CN107912366B

CN107912366B - Non-alcoholic chronic steatohepatitis non-human primate model and construction method and application thereof

Info

Publication number: CN107912366B
Application number: CN201711276545.5A
Authority: CN
Inventors: 庄永杰; 陈继巧
Original assignee: Jiangsu Kemaqi Biotechnology Co ltd
Current assignee: Jiangsu Kemaqi Biotechnology Co ltd
Priority date: 2017-12-06
Filing date: 2017-12-06
Publication date: 2020-12-11
Anticipated expiration: 2037-12-06
Also published as: CN107912366A

Abstract

The invention discloses a non-alcoholic chronic steatohepatitis (NASH) non-human primate model, a construction method and application thereof. The animal model constructed by the invention can be further accompanied with the pathological characteristics of chronic hepatic fibrosis and liver cirrhosis, can be used for research and development of medicaments for preventing or treating non-alcoholic chronic steatohepatitis, hepatic fibrosis and liver cirrhosis, and can be used as an animal model for research on chronic fatty liver, steatohepatitis, hepatic fibrosis, liver cirrhosis, hepatocellular carcinoma, metabolic diseases, diabetes and diabetic complications, and has extremely high application value.

Description

Non-alcoholic chronic steatohepatitis non-human primate model and construction method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a construction method and application of non-alcoholic chronic steatohepatitis (NASH) and a non-human primate model capable of developing chronic hepatic fibrosis and liver cirrhosis.

Background

Non-alcoholic steatohepatitis (NASH) is a stage with poor prognosis of non-alcoholic fatty liver disease (NAFLD), can develop into cirrhosis and even hepatocellular carcinoma, has an annual rising trend of morbidity, and has no obvious effective treatment method at present. The animal model has important functions in the fields of exploring pathogenesis of diseases, evaluating diagnosis methods, screening prevention and treatment medicines and the like. In recent years, many scholars have reproduced and studied a number of pathological models of nonalcoholic steatohepatitis using experimental animals. At present, the non-alcoholic steatohepatitis animal models which are commonly used at home and abroad comprise fatty liver models induced by nutrition imbalance, drug toxicity, special strains and compound factors. The animal model of the nutritional-imbalance fatty liver is the most common non-alcoholic steatohepatitis animal model at home and abroad, and comprises a high-fat diet fatty liver animal model, a high-sugar diet fatty liver animal model and a choline-methionine deficiency fatty liver animal model. The model has good stability, high repeatability, simple method, low fatality rate, progressive pathological changes, slow reversion after the model is stopped and convenient pharmaceutical intervention.

For example, CN201310452707.1 discloses an application of a compound high-fat feed in constructing a non-alcoholic fatty liver disease rat model. CN201710468378.8 discloses a model establishment method for inducing non-alcoholic fatty liver disease by rat obesity caused by high-fat and high-sugar diet. CN201610188042.1 discloses a method for establishing a mouse model of non-alcoholic fatty liver disease. CN201210533667.9 discloses a modeling method of a non-alcoholic fatty liver disease model mouse. However, because the composition of the feed is different from the actual dietary situation (mainly including saccharides) of clinical patients with nonalcoholic steatohepatitis, the existing mouse or rat NASH, hepatic fibrosis, cirrhosis and liver cancer models have great difference from human diseases, including the difference and inconsistency of steatosis and steatohepatitis, namely, the inflammation of small animal models often appears at the same time as early steatosis, which is far away from the human diseases. Especially in the immunological response to disease. The animal closest to the human immune system is a non-human primate. For the development of macromolecular immunotherapy drugs, rational animal models can reflect the etiology, disease progression and pathological features of human nonalcoholic steatohepatitis and clinical drug development only when established on nonhuman primates and monkeys.

However, to date, there is no ideal non-human primate NASH model. The more common are fatty liver models of non-human primates, and simple chemical induction (CCl)₄) The liver fibrosis model of (1). None of these models accurately reflects the true pathological course and clinical symptoms and outcome of NASH, and few or only a partial recurrence of some of the pathological features of human non-alcoholic steatohepatitis, with many important diseases such as insulin resistance and fibrosis not being reproduced. Meanwhile, there is an increasing demand for new nonalcoholic steatohepatitis based on the development of biomacromolecule drugs.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a non-human primate model for simulating human non-alcoholic steatohepatitis (NASH) and a construction method and application thereof, and the model can be further accompanied with pathological characteristics of chronic hepatic fibrosis and liver cirrhosis. The method can reproduce basic diabetes (blood sugar rise) of model animals, severe fatty liver accompanied with steatohepatitis and hepatic fibrosis, so that basic pathological features of human NASH are intensively presented on one model animal, and reliable model guarantee is provided for better research and development of NASH and development of new drugs, particularly development of macromolecular drugs.

