CN111631187B - Method for rapidly inducing hepatic fibrosis animal model - Google Patents
Method for rapidly inducing hepatic fibrosis animal model Download PDFInfo
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- CN111631187B CN111631187B CN201910736571.4A CN201910736571A CN111631187B CN 111631187 B CN111631187 B CN 111631187B CN 201910736571 A CN201910736571 A CN 201910736571A CN 111631187 B CN111631187 B CN 111631187B
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Abstract
The invention discloses a method for rapidly inducing a hepatic fibrosis animal model, which is characterized in that a non-human primate is drunk with wine containing 10-35% of ethanol according to the ethanol amount of 4.0-5.7g/kg of body weight every day, meanwhile, basic feed is freely fed, and the hepatic fibrosis animal model can be obtained after the non-human primate is fed for 160 days of 120-fold supplementation. The invention can obtain a stable hepatic fibrosis animal model which is close to the natural occurrence of human beings, the compliance of non-human primates is better, operations or animal sacrifice examination is not 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 hepatic fibrosis, the screening of therapeutic drugs, the pharmacodynamics evaluation and the like.
Description
Technical Field
The invention relates to a method for establishing an animal disease model, in particular to a method for rapidly inducing a hepatic fibrosis animal model.
Background
Hepatic Fibrosis (HF) is a common manifestation of many chronic liver diseases such as viral hepatitis, alcoholic liver, etc., and refers to a pathophysiological process in which excessive deposition of extracellular matrix in the liver is finally caused by tissue repair due to liver injury caused by various causes. If liver damage continues to develop, mild hepatic fibrosis will develop into decompensated cirrhosis, but at present, no good treatment means exists for cirrhosis, the survival rate of decompensated cirrhosis in 5 years is only 14%, and meanwhile, the continuous development of hepatic fibrosis may also cause abnormal liver function, even hepatocellular carcinoma, and greatly threatens the life and health of human beings. Therefore, the construction of an ideal animal model capable of simulating the hepatic fibrosis process of a human body becomes a key for exploring early diagnosis, searching effective therapeutic drugs for hepatic fibrosis and improving the disease condition and prognosis.
The existing hepatic fibrosis animal model modeling methods include a chemical modeling method, an ethanol modeling method, an immune modeling method, a viral modeling method and the like. Among them, the carbon tetrachloride-induced hepatic fibrosis model in the chemical modeling method is most commonly used, and the method is to inject 1:1 CCl into the abdominal cavity according to the quality of 0.5-2mL/kg body4Olive oil solution, 6-9 weeks later can lead to liver fibrosis. Pure CCl4The method has simple operation and low cost, the hepatic fibrosis degree gradually reaches the peak of average 9.2% in 8 weeks from average 4.9% in 4 weeks, the model success rate reaches 89.33%, but CCl4Severe hepatotoxicity and high animal mortality, and is not suitable for experimental studies with a long period. There are mainly 2 ethanol method models: firstly, Lieber-Decali model, rodent is allowed to freely drink liquid feed containing ethanol. The model can form fatty liver, but does not have hepatic fibrosis related expression, and simultaneously, experimental animals are tired of wine and have low induction success rate, thus bringing great challenge to model building. Tsukamoto-French model: rodent is perfused with high-fat liquid diet containing alcohol continuously in stomach, and liver is injured to induce hepatic fibrosis. For example, the stomach of an experimental rat was continuously perfused with a solution containing 60% ethanol (1mL/kg body mass), olive oil (2mL/kg body mass) and pyrazole (25mg/kg-1.d-1) for 9 weeks. But the degree of hepatic fibrosis is light, the experimental technical requirement is high, the operation is complicated, and the popularization and the application need to be further improved to aggravate the degree of hepatic fibrosis and reduce the molding cost.
