阅读说明：本技术 单一因素快速诱导酒精性脂肪肝模型的方法 (Method for rapidly inducing alcoholic fatty liver model by single factor ) 是由 冷静 梁丹 唐海波 吴展帅 李晓红 肖健 唐耀平 于 2019-12-05 设计创作，主要内容包括：本发明公开了一种单一因素快速诱导酒精性脂肪肝模型的方法,该方法每天按体重4-5.7g/kg的乙醇量,给非人灵长类动物饮用含10-35％乙醇的酒,同时给予基础饲料自由喂养,喂养50-90天,即可得到稳定的酒精性脂肪肝模型。本发明能获得稳定的接近于人类自然发生的酒精性脂肪肝动物模型,非人灵长类动物依从性较好,试验中无须手术或处死动物检查,且造模时间短、成本低,为酒精性脂肪肝发病机理的研究、治疗药物的筛选、药效学评价等提供良好的模型载体。(The invention discloses a method for rapidly inducing an alcoholic fatty liver model by a single factor, which is characterized in that a stable alcoholic fatty liver model can be obtained by drinking wine containing 10-35% of ethanol for a non-human primate according to the ethanol amount of 4-5.7g/kg of body weight every day and freely feeding the non-human primate with basic feed for 50-90 days. The invention can obtain a stable animal model of the alcoholic fatty liver which is close to the natural occurrence of human beings, the compliance of non-human primates is better, no operation or animal sacrifice examination is needed in the test, the modeling time is short, the cost is low, and a good model carrier is provided for the research of the pathogenesis of the alcoholic fatty liver, the screening of therapeutic drugs, the pharmacodynamics evaluation and the like.)

1. A method for rapidly inducing an alcoholic fatty liver model by a single factor is characterized by comprising the following steps: drinking wine containing 10-35% ethanol for non-human primate with ethanol content of 4.0-5.7g/kg per day, feeding with basal feed freely, and feeding for 50-90 days to obtain stable alcoholic fatty liver model.

2. The method for rapidly inducing alcoholic fatty liver model with single factor according to claim 1, wherein the non-human primate drinks wine by the following way: the ethanol concentration in the wine and the daily ethanol amount are gradually increased.

3. The method for rapidly inducing alcoholic fatty liver model with single factor according to claim 2, wherein the non-human primate drinks wine by the following way: the concentration gradient of ethanol in the wine is increased to 35 percent, the ethanol amount gradient in each day is increased to 5.7g/kg, the maintenance time is more than or equal to 20 days, and the total feeding time is 50-70 days, thus obtaining the stable alcoholic fatty liver model.

4. The method for rapidly inducing alcoholic fatty liver model with single factor according to claim 2 or 3, wherein the non-human primate drinks wine by the following way: the concentration of ethanol in the wine is increased by 1-5% every 1-5 days, and the amount of ethanol in the wine is increased by 0.03-0.31g/kg every 1-5 days.

5. The method for rapidly inducing alcoholic fatty liver model by single factor according to claim 1, wherein: the wine is distilled wine.

6. The method for rapidly inducing alcoholic fatty liver model with single factor as claimed in claim 1, wherein the non-human primate is at least 6 years old.

7. The method for rapidly inducing the alcoholic fatty liver model with the single factor as claimed in claim 1, wherein the non-human primate is cynomolgus monkey, rhesus monkey or macaque.

8. The method for rapidly inducing the alcoholic fatty liver model by the single factor according to claim 5, which is characterized in that: the distilled liquor is Chinese liquor or brandy.

9. A single-factor rapid induction alcoholic fatty liver model established by the method of any one of claims 1 to 8.

10. The use of the single-factor rapid induction alcoholic fatty liver model of claim 8 for: research on pathogenesis of alcoholic fatty liver, screening of drugs for preventing or treating alcoholic fatty liver, and pharmacodynamic evaluation.

Technical Field

The invention relates to a method for establishing an animal disease model, in particular to a method for rapidly inducing an alcoholic fatty liver model by a single factor.

Background

Alcoholic Fatty Liver Disease (AFLD) is a type of fatty liver disease, and is a special one of clinical liver diseases, most of patients are long-term drinkers, generally for more than 5 years, and because of long-term and large-amount drinking, liver cells produce a large amount of toxic substances such as acetaldehyde, and secrete a large amount of free radicals and hydroxyl radicals, so as to cause metabolic disorders. In recent years, with the change of life style, liver diseases and related diseases induced by alcohol and/or metabolic factors are rapidly increased, the incidence rate of fatty liver is increased year by year and is in a youthful state, and the fatty liver in China is increased to the second place in the liver diseases. In people who drink excessively for a long time, the alcoholic fatty liver accounts for about 90-100%, the alcoholic hepatitis accounts for 10-35%, and the alcoholic cirrhosis finally develops to 8-20%. The existing research shows that the 5-10-year survival rate of the nonalcoholic cirrhosis patients is 23%, while the survival rate of the alcoholic cirrhosis patients is only 7%. The alcoholic hepatitis patients should pay high attention to 10-20% of those who develop liver cirrhosis every year and finally about 70% of those who develop liver cancer. In order to deeply research the pathogenesis of alcoholic fatty liver and find effective preventive and therapeutic drugs, an animal model which is similar to human beings, simple to operate and stable needs to be established.