The specific technical scheme of the invention is as follows:

a method for constructing a non-human primate model of non-alcoholic chronic steatohepatitis comprises the following steps:

(1) providing high-fat feed and alcohol drinking water for adult non-human primates, and feeding for 1-12 months;

(2) the non-human primate model of non-alcoholic chronic steatohepatitis is constructed by administering carbon tetrachloride to animals for 1-16 weeks while continuing to administer high fat feeding and alcoholic drinking water.

The non-human primates include but are not limited to female or male experimental non-human primates such as cynomolgus monkey and macaque.

The fat content of the high-fat diet is 30-60 Kcal%, but is not limited thereto.

The alcohol concentration of the alcohol drinking water is preferably 5-20%, more preferably 10% (V/V).

Preferably, the feeding period of the high-fat feed and the alcohol drinking water in the step (1) is 4-6 months.

Preferably, step (2) is carried out with the animal being administered a dose of carbon tetrachloride of 0.5 to 1.5mL/kg, more preferably 1 mL/kg. Preferably CCl₄: the edible olive oil is 1:1 (volume ratio).

Preferably, the route of step (2) of administering carbon tetrachloride to the animal includes, but is not limited to, oral administration. The administration route also comprises subcutaneous injection and intraperitoneal injection according to experimental requirements.

Preferably, step (2) is performed by administering carbon tetrachloride to the animal for 4 to 10 weeks.

Another objective of the invention is to provide a non-human primate model of non-alcoholic chronic steatohepatitis, which is constructed by the method of the invention and can be accompanied with pathological characteristics of chronic hepatic fibrosis and early cirrhosis.

The invention also aims to provide application of the non-alcoholic chronic steatohepatitis non-human primate model in screening medicines for preventing or treating non-alcoholic chronic steatohepatitis, hepatic fibrosis, liver cirrhosis or liver cancer and application of the non-alcoholic chronic steatohepatitis non-human primate model as an animal model in research on chronic steatohepatitis, hepatic fibrosis, hepatic cirrhosis, hepatocellular carcinoma, metabolic diseases, diabetes and diabetic complications.

The invention has the advantages that: the invention adopts high-fat feed and alcohol drinking water for breeding, and gives secondary hit to the liver for inducing NASH in a certain period, thereby conforming to the current basic pathological process of human NASH and hepatic fibrosis. Can truly simulate the pathological process of human NASH. The success rate of the model is one hundred percent, the stability is good, and the death rate is low. Fills the gap of non-human primate animal models in the field, provides an essential animal model for the research of human NASH and hepatic fibrosis, particularly for the research and development of new drugs of macromolecular antibody drugs, and makes the preclinical research and development of NASH macromolecular drugs possible. The method is simple and easy to implement, can be widely popularized, and can truly simulate the pathological process of NASH and the conversion of NASH into hepatic fibrosis and liver cirrhosis. Because the pathological process of the liver cancer is highly similar to the pathological process of human NASH, hepatic fibrosis and liver cirrhosis, the liver cancer has very high etiology and pathology research value and has important significance in the evaluation of medicaments.

Drawings

FIG. 1 shows CCl alone₄Liver histopathological change H for inducing liver fibrosis&And E, dyeing results. (A: normal liver, B: CCl)₄2 weeks, C: CCl₄6 weeks).

FIG. 2 shows CCl alone₄Inducing hepatic fibrosis to result in stellera red staining. (D: Normal liver, E: CCl)₄2 weeks, F: CCl₄6 weeks).

FIG. 3 shows CCl alone₄Induce serological ALT and AST changes in animals with liver fibrosis.

FIG. 4 is a general diagram of animal liver after 5 months of high fat diet + alcohol drinking water feeding. (G & H: normal liver, I & J: fatty liver after 5 months of high fat + alcohol).

Fig. 5 is a histological picture of HE staining of animal livers after 5 months of high fat diet + alcoholic drinking water feeding. (K: normal liver, L: high fat + alcohol for 2 months, M: high fat + alcohol for 5 months)

Fig. 6 shows the changes in animal serology ALT and AST after 5 months of high fat diet plus alcohol drinking water feeding.

FIG. 7 shows that CCl is added to high-fat feed and alcohol drinking water for 5 months₄Animals serological ALT and AST changes after the first week of the second hit.

FIG. 8 shows that the feed is fed with high fat feed and alcohol drinking water for 5 months and CCl₄Second, fourth and sixth week of Secondary hit liver histology Change H&And E, dyeing results. (N: CCl)₄2 weeks, O: CCl₄4 weeks, P: CCl₄6 weeks).

FIG. 9 shows that the feed is fed with high fat feed and alcohol drinking water for 5 months and CCl₄Second, fourth and sixth weeks of the second stroke, liver histological changes, Masson Trichrome staining results. (Q: CCl)₄-2 weeks, R: CCl₄4 weeks, S: CCl₄6 weeks).

FIG. 10 shows that the feed is fed with high fat feed and alcohol drinking water for 5 months and CCl₄Secondary hits resulted in changes in liver histology at week two, week four and week six, sirius red staining. (T: CCl)₄2 weeks, U: CCl₄4 weeks, V: CCl₄6 weeks).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Examples adult cynomolgus monkeys were used as an example to prepare non-alcoholic steatohepatitis (NASH) and animal models further associated with pathological features of chronic liver fibrosis and early cirrhosis.