Although there are many existing animal models of hepatic fibrosis, because the causes of hepatic fibrosis are complex and various, and the species of animals are different from human, so far, animal models which can completely reflect hepatic fibrosis of human have not been completely successful, and in the concrete work, the most suitable animal model is selected by fully considering the causes and mechanisms in the process of hepatic fibrosis formation, species difference between human and animals, natural recovery rate of model-making animals and other factors. The non-human primate animal has high homology with human genes, the immune response is similar to human, the physiological and biochemical indexes of all animals are most similar to those of human, and the non-human primate animal is taken as an object to construct an ideal hepatic fibrosis animal model and needs further intensive research.
Disclosure of Invention
Aiming at the defects of the hepatic fibrosis animal model, the invention provides a method for rapidly inducing the hepatic fibrosis animal model of the non-human primate by adopting a wine singles factor, the method takes the non-human primate as an object, obtains the animal model which has obvious B ultrasonic and pathological detection results and can completely simulate the occurrence and development of the hepatic fibrosis animal model by adopting the wine singles factor induction, and provides a good model carrier for the research of the pathogenesis of the hepatic fibrosis, the screening of preventive or therapeutic drugs, the pharmacodynamic evaluation and the like.
The invention is realized by the following technical scheme:
a method for rapidly inducing hepatic fibrosis animal model comprises drinking wine for non-human primate according to ethanol amount of weight more than or equal to 4.0g/kg every day to obtain hepatic fibrosis animal 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 should meet the quality standards stipulated by the state or relevant departments.
In the method for rapidly inducing an animal model of liver fibrosis, the drinking time of the wine in the non-human primate is preferably 120-160 days.
In the method for rapidly inducing an animal model of hepatic fibrosis, the wine is preferably a wine containing 10-35% ethanol. 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.
In the method for rapidly inducing an animal model of liver fibrosis, the non-human primate preferably drinks wine in a way that: the alcohol concentration in the wine and/or the alcohol amount per day are gradually increased.
In the method for rapidly inducing an animal model of liver fibrosis, the non-human primate preferably drinks wine in a way that: the concentration gradient of the ethanol in the wine is increased to 35 percent, the maintenance time is more than or equal to 80 days after the ethanol amount gradient in each day is increased to 5.7g/kg, and the total feeding time is 120-135 days, so that the hepatic fibrosis animal model can be obtained.
The method for the hepatic fibrosis animal model performs tracking observation on the non-human primates through liver imaging after feeding for 100 days, and the hepatic fibrosis model can be judged when obvious hepatic fibrosis signs are observed. In the experimental condition phase of the present invention, in the investigation phase, after 90 days of feeding, B-ultrasound imaging examination of non-human primates was started, and then every 10-15 days, when: the liver is reduced or unchanged, the liver envelope is thickened, the surface of the liver is uneven and is in a saw-toothed shape, the edge of the liver becomes irregular due to blunting, the far-end echoes of the liver parenchyma are increased and are in a dot shape with different and dense sizes, high echo light spots or patch cords are irregularly scattered, a hepatic fibrosis model can be judged, and further verification of histopathology detection of the liver can be carried out.
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.
In the method for rapidly inducing an animal model of liver fibrosis, the non-human primate preferably drinks wine in a way that: the ethanol concentration in the wine and the daily ethanol amount are gradually increased.
The method for rapidly inducing the hepatic fibrosis animal model is characterized in that the wine drinking mode of the nonhuman primate is as follows: the concentration gradient of the ethanol in the wine is increased to 35 percent, the maintenance time is more than or equal to 80 days after the ethanol amount gradient in each day is increased to 5.7g/kg, and the total feeding time is 120-135 days, so that the stable hepatic fibrosis animal model can be obtained.
The method for rapidly inducing the hepatic fibrosis animal model is characterized in that the wine drinking mode of the nonhuman primate is 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.
In the method for rapidly inducing an animal model of liver fibrosis, the nonhuman primate is preferably an adult nonhuman primate, and more preferably a nonhuman primate aged 6 or older.
