crude fat, and about 1-10% crude fiber. Preferably, the main nutritional ingredients of the common feed for golden hamster can be (by weight percent): about 40-50% carbohydrate, about 20-30% crude protein, about 4-10% crude fat, and about 2-6% crude fiber. For example, the nutrient components of the common feed may be (in weight percent): about 45% carbohydrate, about 22% crude protein, about 4.5% crude fat, about 3.5% crude fiber.

The high-fat high-cholesterol feed comprises the following components: adding 1-10g cholesterol and 10-200g corn oil and/or olive oil into per kilogram of common feed. As the cholesterol, commercially available edible cholesterol such as cholesterol produced by Schbefu (Beijing) Biotechnology Co., Ltd can be used. As the corn oil or olive oil, commercially available edible corn oil or olive oil may be used. In the present invention, corn oil and olive oil may be used alone or in combination.

For example, the feed used in the present invention can be manufactured by sbefu (beijing) biotechnology limited by mixing the respective components in a certain ratio, and then compressing and granulating the mixture.

For example, general feeds: hamster maintenance feed, purchased from sbefu (beijing) biotechnology limited, production license number: [ SCXK (Jing) 2019-.

High-fat high-cholesterol feed: adding common feed ingredients, corn oil (and/or olive oil) and cholesterol according to the mixture ratio, entrusting the production and manufacture of Beefu (Beijing) biotechnology limited company, and producing a license number: [ SCXK (Jing) 2019-. For example, a high-fat high-cholesterol diet may comprise 90 wt% common feed ingredients +9 wt% corn oil (and/or olive oil) +1 wt% cholesterol. In some embodiments, for high-fat high-cholesterol diet, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10g of cholesterol may be added per kg of common diet; and 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200g of corn oil and/or olive oil.

In the step 2), the golden pheasant is fed at a daily dose of 15g-20 g/day per rat, and the animals eat and drink water freely.

In a preferred embodiment, the golden hamster is bred under the following conditions: and (3) a clean-grade animal house, wherein the temperature is kept at 24 +/-2 ℃, the relative humidity is 40-60%, and the environment is kept clean and the air is circulated. For example, clean-grade animal houses are kept at 24 + -2 deg.C and 40-60% relative humidity for 12 hours artificial lighting/dark cycles with 15 air changes/hour. In addition, fresh grains are replaced every two days, padding is replaced every week, and the mouse cage, the tray and the like are cleaned.

In the present invention, the drinking water uses sterile water.

In the present invention, the model groups can be further classified into a corn oil model group, an olive oil model group and a mixed model group. In the corn oil model group, the high-fat high-cholesterol diet comprises cholesterol and corn oil, but does not contain olive oil. In the olive oil model group, the high-fat high-cholesterol diet contained cholesterol and olive oil, but did not contain corn oil. In the mixed model group, the high-fat high-cholesterol diet comprises both corn oil and olive oil.

For the successful establishment of non-alcoholic simple fatty liver animal models, the gold standard is a pathological section examination, which is usually presented as HE staining results. In a pathological section stained by HE, when fatty oil drops of liver tissues, liver cell steatosis, cell swelling, easy fat vacuole of cytoplasm or pathological results such as inflammatory change of liver cells, liver cell necrosis and the like appear, the non-alcoholic simple fatty liver golden hamster animal model is considered to be successfully constructed.

In addition, the establishment of the non-alcoholic simple fatty liver animal model can refer to one or more of the following: serum biochemical index, liver morphological observation and the like. Examples of the lipid include blood lipids (including at least serum total Triglycerides (TG), serum Total Cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C)), liver organ index, and liver lipid content. These parameters can all be determined using relevant tests in the art. Without wishing to be bound by any theory, when the relevant parameters in the model group are statistically different from those of the control group, the model group can be used for supporting the establishment of the non-alcoholic simple fatty liver animal model.

