CN107519153B

CN107519153B - Method for establishing rhesus monkey hepatic fibrosis model by gastric lavage method

Info

Publication number: CN107519153B
Application number: CN201710909300.5A
Authority: CN
Inventors: 黄孝伦; 冯天航; 赖春友; 魏玲玲; 施静; 袁可; 张恬莹
Original assignee: Sichuan Provincial Peoples Hospital
Current assignee: Sichuan Provincial Peoples Hospital
Priority date: 2017-09-29
Filing date: 2017-09-29
Publication date: 2020-07-31
Anticipated expiration: 2037-09-29
Also published as: CN107519153A

Abstract

The invention provides a method for establishing a rhesus monkey hepatic fibrosis model by a gavage methodThe method comprises the following steps: selecting male rhesus monkeys of 2-3 years old and 5-9kg weight as model animals, feeding high-fat feed with 9-11% alcohol water solution in molding process, and feeding CCl to each rhesus monkey in intragastric administration₄The solution is used for 2 times per week, the dosage is 0.8-1.5m L/kg each time, the duration is 20-25 weeks, and the modeling is successful.

Description

Method for establishing rhesus monkey hepatic fibrosis model by gastric lavage method

Technical Field

The invention belongs to the technical field of animal model construction, and particularly relates to a method for establishing a rhesus monkey hepatic fibrosis model by a gastric perfusion method.

Background

Chronic liver disease is a serious disease that endangers the health of people. On the basis that billions of people in China carry hepatitis viruses, factors such as environmental pollution, food mildew, general food additives, alcoholic and non-alcoholic excessive diet and the like are mutually superposed to cause inflammatory reaction, fatty liver and hepatic fibrosis, wherein millions of people develop into cirrhosis and liver cancer every year, and huge social and family burden is caused. The current traditional clinical drug therapy does not improve the prognosis of end-stage liver disease, but the serious shortage of global donor organs, the increasing prominence of the contradiction between supply and demand (supply: demand/1: 200), makes most patients die only in the process of waiting. Therefore, at present, when the serious shortage of donor organs is contradicted with the increasingly outstanding treatment requirement of patients with end-stage liver diseases, how to expand new organ sources or open up new alternative therapies is a challenge and urgent problem to be solved currently. Therefore, the research on pathogenesis, pathogenic factors and treatment means of the chronic liver disease is an effective way for fundamentally solving the chronic liver disease, and the ideal animal model is the key.

At present, widely applied hepatic fibrosis animal models comprise a chemical hepatic fibrosis model, an alcoholic hepatic fibrosis model, an immune hepatic fibrosis model, a bile duct obstructive hepatic fibrosis model and a compound factor hepatic fibrosis model, and rats and other common small animal models are mature and convenient animal models, but the established models are difficult to directly correspond to human diseases due to the large genetic background and physiological difference between rats and other small animals and human beings. At present, there are experiments that demonstrate that the study of disease mechanisms using some mouse models is not the same as that of human. The use of primates began in the early 20 th century and was not used in the general research institutes until 1960. This is because primates are closest to humans in terms of relativity, are highly similar to humans in terms of tissue structure, immunity, physiology, metabolism, etc., have homology of 75% to 98.5% with human genetic genes, are ideal animal models for studying human health and diseases, and have far higher application value than animals of other species. In recent years, experimental monkey animal model research is widely applied to pathological researches such as infectious diseases, endocrine diseases, cardiovascular diseases, senile diseases, reproductive physiology and the like, and provides a good research platform for more clearly disclosing human disease pathogenesis and screening appropriate treatment means and medicines. The choice of animal models is critical, and large animal experiments are the bridge between grafting transformation medicine (from the bench to composites). Therefore, constructing animal models similar to the genetic background, physiology, metabolism and disease process of human beings is an important method for studying hepatic fibrosis, exploring new therapeutic methods and screening effective therapeutic methods.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for establishing a rhesus monkey hepatic fibrosis model by a gastric perfusion method, which has the advantages of short molding time, high molding success rate and high molding efficiency.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a method for establishing a rhesus monkey hepatic fibrosis model by a gastric perfusion method comprises the following steps: selecting the weight of 2-3 years old5-9kg of male rhesus macaques are used as model animals, the feed used in the molding process is high-fat feed, alcohol water solution with volume concentration of 9% -11% is used as unique drinking water, and CCl is fed to each rhesus macaque in the gastric lavage process₄The solution is used 2 times per week, the dosage is 0.8-1.5m L/kg per time, the duration is 20-25 weeks, and the molding is successful.

