CN111248152A - Cirrhosis ascites animal model and construction method thereof - Google Patents
Cirrhosis ascites animal model and construction method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
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- Life Sciences & Earth Sciences (AREA)
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Abstract
The invention relates to an animal model of ascites due to cirrhosis. The animal model is prepared by the following method: after the weight of the rat reaches 220g, the model is started to be molded, 0.5% DMN solution is used for intraperitoneal injection according to 0.2ml/100g of the weight of the rat, three, four and five doses are administered once every week, the rest six, one, two doses are not interfered in the rest weeks, the injection is performed for 4 weeks, the injection is performed according to the original dose in the first two weeks, the administration dose is adjusted according to the weight of the rat in the injection third week, the amplitude is adjusted, and the following scheme is adopted: the body weight is not changed or reduced by less than 10 g, and the original dose is used for administration; the original dosage of 75 percent is used when the weight is reduced by 10 to 30 grams; the weight is reduced by 31-50 g, and the original dosage of 50% is used; when the weight loss exceeds 50g, the injection is suspended once, and the dosage is adjusted according to the weight change after 1 week. The stable, reproducible and strong-operability cirrhosis ascites rat model is successfully prepared by improving the scheme. Lays a foundation for the subsequent ascites due to cirrhosis animal experiments.
Description
Technical Field
The invention relates to the technical field of animal models, in particular to an ascites due to cirrhosis animal model and a construction method thereof.
Background
Currently, the most common methods for preparing the ascites due to cirrhosis include a carbon tetrachloride (CCL4) modeling method (including a comprehensive preparation method of CCL4 alone and combined phenobarbital, ethanol, improved diet and the like), an alcoholic modeling method, an immune modeling method, a common bile duct ligation induction method and a chemical carcinogen induction method (DMN modeling method). Among them, the CCL4 molding method is the earliest and most typical molding method. The main action mechanism of the method is oxidative stress reaction, CCL4 is decomposed into trichloromethyl and trichloromethyl free radicals through cytochrome P450, so that lipid peroxidation of liver cells is caused, degeneration damage and degeneration necrosis of cell membranes are caused, and liver cirrhosis is finally developed through continuous damage-repair-damage processes because liver cells have a repair function. But the method has long molding period and high death rate. The main mechanism of alcoholic modeling is that on one hand, alcohol is oxidized and metabolized in liver to generate acetaldehyde which has direct hepatotoxicity, and on the other hand, alcohol can stimulate in-vivo immune reaction system, induce tissue inflammatory reaction and start tissue hepatic fibrosis program. Eventually, the liver disease is further aggravated if alcohol is not given up. The alcohol molding method has simple operation and low cost, can assist the carbon tetrachloride method, reduce the molding period, but has defects because animals refuse to drink alcohol and the alcohol can cause the animals to die due to water shortage, electrolyte disorder and even dehydration. The immune modeling method adopts allogenic serum to repeatedly inject animals to generate organism allergic reaction, and a large amount of immune complexes are deposited, so that hepatic fibrosis is generated, and then, cirrhosis is continuously developed along with the course of disease. However, the method has long molding time and high mortality rate, and the rat has certain self-healing tendency. The choledocholithiasis can cause expansion of bile ducts and intrahepatic cholestasis of animal models, and can also cause expansion and rupture of intrahepatic tubules, so that ischemic necrosis of liver cells (hepatocytes) slowly progresses to cholestasis type cirrhosis. Has certain similarity with the development of human primary biliary cirrhosis, but has great operation difficulty, less influence on liver histology, high death rate of rats and certain self-healing tendency. DMN induction is the most common in chemical carcinogen induction, and DMN has hepatotoxicity, genotoxicity and immunotoxicity. DMN toxicity is generated by the activation of in vivo metabolism, and the formation of strong alkylate through microsome transformation can cause the damage of intracellular macromolecules, induce the activation of Hepatic Stellate Cells (HSC) of hepatic sinus walls of rats and convert the HSC into myofibroblast-like cells. Activation of HSCs is involved on the one hand in the reconstruction of intrahepatic structures and the formation of liver fibrosis; on the other hand, the pressure in the liver sinus is increased. Therefore, the method is mainly used for the research of liver fibrosis, liver cirrhosis formation, portal hypertension mechanism and liver cancer. The method has the following defects: the mortality rate of molded animals is high due to the high toxicity of DMN agents and the difficulty in dose control, and therefore dose control is particularly important.