The serum biochemical indexes mainly comprise: serum Total Cholesterol (TC), serum total Triglyceride (TG), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C). Biochemical indexes of liver mainly include: liver Total Cholesterol (TC) and liver total Triglycerides (TG).

The liver morphology refers to the liver tissue morphology of the golden hamster after dissection.

Liver organ index is liver wet weight/animal body weight.

Liver lipid content was determined by oil red O fat staining (hereinafter referred to as oil red staining).

For example, HE staining in liver pathology section examination is performed by: dissecting golden hamster after the model building is finished, fixing a part of liver tissue, rinsing, dehydrating, embedding paraffin and other steps, slicing with a slicer at the thickness of 8 μm, preparing pathological sections for HE staining, and observing the fatty degeneration condition of liver cells by a Nikon microscope.

The invention has the following advantages and excellent effects:

the mechanisms by which golden hamster synthesizes cholesterol and bile acids in response to the lipid morphology of the diet and the liver are similar to those of humans. For example, 90% of human endogenous cholesterol is synthesized by extrahepatic tissues, and the extrahepatic synthesis of cholesterol in male golden hamster is about 85%, whereas this proportion is only 35% in male rats. The golden hamster is closer to human beings in the aspects of cholesterol synthesis, transportation, metabolism, reaction to lipid in food and functional components for regulating blood fat, lipoprotein metabolism and the like, and can better reflect the lipid metabolism condition of the human beings. In addition, the non-alcoholic simple fatty liver model established by selecting vegetable oil is more in line with the dietary structure of people at present.

The method for constructing the simple fatty liver animal model by using the golden hamster is safe and reliable, has short molding time, high molding success rate and molding efficiency, and has good application prospect. The constructed nonalcoholic simple fatty liver golden hamster model can be used for researching the generation mechanism of fatty liver, screening therapeutic drugs and evaluating the drug effect of related drugs on the disease, and has very important significance.

Drawings

Fig. 1 shows representative liver morphology of golden hamster in control groups of example 1 and example 2, wherein the liver of golden hamster in control group is ruddy, tough and sharp-edged.

Fig. 2 shows representative liver morphology maps of golden hamsters in the corn oil model group of example 1, wherein the corn oil model group golden hamsters have increased liver volume, a yellow, greasy feel, and blunted edges.

Fig. 3 shows a representative liver morphology map of golden hamsters in the olive oil model group of example 2, wherein the livers of the olive oil model group golden hamsters are enlarged, yellow, greasy and blunted.

Fig. 4 shows a graph comparing the liver organ indexes of golden hamster with respect to corn oil model group and olive oil model group and control group. Wherein, the corn oil model group and the olive oil model group have very significant difference (P < 0.05) with respect to liver organ index.

FIGS. 5-8 show graphs comparing the results of serum biochemical indicators TC, TG, HDL-C, LDL-C for the corn oil model group and the olive oil model group with the control group.

Fig. 9-10 show graphs comparing results of biochemical indices TC, TG of liver with respect to corn oil model group and olive oil model group and control group.

Fig. 11 shows a view of HE stained normal liver sections (200 ×), which are exemplary pictures from the control groups of example 1 and example 2. The liver tissue capsule is composed of elastic fiber-rich dense connective tissues with uniform thickness, liver lobules have obvious demarcations and regular arrangement, the center of the liver lobules is a central vein, and hepatic cells and hepatic blood sinuses which are approximately radially arranged are arranged around the liver lobules, and the hepatic cells are round and full; the liver plates are regularly and tidily arranged, and liver sinuses are not obviously expanded or extruded; no obvious abnormality in the portal area between adjacent hepatic lobules; no significant inflammatory changes were seen.

Fig. 12 shows a mild adipose-like lesion map (200 x) of HE staining, which is an exemplary picture of the model groups from example 1 and example 2. A large amount of hepatic cell steatosis and cell swelling can be widely seen in liver tissues, and tiny round vacuoles can be seen in cytoplasm; foam cell and lymphocyte infiltration can be seen locally.