Further, the high-fat feed comprises the following components in percentage by weight: 16 to 18 percent of crude protein, 21 to 23 percent of crude fat, 9.5 to 11.5 percent of water, 6.5 to 7.0 percent of ash, 3 to 4 percent of crude fiber, 0.9 to 1.0 percent of calcium, 0.7 to 0.8 percent of phosphorus and 0.3 to 0.4 percent of cholesterol.

Further, the high-fat feed comprises the following components in percentage by weight: 16.78% of crude protein, 21.35% of crude fat, 10% of water, 6.8% of ash, 3.2% of crude fiber, 0.92% of calcium, 0.71% of phosphorus and 0.32% of cholesterol.

Further, the alcohol concentration of the alcohol aqueous solution was 10% by volume.

Further, CCl₄The solution was prepared by the following method: mixing CCl₄Adding the stock solution into olive oil to make CCl₄Sterilizing at concentration of 400m L/L with ultraviolet irradiation for 30 min.

Further, CCl was administered every gavage₄The dosage of the solution is 1.0m L/kg.

Further, the intragastric injection mode is as follows: the normal anesthesia is carried out before the injection, then oral secretion is cleaned, the operation of indwelling stomach tube is carried out, the stomach tube is injected after being butted with the needle cylinder, the injection is slowly carried out, and the tube flushing is carried out by using 0.9% physiological saline after the injection is completed.

Further, the molding time was 120 days.

The method for establishing the rhesus monkey hepatic fibrosis model by the gavage method has the following beneficial effects: the method for establishing the animal model is simple, safe and reliable, short in modeling time and high in success rate, and can be used for researching the occurrence mechanism of the liver disease.

The specific high-fat feed provided by the invention can provide nutrition for rhesus monkeys, and then the rhesus monkeys can drink alcohol water solution with specific concentration and the alcohol water solutionThe concentration of the solution does not need to be increased gradually, the process is simple, and then CCl is injected₄The injection has the advantages that after 20 times of administration, the basic structure of the liver is abnormal, the inflammatory reaction is severe, the periantral and the tract area fibrosis can be seen after 40 times of administration, the incomplete fibrosepta in the liver can be formed, the L aennec liver puncture tissue fibrosis grading score indicates that the liver fibrosis trend is very obvious, and after 60 times of administration, the liver fibrosis is severe, the modeling time is short, and the efficiency is high.

In the preparation of CCl₄The injection adopts oleum Olivarum as CCl₄The solvent of the stock solution has good compatibility, is beneficial to injection and is also beneficial to CCl₄Damaging plasma membranes, destroying membranous structures and functions of hepatocytes, causing degeneration and necrosis of hepatocytes, and promoting hepatic fibrosis; in addition, olive oil does not cause damage to rhesus monkeys.

Drawings

FIG. 1 is a graph showing the staining results of the liver tissue puncture samples of the intragastric administration group.

Detailed Description

The experimental principle is as follows: CCl₄Is the most widely used chemical liver poison, CCl₄Free radicals generated by metabolism after being activated by cytochrome 2E1(CYP2E1) in liver cells in endoplasmic reticulum of the liver can directly damage plasma membranes, initiate lipid peroxidation, destroy membranous structures and functions of the liver cells, cause degeneration and necrosis of the liver cells, activate static lipid storage cells, release type IV collagenase, degrade type IV collagen, synthesize and secrete type I collagen, promote hepatic fibrosis, and inflammation and necrosis in the whole process are usually preceded by fibrosis.

Example 1

Selecting 10 male rhesus monkeys of 2-3 years old and 5-9kg weight as model animals, feeding with conventional feed for 15 days in molding process, wherein the drinking water is normal tap water, changing the conventional feed into high-fat feed after 15 days, the drinking water is 10% alcohol water solution (prepared by adding absolute ethanol into purified water), and filling into stomach to administer CCl₄The solution, 2 times a week, at a dose of 1.0m L/kg for 20-25 weeks, was successfully molded and showed severe fibrosis when it lasted for 30 weeks.

The high-fat feed comprises the following components in percentage by weight: 16.78% of crude protein, 21.35% of crude fat, 10% of water, 6.8% of ash, 3.2% of crude fiber, 0.92% of calcium, 0.71% of phosphorus and 0.32% of cholesterol.

The 10% alcohol water solution is prepared by mixing absolute ethyl alcohol and purified water according to the volume ratio of 1: 9.

CCl₄The solution was prepared by the following method: mixing CCl₄Adding the stock solution into olive oil to make CCl₄Sterilizing at concentration of 400m L/L with ultraviolet irradiation for 30 min.

The gastric perfusion method is that the conventional anesthesia is carried out before the injection, then the oral secretion is cleaned, the operation of indwelling gastric tube is carried out, the gastric tube is butted with the needle cylinder and then injected, the injection is slowly carried out, and the tube flushing is carried out by 0.9 percent of physiological saline of 2-5m L after the injection is finished.