Disclosure of Invention
The first purpose of the present invention is to provide an animal model of ascites due to cirrhosis, which addresses the deficiencies in the prior art.
The second purpose of the invention is to provide a method for constructing the ascites due to cirrhosis animal model.
In order to achieve the first purpose, the invention adopts the technical scheme that: an animal model of ascites due to cirrhosis is prepared by the following method: after the weight of the rat reaches 220g, the model is started to be molded, 0.5% DMN solution is used for intraperitoneal injection according to 0.2ml/100g of the weight of the rat, three, four and five doses are administered once every week, the rest six, one, two doses are not interfered in the rest weeks, the injection is performed for 4 weeks, the injection is performed according to the original dose in the first two weeks, the administration dose is adjusted according to the weight of the rat in the injection third week, the amplitude is adjusted, and the following scheme is adopted: the body weight is not changed or reduced by less than 10 g, and the original dose is used for administration; the original dosage of 75 percent is used when the weight is reduced by 10 to 30 grams; the weight is reduced by 31-50 g, and the original dosage of 50% is used; when the weight loss exceeds 50g, the injection is suspended once, and the dosage is adjusted according to the weight change after 1 week.
In order to achieve the second object, the invention adopts the technical scheme that: a method for constructing an animal model with ascites due to cirrhosis comprises the following steps: the construction method comprises the following steps: after the weight of the rat reaches 220g, the model is started to be molded, 0.5% DMN solution is used for intraperitoneal injection according to 0.2ml/100g of the weight of the rat, three, four and five doses are administered once every week, the rest six, one, two doses are not interfered in the rest weeks, the injection is performed for 4 weeks, the injection is performed according to the original dose in the first two weeks, the administration dose is adjusted according to the weight of the rat in the injection third week, the amplitude is adjusted, and the following scheme is adopted: the body weight is not changed or reduced by less than 10 g, and the original dose is used for administration; the original dosage of 75 percent is used when the weight is reduced by 10 to 30 grams; the weight is reduced by 31-50 g, and the original dosage of 50% is used; when the weight loss exceeds 50g, the injection is suspended once, and the dosage is adjusted according to the weight change after 1 week.
The invention has the advantages that: the method of the invention is based on the existing Alakokko modeling method to construct a rat model with ascites due to cirrhosis. The stable, reproducible and strong-operability cirrhosis ascites rat model is successfully prepared by improving the scheme. Lays a foundation for the subsequent ascites due to cirrhosis animal experiments.
Drawings
FIG. 1 general phenomenon observation of rats. A: a model group; b: a normal group; c: the abdominal cavity is opened to show light yellow and clear ascites.
Figure 2 liver morphology observation. C: a normal group; d: and (4) model groups.
The anechoic zone is visible in the abdominal cavity of the model group of fig. 3.
Figure 4 rat liver HE staining: liver light microscopy 10X: normal group L-1; control group L-2; liver light microscopy 20X: normal group M-1.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
First, experimental materials, methods and results.
Experimental materials: selecting 45 healthy male cleaning grade Wistar rats, 6-8 weeks old, body weight (200 + -20) g, purchased from Shanghai Si Laike laboratory animals, Inc., animal production certification number: 2015000557591.
the experimental method comprises the following steps: preparing a DMN induced cirrhosis ascites rat model, adaptively feeding 45 male Wistar rats for 1 week, starting modeling after the weight is increased to about 240g, taking 5 rats as a normal group, carrying out intraperitoneal injection on 0.5 percent DMN solution according to the weight of 0.2ml/100g of rats by taking the rest rats as a normal group according to an Alakokko modeling method and improving, carrying out administration once every three, four and five weeks in the week, carrying out injection for 4 weeks in the total time without intervention on the rest rats in the six, four, one and two weeks, carrying out injection according to the original dose in the first two weeks, regulating the dose according to the weight of the rats in the third week of injection, and regulating the amplitude according to the following scheme: no change or less than 10 grams of body weight-the original dose administered; the weight is reduced by 10-30 g-75% of the original dose; the weight is reduced by 31 to 50 grams, namely the original dose of 50 percent; weight loss was over 50 g-one injection was suspended and after 1 week the dose was adjusted to weight change. During the period, people eat the food normally and drink water freely. During modeling, the weight, abdominal circumference, body weight: daily measurements and recordings; abdominal circumference: the first three weeks are measured on two and five weeks each, and the last three weeks are measured and recorded on a daily basis. And (3) adjusting the dosage according to the weight of the rat, and constructing a rat model of ascites due to cirrhosis.