Fig. 13 shows a moderate adipose-like lesion map (200 x) of HE staining, which is an exemplary picture of the model groups from example 1 and example 2. A large amount of hepatic cell steatosis and cell swelling can be widely seen in liver tissues, and tiny round vacuoles can be seen in cytoplasm; the infiltration of foam cells and lymphocytes at a plurality of positions can be seen, and hepatic cells are rarely necrotized in a punctate manner, and the nucleus is cracked; mild connective tissue hyperplasia is seen locally.

Fig. 14 shows a severe adipose-like lesion map (200 x) of HE staining, which is an exemplary picture of the model set from example 1 and example 2. A large amount of hepatic cell steatosis and cell swelling can be widely seen in liver tissues, and tiny round vacuoles can be seen in cytoplasm; most of the cells were infiltrated with lymphocytes, accompanied by punctate necrosis of hepatocytes and nuclear fragmentation.

Fig. 15 shows a normal liver image (200 ×) stained with oil red, which is an exemplary image of the control group from example 1 and example 2.

Fig. 16 shows a mild adipocyte positive image of oil red staining (200 ×), which is an exemplary picture from the model groups of example 1 and example 2.

Fig. 17 shows a moderate adipocyte positive plot (200 ×) of oil red staining, which is an exemplary picture from the model groups of example 1 and example 2.

Fig. 18 shows a positive image (200 ×) of oil red stained severe adipocytes, which is an exemplary picture from the model groups of example 1 and example 2.

The present invention will be further described with reference to the accompanying drawings, and the present invention includes, but is not limited to, the following examples, such as simply expanding the number of animals, and modifying the amount of lipid added to the feed should be considered as the protection scope of the present invention.

Detailed Description

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials, instruments and the like used in the following examples are commercially available unless otherwise specified.

Clean grade 8 week old male Syrian Golden yellow Hamster (LVG Golden Syrian Hamster), initial body weight 110-: [ SCXK (Jing) 2016-. The animals were bred in clean-grade animal houses of Beijing Huafukang biotech GmbH (production license number: [ SCXK (Jing) 2019-. The experimental animals were used in compliance with the 3R principle and were approved by the Committee for use and management of experimental animals (approval No.: IACUC-20201110) from Beijing Huafukang Biotech GmbH.

Example 1

The clean-grade animal house feeding conditions comprise room temperature of 24 +/-2 ℃, relative humidity of 40-60%, 12-hour artificial lighting/dark cycle and ventilation frequency of 15 times/hour; the grains are replaced once every two days, the padding is replaced every week, and the mouse cage, the tray and the like are cleaned. During the rearing period, the animals are guaranteed to freely eat and drink water.

20 adult male golden hamster with age of 8 weeks are selected, the weight is 110g-130g, wherein 10 animals are randomly selected as a corn oil model group animal, and 10 animals are selected as a control group animal. Golden hamsters in both corn oil model group and control group can freely eat feed and sterile water. Wherein, the control group is fed with common feed, and the nutrient components are as follows: about 45% carbohydrate, about 22% crude protein, about 4.5% crude fat, about 3.5% crude fiber. The corn oil model group is fed with high-fat high-cholesterol feed, and the components of the high-fat high-cholesterol feed are as follows: each kilogram of common feed is added with 10g of cholesterol and 90g of corn oil. The food is taken at a dose of 20 g/day for 10 weeks.

After 10 weeks of modeling, 100% of golden hamster all develop obvious symptoms of nonalcoholic simple fatty liver.

No animal death occurred during the entire modeling experiment.