It should be noted that in the molding process, if the body weight of the model animal is reduced by about 1kg within one week, or there is any adverse reaction after the injection of the drug or other situations that may threaten the safety of the experimental animal, the experiment should be stopped.

After the model animal is dosed for every 20 times, the hepatic fibrosis condition is judged by color Doppler ultrasound on the abdomen, the downward puncture sampling is carried out on the color Doppler ultrasound, and the hepatic fibrosis condition is judged by HE, Masson and sirius red staining on the obtained sample.

Experimental example rhesus monkey liver injury diagnosis mode evaluation method and result

1. Serum biochemical marker detection

The serum biochemical markers mainly comprise glutamic-pyruvic transaminase (A L T), glutamic-oxalacetic transaminase (AST), albumin (A L B), bilirubin (BI L), serum Total Cholesterol (TCH), Triglyceride (TG), blood coagulation time (PT) and creatinine (Cr).

After 10 weeks and 20 weeks of model building, the experimental rhesus monkey is fasted for 12 hours, then the muscle anesthesia is carried out by injecting Shutai 0.5m L, the hind limb vein is collected for blood 5m L, the centrifugation is carried out for 15min at 3000r/min, the serum is separated, the biochemical marker content in the serum is tested, and the difference between the marker content and the control group is found to be obvious (P is less than 0.05), wherein the control group is fed with conventional feed and drinking tap water in the whole process.

2. Histopathological analysis process:

sutai 0.5m L was injected for muscle anesthesia, and the liver tissue samples were obtained in the supine position under the guidance of Supersonic color Doppler ultrasound diagnostic equipment using a BARD full-automatic biopsy gun (MG15-22, USA) and then subjected to the following tests:

(1) tissue paraffin embedded section experiment

Material taking: fixing fresh tissues in 4% paraformaldehyde for more than 24h, taking out the tissues from the fixing solution, flattening the tissues of a target part in a fume hood by using a scalpel, and placing the trimmed tissues and corresponding labels in a dehydration box.

And (3) dehydrating: placing the dewatering box into a hanging basket, and placing the hanging basket into a dewatering machine to dewater according to the following gradient alcohol and procedures in sequence: 75% alcohol 4 h-85% alcohol 2 h-90% alcohol 2 h-95% alcohol 1 h-absolute ethanol 30 min-alcohol benzene 5-10 min-xylene 5-10 min-wax 1 h.

Embedding: embedding the wax-soaked tissue in an embedding machine. Firstly, molten wax is put into an embedding frame, tissues are taken out from a dehydration box and put into the embedding frame according to the requirements of an embedding surface before the wax is solidified, corresponding labels are attached to the tissues, the tissues are cooled on a-20-degree freezing table, the wax is taken out after the wax is solidified and put into the embedding frame according to the requirements of the embedding surface, corresponding labels are attached to the tissues, the tissues are cooled on a-20-degree freezing table, the tissues are taken out, the wax is taken out from the embedding frame according to the requirements of the embedding surface, the wax blocks are cooled on a-20.

Slicing: placing the trimmed wax block on a paraffin slicer to be sliced, wherein the slice thickness is 4 mu m, floating the sliced section on warm water at 40 ℃ of a slice spreading machine to flatten the tissue, taking out the tissue by using a glass slide, placing the tissue into a baking oven at 60 ℃ to bake the slice, and taking out the sliced section to be stored at normal temperature for later use after the water is used for baking the wax.

(2) HE staining

Paraffin section dewaxing to water washing: sequentially placing the slices in xylene for 20 min-absolute ethyl alcohol for 10 min-95% ethyl alcohol for 5 min-90% ethyl alcohol for 5 min-80% ethyl alcohol for 5 min-70% ethyl alcohol for 5 min-distilled water washing.

Hematoxylin staining of cell nucleus: slicing into Harris hematoxylin, staining for 3-8min, washing with tap water, differentiating with 1% hydrochloric acid alcohol for several seconds, washing with tap water, returning blue with 0.6% ammonia water, and washing with running water.

Eosin staining of cytoplasm: the sections were stained in eosin stain for 1-3 min.

Dewatering and sealing: placing the slices in 95% alcohol for 5 min-anhydrous alcohol for 5 min-xylene for 5min, dehydrating, removing the slices from xylene, air drying, and sealing with neutral gum. Microscopic examination and image acquisition and analysis.

And (3) dyeing results: blue in nucleus and red in cytoplasm.