The non-human primate animal 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 the non-human primate animal is selected as the hepatic fibrosis animal model. 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 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 rapid hepatic fibrosis induction animal model is established by adopting the method. The model is preferably applied to: research on the pathogenesis of hepatic fibrosis, screening of medicaments for preventing or treating hepatic fibrosis and pharmacodynamic evaluation.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention naturally induces non-human primates by the diet of single factor of wine, and freely feeds basic feed, thus obtaining the hepatic fibrosis animal model which is close to the hepatic fibrosis naturally occurring in human beings, having stable model, and providing good model carrier for the research of the pathogenesis of the hepatic fibrosis, the screening of therapeutic drugs, the pharmacodynamics evaluation and the like.
(2) The invention adopts wine and water to prepare wine containing 10-35% ethanol by volume percentage, and the wine with the concentration and the dosage is drunk by each non-human primate according to the ethanol amount of 4-5.7g/kg of body weight every day, the wine induces, stimulates and accelerates the formation of a hepatic fibrosis model, the animal is fed for 120-160 days, and the stable hepatic fibrosis animal model can be obtained, and the success rate of modeling is 100%. Through test optimization, the concentration gradient of the ethanol in the wine is increased to 35 percent, the maintenance time is more than or equal to 80 days after the ethanol amount gradient in each day is increased to 5.7g/kg, and the total feeding time is 120-135 days, so that the stable hepatic fibrosis animal model can be obtained. The molding time is 10-40% 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 savory 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 non-human primates, stimulates and accelerates the formation of hepatic fibrosis, and is convenient and easy to operate. The invention adopts a life style which is similar to that of alcoholism or alcoholism of the human to continuously feed, can construct a real natural biological environment for simulating human to ingest the alcohol by the non-human primate, and the obtained hepatic fibrosis animal model is more similar to the human.
(4) In the test, liver biochemical indexes, B-ultrasonic imaging and histopathology are adopted to check the liver of the non-human primate so as to check whether the non-human primate has hepatic fibrosis. The stable hepatic fibrosis model can be determined and obtained through B-ultrasonic imaging, and further verification through histopathology experiments shows that the results are consistent. Therefore, the detection method has high sensitivity, strong specificity and simple operation, can be used for biopsy, has small harm to animals, and can still carry out pharmacodynamic tests on the animals after stable hepatic fibrosis animal models are obtained.
(5) The non-human primate animal selected by the invention has high homology with human genes, the immune response is similar to human, the liver and physiological and biochemical indexes of all animals are most similar to human, and the degree of similarity between the occurrence and pathological processes of the diseases of the established hepatic fibrosis animal model and the human is high, so that the occurrence and development of the hepatic fibrosis of the human 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 the staining (100 ×) (200 ×) of the liver tissue structure of a blank group of cynomolgus monkeys after 90 days of feeding
FIG. 7 is a B-ultrasonic photograph of liver of cynomolgus monkey fed for 135 days in example 6
FIG. 8 is the anatomical diagram of the liver tissue of the cynomolgus monkey fed for 135 days in example 6
FIG. 9 is the HE staining pattern of the liver tissue structure of 135-day-old cynomolgus monkey fed in example 6
FIG. 10 is a graph showing the staining of masson pine (100 ×) (200 ×) in the liver tissue structure of 135-day-old cynomolgus monkeys fed with the feed of example 6
FIG. 11 is B ultrasonic picture of liver of macaque fed for 120 days in example 2
FIG. 12 is the HE staining pattern of the liver tissue structure of macaque fed for 120 days in example 2
FIG. 13 is a graph showing the staining (200X) of the liver tissue structure of macaque (Macaran) by 120 days of feeding in example 2
FIG. 14 is a B-ultrasonic diagram of liver of rhesus monkey fed for 160 days in example 3
FIG. 15 is a graph showing the staining (200X) of masson in the liver tissue structure of rhesus monkey fed for 160 days in example 3
FIG. 16 is B ultrasonic picture of liver of macaque fed for 135 days in example 10
FIG. 17 is a graph showing the staining (200X) of the liver tissue structure of macaque by Masson in 135 days of feeding in example 10
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 feeding with normal drinking 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 and masson staining of cynomolgus macaque fed for 90 days are shown in figures 4-6. 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. The lobule structure of the liver tissue is clear, the liver cells and the liver blood sinuses are arranged in a radial shape 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. 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 and hepatic fibrosis are not shown.