At present, the traditional alcoholic fatty liver model usually takes rats and mice as experimental objects, adopts alcohol or wine, and injects alcohol into the stomach or abdominal cavity, and the experimental period is about 1-16 weeks. For example, after the Wanhaihui rats and other rats are administrated with 8ml/kg of wine and are perfused for 2 times/day for 5 consecutive days, the pathological condition shows that the liver cytoplasm of the rats has lipid drops vacuoles with different sizes. Rogory et al uses 56 degree red star Erguotou wine to perfuse the stomach at a ratio of 10ml/kg, each for 1 time in the morning and afternoon, and perfuse the stomach continuously for 8 weeks to establish a rat alcoholic fatty liver model. Rogory et al also mixed a high fat formulation containing fat emulsion (lard 25g, cholesterol lOg, propylthiouracil 1g, Tween 20ml, 50% ethanol) with ethanol and gavage at 15ml/kg for 4 weeks. The results show that the fatty degeneration of the liver tissue of the rat in the model group is obvious, and the serum ALT, AST, Total Cholesterol (TC) and Triglyceride (TG) are all obviously increased. The traditional alcoholic fatty liver model method is simple and easy to implement, short in modeling period and low in cost, but is more complicated in work, high in animal mortality, and not in line with the drinking habit of human beings due to the adoption of the gastric lavage operation.

The non-human primate animal has high homology with human genes, the immune response is similar to that of human, the physiological and biochemical indexes of all animals are most similar to those of human, and in recent years, the non-human primate animal is adopted to establish an alcoholic fatty liver animal model. For example, the invention relates to a Chinese patent with application number 201010200583.4, named as a method for constructing an animal model of liver injury of macaque, which uses macaque as a model animal, adds absolute ethyl alcohol into purified water for drinking by macaque, increases the concentration by 5% per week until the concentration reaches 20%, lasts for 20-30 weeks, and feeds a maintenance period feed to obtain the animal model of alcoholic fatty liver. For example, the invention discloses a Chinese patent with application number of 201610793182.1, entitled "construction method of a cynomolgus monkey alcoholic liver disease model", which adopts aspartame drinking water, adds 10% of alcohol every week to enable the drinking water to be freely drunk, screens out test animals, replaces the aspartame in the alcohol with 10% of cane sugar, continuously feeds 30% of alcohol and additionally adds 20% of lard into basic feed, and obtains an alcoholic fatty liver animal model at the end of 12 weeks. The method uses alcohol which is not normally drunk by people, adopts high-sugar high-fat feeds such as aspartame, cane sugar, lard and the like, has the defects of long molding period, complex operation and the like due to the molding time of 84-238 days, and limits the application of the method in the aspects of research on pathogenesis of alcoholic fatty liver, screening of medicines for preventing or treating alcoholic fatty liver, pharmacodynamic evaluation and the like.

Disclosure of Invention

Aiming at the defects of the alcoholic fatty liver model, in particular to the defects of an alcoholic fatty liver disease model induced by single factor wine similar to human, the invention provides a method for rapidly inducing the alcoholic fatty liver model of a non-human primate by adopting a wine list factor.

The invention is realized by the following technical scheme:

a method for rapidly inducing alcoholic fatty liver model by single factor comprises drinking wine containing 10-35% ethanol for non-human primate with ethanol content of 4.0-5.7g/kg per day, feeding with basal feed freely, and feeding for 50-90 days to obtain stable alcoholic fatty liver model.

The wine is an ethanol-containing beverage prepared by fermenting starch-containing or sugar-containing substances such as grains, fruits and the like by people, and is mainly divided into fermented wine, distilled wine, prepared wine and the like according to a production process; the wine is required to meet the quality standard specified by the state or related departments, and the ethanol concentration is more than 10 percent Vol. The invention prefers distilled liquor, namely liquor obtained by adopting distillation technology after raw material alcohol fermentation, namely liquor with the alcohol content improved by distillation by using fermented liquor, the alcohol content is higher, such as: brandy, whisky, gold wine, vodka, rum, white spirit, and the like. The distilled spirit of the present invention is preferably white spirit or brandy. The white spirit is prepared by preparing fermented grains or fermented mash from starch or sugar raw materials and distilling the fermented grains or the fermented mash, wherein the sugar raw materials are mainly grains. The white spirit is divided into Maotai-flavor type, Luzhou-flavor type, fen-flavor type, rice-flavor type and the like according to the national relevant standards, and is divided into the following components according to the alcohol content or the volume percentage of ethanol in the white spirit: 56% Vol, 53% Vol, 48% Vol, 35% Vol, and the like. Brandy is made from fruits by fermentation, distillation, storage and brewing. The wine containing 10-35% of ethanol is the percentage of the volume of ethanol in the wine to the volume of the wine, and is the volume percentage concentration.