(3) Masson staining

Hematoxylin staining of cell nucleus: weigert's ferrohematein staining in the massson staining kit is carried out for 5min, washed by tap water, 1% hydrochloric acid alcohol is differentiated for a plurality of seconds, washed by tap water and washed by running water for a plurality of minutes to return blue.

Ponceau red staining: and dyeing the ponceau acid fuchsin liquid in the massson dyeing kit for 5-10min, and quickly rinsing with distilled water.

Phosphomolybdic acid treatment: and (3) treating the phosphomolybdic acid aqueous solution in the massson staining kit for about 3-5 min.

And (3) aniline blue dyeing: without washing, the reagent box is directly re-dyed with aniline blue liquid in a massson dyeing reagent box for 5 min.

Differentiation: treating with 1% glacial acetic acid for 1 min.

And (3) dyeing results: collagen fibers, mucus and cartilage are blue; myofibers, cellulose and red blood cells are red; the nucleus appears blue-black.

(4) Tianlang scarlet staining

Dyeing with a sirius red staining solution: the sections were stained in saturated picric acid sirius red staining solution for 8 min.

Rinsing: sections were rinsed in absolute alcohol for several minutes until satisfactory observation under a microscope.

Sealing: slicing, oven drying at 60 deg.C, clearing in xylene for 5min, and sealing with neutral gum. Microscopic examination and image acquisition and analysis.

And (3) dyeing results: collagen fibers were red under light microscopy and the background was yellow.

The staining results of the liver tissue puncture samples of the gavage group are shown in FIG. 1.

As can be seen from figure 1, rhesus liver tissues have normal structures and regular cell arrangement and obvious Dirichan gaps before administration, after 20 times of administration, the basic liver structures of the intragastric administration group are abnormal, the inflammatory reaction is severe, vacuoles with different sizes are visible in liver cells, part of cell nuclei are biased to one side of the cells, the liver has a tendency of fatty sample change, after 40 times of administration, the liver cells are disorganized, the central vein is absent, the hepatic lobules disappear, the inflammatory reaction is severe, fibrosis of periantral and periportal areas can be seen, incomplete fibrosis in the liver can be formed, L aennec liver puncture tissue fibrosis grading score is 2, the trend of hepatic fibrosis is obvious, after 60 times of administration, the intrahepatic fibrosis grading score is obvious, the trend of pseudolobules can be seen, L aennec score is 4, the severe hepatic fibrosis is shown, and L aennec liver puncture tissue fibrosis grading score is shown in Table 1.

TABLE 1L aennec hepatic puncture tissue fibrosis grading score criteria

Claims

1. A method for establishing a rhesus monkey hepatic fibrosis model by a gastric perfusion method is characterized by comprising the following steps: selecting male of 2-3 years old and 5-9kg body weightFeeding high-fat feed as model animal in molding process, drinking 9-11% alcohol water solution, and feeding CCl to each rhesus monkey in intragastric administration₄The solution is taken 2 times a week, each dosage is 1.0m L/kg, after 10 weeks, the liver shows a tendency of lipoid change, lasts for 20-25 weeks, shows moderate hepatic fibrosis, and is successfully molded, wherein the high fat feed comprises the following components, by weight, 16% -18% of crude protein, 21% -23% of crude fat, 9.5% -11.5% of water, 6.5% -7.0% of ash, 3% -4% of crude fiber, 0.9% -1.0% of calcium, 0.7% -0.8% of phosphorus, and 0.3% -0.4% of cholesterol.

2. The method for establishing the rhesus monkey liver fibrosis model by the gavage method according to claim 1, wherein the high-fat diet comprises the following components in percentage by weight: 16.78% of crude protein, 21.35% of crude fat, 10% of water, 6.8% of ash, 3.2% of crude fiber, 0.92% of calcium, 0.71% of phosphorus and 0.32% of cholesterol.

3. The method for establishing a rhesus monkey liver fibrosis model according to claim 1, wherein the alcohol concentration in the alcohol-water solution is 10% by volume.

4. The method for establishing rhesus monkey liver fibrosis model by gavage according to any one of claims 1-3, wherein CCl is₄The solution was prepared by the following method: mixing CCl₄Adding the stock solution into olive oil to make CCl₄Sterilizing at concentration of 400m L/L with ultraviolet irradiation for 30 min.

5. The method for establishing the rhesus monkey liver fibrosis model by the intragastric perfusion method according to claim 1, wherein the intragastric perfusion method comprises the following steps: the normal anesthesia is carried out before the injection, then oral secretion is cleaned, the operation of indwelling stomach tube is carried out, the stomach tube is injected after being butted with the needle cylinder, the injection is slowly carried out, and the tube flushing is carried out by using 0.9% physiological saline after the injection is completed.