2. Modeling methods and results analysis for each example of the experimental group.
(1) Modeling methods of examples 1-4.
The healthy cynomolgus monkeys (half each female and half) without hepatotropic virus infection, drug or toxic liver injury and autoimmune liver disease were randomly selected and divided into 2 groups: example 1 and example 4 each contain 8 (hermaphrodite half). The group of example 2 was prepared by randomly selecting 4 healthy macaques (male and female halves) without hepatotropic virus infection, drug or toxic liver injury, and autoimmune liver disease. The healthy rhesus monkeys 8 (female and male half) without hepatotropic infection, drug or toxic liver injury, and autoimmune liver disease were randomly selected as the group of example 3.
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. After 90 days of feeding, B-ultrasound imaging examinations of non-human primates were started, and every 10-15 days thereafter, when: the liver is reduced or unchanged, the liver envelope is thickened, the surface of the liver is uneven and is in a saw-toothed shape, the edge of the liver becomes irregular due to blunting, the far-end echoes of the liver parenchyma are increased and are in a dot shape with different and dense sizes, high echo light spots or patch cords are irregularly scattered, a hepatic fibrosis model can be judged, and further verification of histopathology detection of the liver can be carried out.
Table 1 examples 1-4 modeling conditions and results
(2) Modeling methods for examples 5-10.
The healthy cynomolgus monkeys 32 (half of female and male) 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 example 8 (hermaphrodite halves). The healthy rhesus monkeys 8 (female and male half) without hepatotropic infection, drug or toxic liver injury, and autoimmune liver disease were randomly selected as the group of example 7. The group of example 10 was randomly selected from 4 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. After 90 days of feeding, B-ultrasound imaging examinations of non-human primates were started, every 10-15 days, and observed: the liver is reduced or unchanged, the liver envelope is thickened, the surface of the liver is uneven and is in a saw-toothed shape, the edge of the liver becomes irregular due to blunting, the far-end echoes of the liver parenchyma are increased and are in a dot shape with different and dense sizes, high echo light spots or patch cords are irregularly scattered, a hepatic fibrosis model can be judged, and further verification of histopathology detection of the liver can be carried out.
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 in that after 160 days of feeding and observation and detection: in 10 experimental groups, a total of 72 non-human primates (including cynomolgus monkey, rhesus monkey and macaque) are observed through abdominal B-ultrasound imaging detection: the liver is reduced or unchanged, the liver envelope is thickened, the surface of the liver is uneven and is in a saw-toothed shape, the edge of the liver is blunted to be irregular, the far-end echoes of the liver parenchyma are increased to be in a dot shape with different and dense sizes, and high echo light spots or patch cords are irregularly scattered, so that a hepatic fibrosis model can be judged, and the hepatic fibrosis model is further verified through the histopathological detection of the liver. Liver histopathological examination shows that the liver fibrosis lesion is obvious. The successful modeling time is 120-160 days, the success rate is 100%, the obtained hepatic fibrosis animal model is stable, the examination is carried out 8 months after the drinking, and the hepatic fibrosis of the animal model in the examples 1-10 can not be recovered to be normal. 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 maintenance time is more than or equal to 80 days after the ethanol amount gradient in each day is increased to 5.7g/kg, and the total feeding time is 120-135 days, so that the stable hepatic fibrosis animal model can be obtained, the modeling 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 hepatic fibrosis animal model established by the method can be applied to the research of the pathogenesis of hepatic fibrosis, the screening of medicaments for preventing or treating hepatic fibrosis and the pharmacodynamic evaluation, in particular to the research of the pathogenesis of hepatic fibrosis caused by drinking, the screening of the medicaments for preventing or treating hepatic fibrosis and the pharmacodynamic evaluation. The experimental conditions of the present invention will be briefly described with reference to the accompanying drawings:
(1) the experimental group of the invention, the liver fibrosis animal model of example 6, is shown in FIGS. 7-10.