The method for rapidly inducing the alcoholic fatty liver model by the single factor is characterized in that the non-human primates are tracked and observed by liver biochemical indexes, imaging and histopathological detection after being fed for 30 days. Carrying out blood index detection 1 time every week, and when serum ALT and AST are observed to be increased; the imaging examination can be started, and when the diffuse enhancement of the liver near-field echo is observed and the echo is stronger than the kidney, the histopathological examination of the liver can be carried out. When B-ultrasonic observation shows that the liver near-field echo is enhanced in a diffuse manner, the echo is stronger than the kidney, and the liver far-field echo is gradually attenuated, so that a stable alcoholic fatty liver model can be judged.

The basic feed is common feed for daily feeding of the non-human primates, and can ensure energy required by growth and activity of the primates, such as commercially available non-human primate feed. Preferably contains the following nutrient components in percentage by weight: crude protein is more than or equal to 15 percent, crude fat is more than or equal to 4.5 percent, crude fiber is less than or equal to 4 percent, and crude ash is less than or equal to 7 percent.

The method for rapidly inducing the alcoholic fatty liver model by the single factor is characterized in that the non-human primate drinking wine preferably comprises the following steps: the ethanol concentration in the wine and the daily ethanol amount are gradually increased.

In the method for rapidly inducing alcoholic fatty liver model by using the single factor, the mode of drinking wine by the non-human primate is further preferably as follows: the concentration gradient of ethanol in the wine is increased to 35 percent, the ethanol amount gradient in each day is increased to 5.7g/kg, the maintenance time is more than or equal to 20 days, and the total feeding time is 50-70 days, thus obtaining the stable alcoholic fatty liver model.

In the method for rapidly inducing alcoholic fatty liver model by using single factor, the mode of drinking wine by the non-human primate is further preferably as follows: the concentration of ethanol in the wine is increased by 1-5% every 1-5 days, and the amount of ethanol in the wine is increased by 0.03-0.31g/kg every 1-5 days.

The non-human primate is preferably an adult non-human primate, and more preferably a non-human primate with the age of more than or equal to 6 years.

The primate has high homology with human genes, the immune response is similar to that of human, and the liver and physiological and biochemical indexes of all animals are most similar to those of human, so that the non-human primate is selected as the animal model of the alcoholic fatty liver. The non-human primate of the present invention may be an ape, a monkey, a chimpanzee, or the like, and more preferably a cynomolgus monkey, a rhesus monkey, a macaque, or the like, which is high in cost performance.

The single-factor rapid induction alcoholic fatty liver model is established by adopting the method. The model is preferably applied to: research on pathogenesis of alcoholic fatty liver, screening of drugs for preventing or treating alcoholic fatty liver, and pharmacodynamic evaluation.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention naturally induces non-human primates by drinking a diet of a single factor, and freely feeds the primates by the basic feed, thereby obtaining the alcoholic fatty liver degeneration animal model which is close to the human natural occurrence, having stable model, and providing a good model carrier for the research of the pathogenesis of the alcoholic fatty liver, the screening of therapeutic drugs, the pharmacodynamics evaluation and the like.

(2) The invention adopts wine and water to prepare wine containing 10-35% of ethanol by volume percentage, and the wine with the concentration and the dosage is drunk by each non-human primate according to the weight of 4-5.7g/kg of ethanol every day, the wine induces, stimulates and accelerates the formation of an alcoholic fatty liver model, and the animal is fed for 50-90 days, so that the stable alcoholic fatty liver disease model can be obtained, and the success rate of modeling is 100%. Through test optimization, the concentration gradient of ethanol in the wine is increased to 35 percent, the ethanol amount gradient in each day is increased to 5.7g/kg, the maintaining time is more than or equal to 20 days, and the total feeding time is 50-70 days, so that the stable alcoholic fatty liver model can be obtained. The molding time is 25-50% of that of the prior art, the molding speed is greatly increased, and the molding cost is reduced.