Example 2

Adult male golden

yellow hamster

20, 8 weeks old and 110-130g in weight were selected, 10 of which were olive oil model animals and 10 of which were control animals. Golden pheasants in both the olive oil model group and the control group were allowed to freely ingest feed and sterile water. Wherein, the control group is fed with common feed, and the nutrient components are as follows: about 45% carbohydrate, about 22% crude protein, about 4.5% crude fat, about 3.5% crude fiber. The olive oil model group is fed with high-fat high-cholesterol feed, and the components of the high-fat high-cholesterol feed are as follows: each kilogram of common feed is added with 10g of cholesterol and 90g of olive oil. The food is taken at a dose of 20 g/day for 10 weeks.

After 10 weeks of modeling, 100% of golden hamster all develop obvious symptoms of nonalcoholic simple fatty liver. The main manifestations are that the liver of the animal is obviously enlarged, the weight is increased, and the liver index is obviously higher than that of a control group; the liver surface presents yellow greasy feeling; the content of cholesterol in the liver of the olive oil model group is obviously higher than that of the control group, and the content of total triglyceride of the olive oil model group is also obviously higher than that of the control group.

No animals died throughout the modeling experiment.

Method and result for diagnosing and evaluating golden hamster nonalcoholic simple fatty liver

The method comprises liver morphological observation, liver organ index, serum biochemical marker and histopathological diagnosis.

The liver morphology refers to the liver tissue morphology of the golden hamster after dissection. The livers of the golden hamster of the control groups of example 1 and example 2 were ruddy, tough and sharp-edged (see fig. 1); the corn oil model group and the olive oil model group golden hamster have enlarged liver volume, yellow color, greasy feeling and blunted edges (see fig. 2 and fig. 3).

Liver organ index is liver wet weight/animal body weight. As shown in fig. 4, the liver organ indexes of the corn oil model group and the olive oil model group were significantly different from those of the control group (P < 0.05).

Blood was collected every two weeks during the molding period. Specifically, the test animal golden yellow hamster is fasted for 12 hours, 0.3-0.5mL of blood is taken from orbital venous plexus, centrifuged at 3000rpm for 10 minutes, serum is separated, and the serum levels of TC, TG and HDL-C, LDL-C are measured.

And (3) displaying a detection result: serum levels of TC from week 2 of molding were very significantly different in the corn oil model group and the olive oil model group from the control group (P < 0.05); serum levels of TG were significantly different in the corn oil model group and the olive oil model group from the control group (P < 0.05) from week 2 of molding; the serum levels of HDL-C were very significantly different in the corn oil model group and the olive oil model group from the control group (P < 0.05) from week 2 of molding; serum levels of LDL-C were significantly different (P < 0.05) in the corn oil model group and the olive oil model group from the control group starting at week 2 of molding (see FIGS. 5-8). After the modeling is finished, the total liver cholesterol (TC) of the corn oil model group and the olive oil model group is obviously higher than that of the control group (P is less than 0.05), and the total liver Triglyceride (TG) of the olive oil model group is obviously higher than that of the control group (P is less than 0.05) (see the figure 9-figure 10).

All data results are expressed as mean ± standard deviation, plotted using GraphPad Prism 9.0, medical statistics software and statistically analyzed. Comparing the model group with the control group, performing statistical analysis by adopting t-test when the data conforms to normal distribution; if the distribution is not in accordance with normal distribution, statistical analysis is carried out by adopting nonparametric test.

Histopathological analysis

HE staining of pathological sections

Dissecting golden hamster, fixing a part of liver tissue in 10% neutral formalin, embedding paraffin, slicing at 8 μm, and preparing liver histological section for HE staining; and (5) observing the fatty degeneration condition of the liver cells by using a Nikon microscope.