FIG. 7 shows a B-ultrasonic picture of liver of cynomolgus monkey with stable molding after 135 days of feeding in example 6. B-mode ultrasonography observed: the liver is reduced, the liver envelope is thickened, the surface of the liver is uneven and is in a sawtooth shape, the edge of the liver is blunted to be irregular, the parenchymal echo at the far end of the liver is increased diffusely, the echo light spots are dense and have different sizes, are in a point shape and are occasionally in a spot shape, and the liver fibrosis pathological change is prompted.
FIG. 8 shows the anatomical diagram of liver tissue of a 135-day molded and stabilized cynomolgus monkey fed in example 6. Visually observed: the liver is in moderate to severe steatosis, the volume is increased, the liver envelope is slightly tense, the texture is hard, the local surface is grayish yellow, and large and small nodules are scattered. The liver of normal cynomolgus monkey is reddish brown or dark red, and is soft.
FIG. 9 shows HE staining patterns of liver tissue structures of 135-day cynomolgus monkeys fed 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 in 135 days have disorder arrangement of hepatocyte cords around central veins and unclear lobular boundary, and vacuolar changes, local cell necrosis and the like can be observed in liver tissues, which indicates that liver fibroid changes are obvious.
FIG. 10 is a graph showing the staining (100X) (200X) of the liver tissue structure of cynomolgus monkey fed for 135 days 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 135 days of molding: the lobule structure of the liver tissue is complete, the expression quantity of the collagen fiber of the liver tissue is obviously increased, the regional plaque-like distribution is formed, the blood vessel periphery and the manifold area are taken as the main parts, and the liver tissue lobule structure and the hepatic tissue collagen fiber are adhered to each other into a sheet.
The results show that: obtaining the stable hepatic fibrosis animal model.
(2) The experimental group of the invention has the animal model conditions of liver fibrosis of macaques in example 2 (see figures 11-12) and example 10 (see figures 16-17).
Example 2 observations of ultrasound B after 120 days of feeding are shown in FIG. 11. The liver is reduced, the liver envelope is thickened, the surface of the liver is uneven and is in a sawtooth shape, the edge of the liver is blunted to be irregular, the parenchymal echo at the far end of the liver is increased diffusely, the echo light spots are dense and have different sizes and are in a point shape, and the liver fibrosis pathological changes are prompted.
See fig. 12, which is a graph of HE staining of liver tissue structure of macaque fed for 120 days in example 2. 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. After 120 days of molding: liver tissue slices have disordered liver cell cord arrangement around the central vein and unclear lobular boundary, and vacuolar changes, local cell necrosis and the like can be observed in liver tissues, which indicates that liver fibroid lesions are obvious.
See fig. 13, which is a graph of masson staining (200 ×) of liver tissue structure of macaques fed for 120 days in example 2. 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 120 days of molding: under the light microscope (200X), massson stains extensive necrotic degeneration of liver fibrosis hepatocytes, and collagenation lesion map of the original reticular scaffold in the liver lobules. The hepatic lobule structure is clear, the hepatic cell degeneration is obvious, fat vacuoles with different sizes and different quantities can be seen in most hepatic cells, the fibrous tissue hyperplasia is obvious, the fiber interval is fine and loose, the fusion phenomenon is rare, hepatic fibrosis hepatic cells are subjected to extensive necrosis and degeneration, and the original reticular stent in the hepatic lobule is subjected to collagenation lesion.