(3) The wine adopted by the invention is prepared by fermenting materials containing starch or sugar, such as grains, fruits and the like, for people, and comprises the following steps: yellow wine, white spirit, brandy and the like, wherein the wine contains water and ethanol as well as other substances, such as amino acids, vinegar, organic acids and the like, and the white spirit also contains higher alcohols, polyhydric alcohols, aldehydes, carboxylic acids, esters, acids and the like, and test results show that: the substances make the wine have more fragrant and mellow flavor compared with a mixture of ethanol and water, and the wine is easily accepted and addicted by non-human primates, so that the wine has better compliance to the wine, stimulates and accelerates the formation of alcoholic fatty liver, and is convenient and easy to operate. The invention adopts a life style which is similar to that of heavy drinking or alcohol drinking of people for continuous feeding, can construct a real natural biological environment for simulating human to eat alcohol by using non-human primates, and the obtained alcoholic fatty liver model is closer to that of the human.

(4) The invention inspects the liver of the non-human primate by liver biochemical indexes, B-ultrasonic imaging and histopathology to inspect whether the non-human primate has the alcoholic fatty liver, the used inspection method has high sensitivity, strong specificity and simple operation, can be used for biopsy, has little harm to animals, and can still carry out pharmacodynamic test after a stable alcoholic fatty liver model is obtained.

(5) The non-human primate animal selected by the invention has high gene homology with human, the immune response is similar to human, the liver and physiological and biochemical indexes of all animals are most similar to human, the similarity of the occurrence and pathological processes of diseases of the established alcoholic fatty liver model with human is high, and the occurrence and development of the human alcoholic fatty liver can be simulated.

Drawings

FIG. 1 is a B-ultrasonic diagram of liver before experiment on a blank group of cynomolgus monkeys

FIG. 2 is a B-ultrasonic diagram of liver of a blank group of rhesus monkeys fed for 90 days

FIG. 3 is B ultrasonic diagram of liver of macaque fed for 90 days

FIG. 4 is a schematic diagram showing the liver tissue of a blank set of cynomolgus monkeys after 90 days of feeding

FIG. 5 is a HE staining pattern of liver tissue structure of a blank group of cynomolgus monkeys fed for 90 days

FIG. 6 is a graph showing oil red staining of liver tissue structure of a blank group of cynomolgus monkeys after 90 days of feeding

FIG. 7 is a staining chart of a liver tissue structure of a blank group of Macaranga fascicularis fed for 90 days

FIG. 8 is a B-ultrasonic photograph of liver of cynomolgus monkey fed for 50 days in example 6

FIG. 9 is the anatomical diagram of the liver tissue of cynomolgus monkey fed for 56 days in example 6

FIG. 10 is the HE staining pattern of the liver tissue structure of cynomolgus monkey fed for 56 days in example 6

FIG. 11 is the oil red staining pattern of the liver tissue structure of cynomolgus monkey fed for 56 days in example 6

FIG. 12 is the staining pattern of masson of the liver tissue structure of cynomolgus monkey fed for 56 days in example 6

FIG. 13 is B-ultrasonic diagram of liver of rhesus macaque fed for 63 days in example 2

FIG. 14 is B ultrasonic picture of liver of macaque fed for 70 days in example 10

FIG. 15 is a B-ultrasonic photograph of liver of rhesus monkey fed for 70 days in example 3

Detailed Description

The present invention is further illustrated by the following specific examples, but the scope of the present invention is not limited thereto.

First, main instrument, test material

1. The main apparatus is as follows:

(1) a biochemical analyzer: hitachi 7180 full-automatic biochemical analyzer, purchased from Hitachi, Japan.

(2) Blood cell analyzer: sysmex XN-1000, available from Hesimecon medical electronics (Shanghai) Inc.

(3) B ultrasonic inspection instrument: mindray portable B-ultrasonic apparatus.

2. Test materials:

(1) all are commercially available quality-qualified wine. Respectively 16% of Vol Tu-brand aged tribute wine dry type (yellow wine), 40% of Vol Royal X.O (brandy), 56% of Vol Hongxing Erguotou fen-flavor type (white spirit), and 48% of Vol Xinghua Fencun wine (white spirit).

Preparing the wine concentration: the concentration of the wine is volume percentage concentration, namely the volume percentage of ethanol in the wine to the volume percentage of the wine. The wine and distilled water are prepared according to the following formula:

X2％×(V1+V2)＝V1×X1％，V1+V2＝1000ml

x1 is the alcohol content, X2 is the alcohol content required to be prepared, V1 is the amount (ml) of the alcohol, V2 is the amount (ml) of the added water,

and observing and measuring the temperature and the concentration of the wine by adopting a thermometer and an alcohol meter after the wine is prepared, finding out the temperature which is the same as that of the solution in a temperature column in a conversion table of the alcohol meter, finding out the concentration which is the same as that of the solution in a concentration column, and obtaining the wine by taking the intersection point number of the two columns as the percentage of the dissolution amount of the alcohol to be observed.