Pathological section preparation:

1. material taking: using a scalpel to take the tissue with the thickness of about 2-3mm and the size of 1.5 multiplied by 0.2-0.3 cm as appropriate; 2. fixing: immediately fixing the tissue in 10% formalin (or 4% formaldehyde), and preserving at 4 deg.C; 3. rinsing: flushing with running water for 2-10 h; 4. and (3) dehydrating: placing the tissue in an embedding box, dehydrating the tissue from low-concentration alcohol to high-concentration alcohol, and placing the tissue in xylene for transparency, wherein 70% alcohol (5min) → 80% alcohol (5min) → 90% alcohol (5min) → 95% alcohol (5min) → 100% alcohol (5min) → xylene I (5min) → xylene II (5 min); 5. wax dipping: the process of dipping tissues in molten paraffin after the tissues are transparent is called wax dipping, and the tissues can be dipped for 2 to 3 times generally, the total time is 3 to 4 hours, and the melting point of the paraffin for wax dipping is 52 to 56 ℃; 6. embedding: pouring molten paraffin into an embedding frame, putting the tissue block into the embedding frame by using heated forceps, wherein the embedding surface is required to be flat, and after the paraffin is slightly condensed after embedding, moving the paraffin into cold water or a refrigerator for accelerating and solidifying; 7. slicing: the embedded wax block is trimmed by a scalpel, and then fixed on a slicer to be sliced, wherein the slice thickness is 8 mu m. The cut slices are unfolded in warm water at 45 ℃ by using a brush pen, and then taken out by using a glass slide and dried at 37 ℃.

HE dyeing step:

baking at 1.60 deg.C for 15 min; 2. dewaxing paraffin sections with xylene (10 min/time, 2 times each xylene); 3. gradient alcoholization (100% ethanol, 90% ethanol, 75% ethanol, 50% ethanol, each for 10min), soaking in distilled water for 5 min; soaking in 4.0.01M PBS (pH 7.4) for 5 min/times, 3 times; 5. adding 1 drop or 50 μ L of 1 Xhematoxylin staining solution into each section, and incubating at room temperature for 10 min; 6. washing with tap water and returning to blue; soaking in 7.0.01M PBS for 5 min/3 times; 8. adding 1 drop or 50 μ L of alcohol-soluble eosin dye solution into each slice, and keeping at room temperature for 1 min; 9. washing with tap water; dehydrating gradient alcohol until absolute ethanol is 5min each time, taking out, naturally drying, and passing xylene twice each time for 10 min; 11. sealing with neutral gum; 12. microscopic examination and image acquisition and analysis.

Oil red staining of pathological sections

Dissecting golden hamster after molding, fixing a part of liver tissue, embedding with OCT, slicing at 8 μm, and preparing frozen section for oil red staining; and collecting images on the stained sections by adopting a tissue section digital scanner Pannoramic DESK, reading the tissue area and the positive area in the measurement area by utilizing software Image-pro plus 6.0, and calculating the positive area ratio.

Based on the results of serum biochemical indicators, animals of each group were dissected at week 8 of molding and histopathologically analyzed by performing HE staining and oil red staining. The histopathological research and analysis show that the modeling method of the invention can cause golden hamster to have obvious symptoms of nonalcoholic simple fatty liver in 8 weeks. To ensure that most animals developed non-alcoholic simple fatty liver disease, the remaining animals were extended to 10 weeks. After the end of the 10 th molding cycle, all test animals were subjected to histopathological analysis.

Histopathological results at 8 weeks and 10 weeks show that although fatty liver lesions are varied in degree, mild in some cases and severe in some cases due to differences among animals, histopathological results clearly confirm that the molding method according to the present invention successfully establishes non-alcoholic simple fatty liver golden hamsters animal models as early as 8 weeks, and all experimental golden hamsters show obvious non-alcoholic simple fatty liver lesions at 10 weeks.

HE pathology analysis results are as follows (exemplary plots from examples 1 and 2):

liver sections from the control group showed normal hepatocytes with obvious nuclei, cytoplasm, and membranes visible (exemplary picture see fig. 11(200 ×), HE staining).

Mild steatosis, clearly visible adipocytes, and also a large number of normal cells [ see fig. 12(200 ×), HE staining ].