15 adult cynomolgus monkeys of 4-6 years old, weighing 3.8-4.0 kg, were selected for this experiment.

Grouping experiments:

pure CCl₄Induced hepatic fibrosis model group of 5 animals, fed with normal diet, orally administered CCl₄（1ml/kg，CCl₄: olive oil =1: 1), three times a week for 6 weeks;

high fat + alcohol + CCl₄Induced NASH model group 10 animals were first given high fat diet (fat 40%) + low alcohol (10%) drinking water and after 5 months of ad hoc feeding, 5 animals were randomly selected for liver function and liver pathology test, and the remaining 5 animals were continuously given high fat feeding and alcohol drinking water, and CCl was orally administered₄（1ml/kg，CCl₄: olive oil =1: 1) two hits, three times a week for 6 weeks.

Periodically performing an animal liver function test (ALT, AST) on the animal to monitor the clinical progression of hepatic fibrosis and NASH; the regular animal liver biopsy pathological detection judges different pathological change periods of NASH, including a fatty liver period, a steatohepatitis period and a pathological process of steatohepatitis accompanied with a hepatic fibrosis period.

Results of the experiment

FIGS. 1-3 show CCl alone₄Inducing liver function of liver fibrosis model group and liver biopsy pathological research result. The research result shows that CCl is simple₄Only hepatocyte injury including degeneration, necrosis and inflammatory cell infiltration (fig. 1) was induced at different stages with significant liver fibrosis (fig. 2) and with elevation of ALT, AST (fig. 3). However, the animals have no fatty degeneration and severe fatty liver, and are in line with the pathological change of the liver fibrosis induced by chemistry.

Fig. 4-6 are liver function and liver biopsy pathology findings for the high fat + alcohol 5 month model group. The results of the study showed that animals were severely fatty liver after 5 months of ad hoc feeding of high fat diet (40% fat) plus low alcohol (10%) with water (fig. 4), but without inflammatory response and fibrotic pathological changes (fig. 5) and without enzymatic changes in liver function, i.e. ALT, AST, were maintained in the normal range (fig. 6).

The above results indicate that CCl alone is₄Induced liver fibrosis is only that of the lesion, not NASH. Simple administration of high fat + alcohol only induced fatty liver, without pathological changes in NASH.

FIG. 7-10 shows 5 animals fed with high fat feed and alcohol drinking waterMonth + CCl₄Liver function and liver biopsy pathology results for the secondary hit model group. The research result shows that CCl₄Significant liver function impairment appeared after the second stroke, as evidenced by significant elevations in ALT and AST (fig. 7). With CCl₄By continuous administration of (A), liver fibrosis gradually occurs to CCl₄Severe fatty liver, inflammatory cell infiltration (fig. 8) and significant liver fibrosis and early cirrhosis (fig. 9) were seen for six weeks. Its fibrosis is characterized by fibrosis between the hepatocyte cords (fig. 10), which is fully consistent with the hepatic fibrosis characteristic of human NASH.

The invention adopts high-fat feed and alcohol drinking water for breeding, and gives secondary attack to the liver for inducing NASH in a certain period, thereby conforming to the basic idea of the occurrence of NASH and hepatic fibrosis of human beings at present. Can truly simulate the pathological process of human NASH. The success rate of the model is one hundred percent, the stability is good, and the death rate is low. Fills the gap of the non-human primate model in the field, and provides an indispensable animal model for the research of human NASH and hepatic fibrosis, in particular for the research and development of new drugs.

Claims

1. A method for constructing a non-human primate model of non-alcoholic chronic steatohepatitis is characterized by comprising the following steps:

(1) providing high-fat feed and alcohol drinking water for animals to adult non-human primates, wherein the fat content of the high-fat feed is 30-60 Kcal%, the alcohol concentration of the alcohol drinking water is 10%, and the feeding is carried out for 4-6 months;

(2) administering to the animal orally carbon tetrachloride for 4-10 weeks, CCl₄: the volume ratio of the edible olive oil is 1:1, the dosage is 0.5-1.5mL/kg, and high-fat feeding and alcohol drinking water are continuously given to construct the non-alcoholic chronic steatohepatitis non-human primate model.

2. The method of claim 1, wherein the non-human primate is a cynomolgus monkey or a cynomolgus monkey.

3. Use of the non-alcoholic chronic steatohepatitis non-human primate model constructed by the method of claim 1 or 2 in screening of drugs for preventing or treating non-alcoholic chronic steatohepatitis, liver fibrosis and liver cirrhosis.

4. The non-alcoholic chronic steatohepatitis non-human primate model constructed by the method of claim 1 or 2 is used as an animal model for researching chronic fatty liver, steatohepatitis, hepatic fibrosis, cirrhosis, hepatocellular carcinoma, diabetes and diabetic complications.