(2) A basal feed. The basic feed is a common feed for daily feeding of commercial non-human primates. The nutrient composition comprises the following nutrient components in percentage by weight: 18.3% of crude protein, 5.6% of crude fat, 3.5% of crude fiber, 3.8% of crude ash, 1.2% of calcium and 1.0% of phosphorus. The preparation method of the basic feed comprises the following steps: 1. respectively pulverizing rice, soybean and soybean meal, and sieving with 60 mesh sieve to obtain pulverized rice powder, soybean powder and soybean meal powder; 2. uniformly mixing 0.1kg of vitamin E and 8kg of soybean oil, adding flour according to an equivalent incremental method, uniformly mixing, and sieving by a 60-mesh sieve to obtain a uniformly dispersed flour mixture A; 3. taking 1.5kg of choline chloride, 0.2kg of vitamin C and 0.15kg of calcium pantothenate, mixing uniformly, adding 3.6kg of stone powder, mixing uniformly, adding 3.7kg of salt and 12kg of calcium hydrophosphate, and mixing uniformly to obtain a mixture B; 4. mixing soybean flour 45kg, soybean protein concentrate 40kg, fish powder 20kg, chicken powder 50kg and mixture A, B to obtain mixture C; 5. and (3) taking 570kg of rice flour, 150kg of soybean meal and the mixture C, uniformly stirring and mixing in a mixing box, putting into a drying oven at 100 ℃ for drying until the water content is less than or equal to 10%, and taking out to obtain the rice cake.

Second, test animals

The cynomolgus monkey, rhesus monkey and macaque used in the experiment are provided by Guangxi Xiongsen primate breeding center. After conventional quarantine and physical examination and no limb damage, the normal behavior habit of animals is complete, and examination reports comprise clinical examination and laboratory examination contents, which are all executed according to national standards. The study was carried out in the International qualification Laboratory of Biotechnology Ltd of Kangxinuoke, Guangxi Ling, Association for Association and authorization of Laboratory animal.

After blood examination, the cynomolgus monkeys, which are free from hepatotropic virus infection, drug or toxic liver injury or autoimmune liver disease, are all adult monkeys, are aged at the age of more than or equal to 6 years, have the male body weight of more than or equal to 5.5kg and have the female body weight of more than or equal to 3.0kg, and are randomly divided into blank groups and experimental groups, wherein the blank groups comprise 6 monkeys (3 men and one sex), and 10 monkeys (5 men and one sex) in each experimental group of the example.

After blood examination, rhesus monkeys, which are free from hepatotropic virus infection, drug or toxic liver injury or autoimmune liver disease, were all adult monkeys, were aged 6 years old or older, had a male weight of 5.5kg or more and a female weight of 3.0kg or more, and were randomly divided into blank groups, experimental groups, 6 blank groups (3 males and females), and 10 experimental groups per example (5 females and males).

After blood examination, the macaques without hepatotropic virus infection, drug or toxic liver injury or autoimmune liver disease are all adult macaques, the age is more than or equal to 6 years old, the male weight is more than or equal to 6.0kg, and the female weight is more than or equal to 4.0kg, and the macaques are randomly divided into blank groups and experimental groups, wherein the blank groups comprise 4 (2 male and female), and 6 (3 male and female) are respectively used in each example of the experimental groups.

Wherein the B-ultrasonic diagram of liver before experiment of the blank group of cynomolgus monkeys is shown in figure 1: the liver has normal size and shape, smooth capsule, uniform substantial echo, clear texture display of blood vessels in the liver, and internal diameter of the main trunk of portal vein of about 0.8 cm. The gallbladder has normal size, thick wall, smooth surface, good internal sound transmission, and the inner diameter of common bile duct is about 0.7 cm. And (4) conclusion: the liver was normal.

Third, preliminary test

26 healthy cynomolgus monkeys (13 male and female) without hepatotropic virus infection, drug or toxic liver injury and autoimmune liver disease were selected. Adding purified water into 56% Vol fen-flavor Hongxing Erguotou white spirit according to volume percentage to prepare the white spirit with ethanol concentration of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% and 45% respectively; mixing absolute ethanol (commercially available absolute ethanol, analytically pure, and produced by hydration) with purified water to obtain mixture containing ethanol 10%, 20%, 30%, and 40%. The prepared 13 samples are respectively fed to 2 cynomolgus monkeys, the samples are freely drunk by the cynomolgus monkeys within 1-2 hours after eating the basic feed according to the ethanol amount of 4.5g/kg of body weight every day, and the compliance and the influence on the body are observed.