Medium-degree adipose-like pathologic change images show that fat cells are obviously seen, and a large number of normal cells are seen at the same time. But the ratio of the number of adipocytes to normal hepatocytes was significantly increased [ see figure 13(200 ×), HE staining ].

Severe adipose-like lesions, a large range of adipocytes were seen, the proportion of normal hepatocytes was significantly reduced, and about 77% steatosis in hepatocytes was seen [ see fig. 14(200 ×), HE staining ].

The results of the oil red staining pathology analysis are as follows (exemplary figures from examples 1 and 2):

the normal liver oil red staining pattern obtained from the control group indicated that the hepatocytes were normal, with trace positive areas visible after oil red staining (exemplary picture see fig. 15(200 ×), oil red staining).

The mild steatosis map shows that fat cells are obviously visible, and a large number of normal cells are visible at the same time, the positive area of oil red staining is between 30% and 50% (see figure 16(200 x), oil red staining).

The moderate lipoid lesion map clearly shows a large number of fat cells, and the positive area of oil red staining is between 50% and 75% (see figure 17(200 x), oil red staining).

Severe adipose-like lesion map, it can be seen that extensive adipose cells are diffusely distributed around hepatocytes, large area appears after oil red staining, and the positive area is about 77% [ see fig. 18(200 ×), oil red staining ].

Through comparing the modeling group and the comparison group, the non-alcoholic simple fatty liver model of the golden hamster can be established successfully more easily by using the vegetable fat high-fat feed, the required cholesterol and fat amount are less, the modeling time is shorter, and the effect is better.

Claims

1. A method for preparing a non-alcoholic simple fatty liver golden hamster animal model, the method comprising:

2. The method according to claim 1, wherein the golden hamster is an adult male golden hamster, preferably 6-8 weeks old; wherein, the weight of the golden hamster is preferably 110g-130 g.

3. The preparation method according to claim 1 or 2, wherein the golden hamster common feed comprises the following nutritional ingredients in percentage by weight: 30-60% of carbohydrate, 20-50% of crude protein, 2-20% of crude fat and 1-10% of crude fiber; preferably, the common feed for golden hamster comprises the following nutrient components in percentage by weight: 40-50% of carbohydrate, 20-30% of crude protein, 4-10% of crude fat and 2-6% of crude fiber; more preferably, the common feed for golden hamster comprises the following nutrient components in percentage by weight: 45% of carbohydrate, 22% of crude protein, 4.5% of crude fat and 3.5% of crude fiber.

4. The preparation method according to any one of claims 1 to 3, wherein the high-fat high-cholesterol diet comprises the following ingredients: adding 1-10g of cholesterol and 10-200g of corn oil and/or olive oil into per kilogram of common feed; preferably, for high-fat high-cholesterol feed, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10g of cholesterol and 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200g of corn oil and/or olive oil are added to each kilogram of common feed.

5. The preparation method according to any one of claims 1 to 4, wherein, in step 2), the golden hamster is administered at 15g to 20g per day per mouse per day.

6. The method according to any one of claims 1 to 5, wherein the golden hamster is bred under the following conditions: a clean-grade animal house is kept at a temperature of 24 +/-2 ℃ and a relative humidity of 40-60%, and the environment is kept clean and air circulation is kept; preferably, the clean-grade animal house feeding condition is 24 +/-2 ℃, the relative humidity is 40-60%, the artificial illumination/dark cycle is 12 hours, and the ventilation frequency is 15 times/hour.

7. The preparation method according to any one of claims 1 to 6, wherein the drinking water uses sterile water.

8. The method according to any one of claims 1 to 7, wherein the confirmation of the nonalcoholic simple fatty liver is performed by pathological section examination.

9. The method of any one of claims 1 to 8, wherein the confirmation of the non-alcoholic simple fatty liver golden hamsters animal model is further with reference to one or more of: serum biochemical index, liver biochemical index and liver morphology observation result.