See fig. 16, which is a B ultrasonic picture of liver of 135 days macaque fed in example 10. Example 10 observation of type-B ultrasonography after 135 days of feeding:
the liver is reduced, the liver envelope is thickened, the surface of the liver is uneven and is in a sawtooth shape, the edge of the liver is blunted to be irregular, the parenchymal echo at the far end of the liver is increased diffusely, the echo light spots are dense and have different sizes and are in a point shape, and the liver fibrosis pathological changes are prompted by occasional spot patches.
See fig. 17, which is a graph of masson staining (200 ×) of liver tissue structure of macaques fed for 135 days in example 10. 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 135 days of molding: under the light microscope (200 ×), extensive necrosis of the liver cells by massson staining occurred, and the proliferating collagen fibers were mapped to the inside of the liver lobules. The hepatic tissue lobule structure is clear, the hepatic cells are obviously degenerated, fat vacuoles with different sizes and different quantities can be seen in most hepatic cells, the fibrous tissue is obviously proliferated, the fiber intervals are fine and loose, the fusion phenomenon is rare, the hepatic cells are widely necrotized, and the proliferated collagen fibers extend into the hepatic lobules.
The results show that: example 2 and example 10 an animal model of liver fibrosis was obtained.
(3) Experimental group example 3 of the invention shows the condition of rhesus monkey liver fibrosis animal model fed for 160 days, which is shown in FIGS. 14-15.
FIG. 14 shows a B-ultrasonic image of liver of rhesus monkey fed for 160 days in example 3. Example 3B-ultrasound observation after 160 days of feeding: the liver has unchanged size, thickened liver envelope, uneven and jagged liver surface, irregular liver edge, diffuse increase of substantial echo at the far end of the liver, dense and irregular echo light spots, and the liver is gradually in a cord shape with spots and indicates liver fibrosis pathological changes.
See FIG. 15, which is a graph of Marson staining (200X) of liver tissue structure of rhesus monkey fed for 160 days in example 3. 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 160 days of molding, under light (200 ×), massson stained central venous and manifold areas were interconnected to develop necrotic zones and a map was developed extending into the lobules of the liver. The hepatic lobule structure is clear, the hepatic cell degeneration is obvious, fat vacuoles with different sizes and different quantities are visible in most hepatic cells, the fibrous tissue hyperplasia is obvious, the fiber interval is fine and loose, the fusion phenomenon is rare, and the central venous and the manifold area are mutually connected to form a necrotic zone and extend into the hepatic lobule.
The results show that: example 3 an animal model of liver fibrosis is obtained.
Claims (7)
1. A method for rapidly inducing a hepatic fibrosis animal model is characterized in that: drinking wine containing 10-35% ethanol for non-human primate with ethanol content of 4.0-5.7g/kg per day, and feeding with basal feed;
the non-human primate drinks the wine in the following way: the concentration gradient of the ethanol in the wine is increased to 35 percent, the maintenance time is more than or equal to 80 days after the ethanol amount gradient in each day is increased to 5.7g/kg, and the total feeding time is 120-135 days, so that the hepatic fibrosis animal model can be obtained;
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.
2. The method for rapidly inducing an animal model of liver fibrosis according to claim 1, wherein: the wine is distilled wine.
3. The method of claim 1, wherein the non-human primate is aged 6 years or more.
4. The method of claim 1, wherein the non-human primate is a cynomolgus monkey, rhesus monkey or macaque.
5. The method for rapidly inducing an animal model of liver fibrosis according to claim 2, wherein: the distilled liquor is Chinese liquor or brandy.
6. A method for rapidly inducing an animal model of liver fibrosis, which is established by the method of any one of claims 1 to 5.
7. Use of the method for rapidly inducing an animal model of liver fibrosis according to claim 6, wherein the model is used for: research on the pathogenesis of hepatic fibrosis, screening of medicaments for preventing or treating hepatic fibrosis and pharmacodynamic evaluation.
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