Wine pre-test results: (1) when the wine containing 5% and 15% of ethanol is given, the cynomolgus monkey can drink freely, has better compliance and can drink the specified amount freely. (2) The wine containing 20% and 25% ethanol is not suitable for cynomolgus monkey, and can be adapted gradually and drunk freely after 2-3 days. (3) The wine with 30 percent and 35 percent of ethanol concentration is given, the cynomolgus monkey can resist at first, and can gradually adapt to the drinking water and freely drink the specified amount after 3 to 5 days after the drinking water is removed. (4) The wine with the ethanol concentration of 40-45% is refused to be drunk by the cynomolgus monkey, after the drinking water is removed, the wine cannot be adapted and has small free drinking amount within 5 days, the specified amount can be drunk only by assisting manual feeding, and about 10 days, the wine with the ethanol concentration of 45% is found that 1 cynomolgus monkey has damaged oral cavity and digestive tract and listlessness. The results show that: the compliance of the cynomolgus monkey with the wine containing the ethanol with the concentration of below 35 percent is better, and the cynomolgus monkey has certain addiction which is expressed as craving for the wine or emotional irritability after stopping giving the wine.

Ethanol and water mixture pre-test results: (1) when a mixture of 10 percent and 20 percent of absolute ethyl alcohol and water is given, the cynomolgus monkey can drink freely, but can only drink 40 to 50 percent of the specified amount, and can drink the specified amount only by assisting manual feeding. (2) The mixture of 30 percent and 40 percent of absolute ethyl alcohol and water is given, the cynomolgus monkey refuses to drink, after drinking water is removed, the cynomolgus monkey can not adapt to the drinking water in 5 days and can drink a specified amount only by assisting manual feeding, and about 1 week, the cynomolgus monkey is found to have damaged oral cavity and digestive tract and listlessness. The results show that: when the mixture of absolute ethyl alcohol and water with the concentration of less than 20 percent is given, the cynomolgus monkey can drink freely, but cannot drink the specified amount, and has no obvious addiction phenomenon after the specified amount is fed by manual assistance for 20 days.

Fourth, modeling method and result

1. Feeding method and results of blank group.

The method for feeding the cynomolgus monkey blank group comprises the following steps: taking 6 healthy cynomolgus monkeys without hepatotropic virus infection, drug or toxic liver injury and autoimmune liver disease, and 3 male and female monkeys respectively, feeding freely with basic feed, and drinking normal purified water.

Feeding method of rhesus monkey blank group: taking 6 healthy rhesus monkeys without hepatotropic virus infection, drug or toxic liver injury and autoimmune liver disease, 3 monkeys and females respectively, feeding freely with basic feed, and drinking normal purified water.

The feeding method of the macaque blank group comprises the following steps: taking 4 healthy rhesus monkeys without hepatotropic virus infection, drug or toxic liver injury and autoimmune liver disease, and 2 monkeys and males respectively, feeding freely with basic feed, and drinking purified water normally.

Blank group test results: the above cynomolgus monkey blank group, rhesus monkey, macaque blank group, totally 16, follow-up observation 1 time per month: blood tests liver function, kidney function, blood fat and blood routine, B-ultrasonic imaging tests liver, kidney, spleen and abdomen B-ultrasonic. The liver ultrasonography B of rhesus macaque fed for 90 days is shown in figure 2, the liver ultrasonography B of rhesus macaque fed for 90 days is shown in figure 3, and the liver tissue dissection, tissue structure HE staining, oil red staining and masson pine staining of cynomolgus macaque fed for 90 days are shown in figures 4-7. The middle of each hepatic lobule on the section is a central vein, the hepatic cells are radially arranged to the periphery by taking the central vein as the center to form a cell plate with the thickness of a single cell, namely the hepatic plate, and hepatic blood sinuses are connected between the hepatic plates. On the section, liver cords are gathered in the cells, liver blood sinuses are in the blank, the cytoplasm of the liver cells is acidophilic, and HE staining is red. Between the liver lobules, there are lobular vena cava, lobular artery, and lobular bile duct, which are mainly concentrated in the portal or the drainage area. The interlobular artery is small and round, the wall thickness of the vessel; the vein between the lobules is large and irregular, and the wall of the tube is thin; the lobular ducts are small and round, and the walls of the ducts are formed by a single layer of cubic epithelium. And (4) conclusion: the liver was normal.

The results show that: after 90 days, the cynomolgus monkey, rhesus monkey and macaque are basically the same as before the test: peripheral blood tests show that liver function, kidney function, blood fat and blood sugar are not obviously changed, abdominal B ultrasonic tests also do not show the diffuse enhancement phenomenon of liver near-field echoes, and liver and kidney echoes are not different, namely fatty lesions do not appear.

2. Modeling methods and results analysis for each example of the experimental group.

(1) Modeling methods of examples 1-4.

Randomly taking 20 healthy cynomolgus monkeys (half of female and male) without hepatotropic virus infection, drug or toxic liver injury and autoimmune liver disease, randomly dividing the healthy cynomolgus monkeys into 2 groups: example 1 and example 4 each contained 10 (hermaphrodite halves). The group of example 2 was prepared by randomly selecting 6 healthy macaques (male and female halves) without hepatotropic virus infection, drug or toxic liver injury, and autoimmune liver disease. The groups of example 3 were randomly selected from 10 healthy rhesus monkeys (female and male half) without hepatotropic infection, drug or toxic liver injury, and autoimmune liver disease.

According to the conditions of table 1, based on the body weight of each animal, a certain concentration, a certain dose, various kinds and degrees of wine were drunk every day, while a basal feed was given for unlimited free feeding. Carrying out blood index detection 1 time every week, and when serum ALT and AST are observed to be increased; the imaging examination can be started, and when the diffuse enhancement of the liver near-field echo is observed and the echo is stronger than the kidney, the histopathological examination of the liver can be carried out. When B-ultrasonic observation shows that the liver near-field echo is enhanced in a diffuse manner, the echo is stronger than the kidney, and the liver far-field echo is gradually attenuated, so that a stable alcoholic fatty liver model can be judged.

Table 1 examples 1-4 modeling conditions and results

(2) Modeling methods for examples 5-10.

The healthy cynomolgus monkeys 40 (half of male and female) without hepatotropic virus infection, drug or toxic liver injury and autoimmune liver disease were randomly selected and randomly divided into 4 groups: example 5, example 6, example 8 and example 9 each contained 10 (hermaphrodite halves). The groups of example 7 were randomly selected from 10 healthy rhesus monkeys (female and male half) without hepatotropic infection, drug or toxic liver injury, and autoimmune liver disease. The group of example 10 was randomly selected from 6 healthy macaques (female and male half) without hepatotropic virus infection, drug or toxic liver injury, and autoimmune liver disease.

According to the conditions of tables 2 to 7: according to the weight of each animal, a certain concentration and a certain dosage of wine prepared by 56% Vol Hongxing Erguotou fen-flavor white spirit are given according to the conditions of the examples every day, the concentration of ethanol in the wine is gradually increased from 10% to 35%, and the amount of the wine given every day is gradually increased from 4g/kg to 5.7g/kg according to the weight of the contained ethanol. While giving unlimited free feeding of the basal feed. Carrying out blood index detection 1 time every week, and when serum ALT and AST are observed to be increased; the imaging examination can be started, and when the diffuse enhancement of the liver near-field echo is observed and the echo is stronger than the kidney, the histopathological examination of the liver can be carried out. When B-ultrasonic observation shows that the liver near-field echo is enhanced in a diffuse manner, the echo is stronger than the kidney, and the liver far-field echo is gradually attenuated, so that a stable alcoholic fatty liver model can be judged.

Table 2 example 5 modeling conditions and results

Table 3 example 6 modeling conditions and results

Table 4 example 7 modeling conditions and results

Table 5 example 8 modeling conditions and results

Table 6 example 9 modeling conditions and results

Table 7 example 10 modeling conditions and results

(3) Analysis of the modeling results of the experimental group of the examples.

The invention is characterized by feeding, observation and detection for 90 days: in 10 experimental groups, 92 non-human primates (including cynomolgus monkeys, rhesus monkeys and macaques) in total in the experimental group all have elevated serum ALT, AST or GGT, and some serum TBil is elevated; liver near-field echo diffuse enhancement phenomenon appears in abdominal B-ultrasonic imaging detection, and liver and kidney echoes are remarkably distinguished; liver histopathological examination shows that liver fatty lesion is obvious. The successful modeling time is 50-90 days, the success rate is 100%, the obtained alcoholic fatty liver model is stable, and the alcoholic fatty liver of the animal model in the examples 1-10 can not be recovered to be normal after the examination of 6 months after the drinking is stopped. Secondly, compliance of the non-human primates drinking the low-concentration wine containing less than 20% of ethanol is good in the experiment, the wine containing 25% -35% of ethanol and higher concentration needs to be induced and adapted by removing normal drinking water or auxiliary feeding, compliance is good after 2-5 days, the wine can be drunk by self to complete specified drinking amount, and addiction phenomena occur in part of cynomolgus monkeys, rhesus monkeys and macaques after 20 days. Third, the test finds that: the animal compliance is better in a mode of increasing the concentration of wine and the dose gradient of drinking ethanol. And the concentration gradient of the ethanol in the wine is increased to 35 percent, the ethanol amount gradient in each day is increased to 5.7g/kg, the maintaining time is more than or equal to 20 days, and the total feeding time is 50-70 days, so that a stable alcoholic fatty liver model can be obtained, the molding time is accelerated, and the method is a more preferable technical scheme. The test of the invention does not need operation or animal sacrifice examination, and the molding cost is very low. The alcoholic fatty liver model established by the method can be applied to the research of the pathogenesis of the alcoholic fatty liver, the screening of the medicine for preventing or treating the alcoholic fatty liver and the pharmacodynamic evaluation. The experimental conditions of the present invention will be briefly described with reference to the accompanying drawings:

(1) the alcoholic fatty liver model of example 6 of the experimental group of the present invention is shown in FIGS. 8 to 12.

FIG. 8 shows a B-ultrasonic picture of liver of cynomolgus monkey fed for 50 days in example 6, which is stable in molding. The B ultrasonic observation shows that the liver parenchymal echo is enhanced compared with that before drinking, the near-field echo is enhanced in a diffuse manner, the liver and kidney echoes have contrast, the liver far-field echo is gradually attenuated, and the spleen echo is not abnormal.

See fig. 9, the liver tissue profile of a 56-day molded stable cynomolgus monkey fed in example 6. After the model is stable for 56 days, the liver of the model group shows moderate to severe steatosis, the volume is obviously increased, the envelope is tense, the texture is hard, and the part of the liver with greasy surface shows milk yellow. The liver of normal cynomolgus monkey is reddish brown or dark red and soft, as shown in FIG. 4.

FIG. 10 shows HE staining patterns of the hepatic histoarchitecture of the molded-stable liver of 56 days after feeding in example 6. Before the experiment, the outline of liver lobules is clear, liver cells are arranged in a radial mode by taking a lobule central vein as a center, liver cell cords are arranged neatly, the liver cells are polygonal, the cell boundaries are clear, the nucleus is round and located in the center of the cells, cytoplasm is rich and basophilic, the cytoplasm of the liver cells around the central vein is occasionally loose, and the alveolate lipid drops are occasionally seen in 2 or 3 acinus areas of the acinus under a high power microscope. Liver tissue sections after the 56-day stable model formation have disordered liver cell cable arrangement around the central vein, unclear lobular boundary, wide and vacuolar degeneration with different sizes in liver cytoplasm, mainly central vein periphery (namely acinus 3 area), and lobular and portal area focal inflammatory cell infiltration with different degrees without fibroseptal formation.

FIG. 11 shows the oil red staining pattern of the liver tissue structure of example 6 fed for 56 days. Before the experiment, the liver tissue structure is complete and clear, the hepatic lobule structure is normal, the central vein is large and thin, the hepatic cells are arranged into hepatic cords and are radially distributed around the central vein, the cells are polygonal, and red fat particles are not seen. After the model is stable for 56 days, the liver shows moderate to severe steatosis, moderate nephelosis of liver cells, necrosis of a small amount of liver cells, red fat particles with different sizes and different quantities can be seen in most liver cells, and for severe steatosis, the fat drops are vacuolated and fused, and the red fat particles are dense.

FIG. 12 shows Masson staining of the liver tissue structure for 56 days of feeding in example 6. The lobule structure of the hepatic tissue is clear before the experiment, the hepatic cells and hepatic blood sinuses are radially arranged by taking the central vein as the center, the fibrous tissue is not proliferated, and a small amount of blue type I collagen can be seen only in the area of the sink and around the central vein. After the molding is stable for 56 days: the lobule structure of the hepatic tissue is clear, the degeneration of the hepatic cells is obvious, fat vacuoles with different sizes and different quantities can be seen in most hepatic cells, the fibrous tissue can be occasionally proliferated, the fiber interval is fine and loose, and the fusion phenomenon is rare.

The results show that: obtaining a stable macaca fascicularis alcoholic fatty liver model.

(2) The experimental group of the invention, example 2 and example 10, show the alcoholic fatty liver model conditions of macaque, as shown in fig. 13 and 14. The B ultrasonic observation shows that the liver parenchymal echo is enhanced compared with that before drinking, the near-field echo is enhanced in a diffuse manner, the liver and kidney echoes have contrast, the liver far-field echo is gradually attenuated, and the spleen echo is not abnormal. The results show that: example 2 and example 10 stable macaque alcoholic fatty liver model was obtained.

(3) Experimental group example 3 of the present invention shows the condition of rhesus monkey alcoholic fatty liver model after feeding for 70 days, as shown in FIG. 15. The B ultrasonic observation shows that the liver parenchymal echo is enhanced compared with that before drinking, the near-field echo is enhanced in a diffuse manner, the liver and kidney echoes have contrast, the liver far-field echo is gradually attenuated, and the spleen echo is not abnormal. The results show that: obtaining a stable rhesus monkey alcoholic fatty liver model.