II, judging the mold making success standard:
1. general physical signs
The rat is lassitude, thin and yellow in hair quantity, poor in glossiness, swollen abdomen and high in abdominal water quantity, and can feel wave motion.
2. Abdominal puncture
After the assistant fixes the head and the limbs of the rat, the right side is in a lying position, the abdomen is fully exposed, the skin of the right lower abdomen of the rat is prepared by about 2cm multiplied by 2cm, after 75% alcohol is locally sterilized, a 1ml syringe is drawn back, and if orange liquid is drawn out, the molding is finished. Ascites can be extracted by diagnostic puncture when the abdominal water volume of the rat is large.
3. Abdominal B-mode ultrasound
Skin preparation: the rat's entire abdomen (down to pubic symphysis, up to xiphoid process, left and right side to bilateral underarms, respectively, and skin preparation was performed using an animal shaver in combination with depilatory cream). Anesthesia: several wet cotton balls containing isoflurane were placed in a large beaker, the rat was placed in and sealed. Rats were excited and then restrained, falling down by themselves. When the cornea reaction of the animals is slow and the muscle tension is reduced, the rats are taken out. If the rat regains muscle tension again (struggles), the anaesthesia can be repeated once, and the experiment can be started after calmness. The operation is as follows: fixing rat head and limbs with assistant, exposing abdomen, coating coupling agent on skin preparation part, and treating with high frequency (7.5)～10 mhz) probe probes the abdomen of the rat and takes a picture, and the presence or absence of an echogenic dark area in the abdominal cavity is shown on the sonogram, thus diagnosing ascites.
4. Liver pathology
(1) The normal hepatic lobule is damaged and replaced by the pseudolobule, (2) the pseudolobule is ① fibrous tissue surrounding all the pseudolobules, (②) hepatic cells are disorganized and have degeneration, necrosis and regeneration to different degrees, (3) the surrounding fibrous connective tissue has clean moistening of lymphocytes, hyperplasia of small bile ducts and formation of pseudobile ducts.
Thirdly, the experimental result is as follows:
1. general physical signs of rat
Compared with the normal group, the rats in the model group can be seen to be lassitude, yellowish fur, poor glossiness and swollen abdomen, have less sensitivity to external reaction, reduce drinking water and weight loss (see figure 1).
2. Morphological observation of liver
The liver is apparently visible: (see FIG. 2).
Normal group: the liver surface is smooth and bright red.
Model group: the liver has reduced volume, brown or earthy yellow color, rough and uneven surface texture, and can touch round-like nodules of different sizes, and adhesion exists between the liver and omentum and between different liver lobes.
3. B ultrasonic of abdomen: the anechoic zone was visible in the abdominal cavity of the model group (see fig. 3).
4. Liver HE staining (see fig. 4).
Under the mirror can be seen:
normal group: the liver cells are consistent in size and normal in shape, the cells are arranged in a cord shape, the lobular structure of the liver is complete, and the region of the sink is not abnormal.
Model group: the liver cells are disorganized in arrangement, the liver cells are swollen, have degeneration, necrosis and regeneration in different degrees, normal liver lobules are damaged and replaced by false lobules, fibrous tissues surround the false lobules, and a large amount of inflammatory cells infiltrate into a sink region.
5. Rat molding rate
Rat molding rate five weeks after experiment: 90% (40 rats, 36 molded), survival: 97.5%, mortality: 2.5% (1 rat died due to overdose anesthesia). (see Table 1).
TABLE 1 rat molding ratio
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (2)
1. An animal model of ascites due to cirrhosis, which is characterized by being prepared by the following method: after the weight of the rat reaches 220g, the model is started to be molded, 0.5% DMN solution is used for intraperitoneal injection according to 0.2ml/100g of the weight of the rat, three, four and five doses are administered once every week, the rest six, one, two doses are not interfered in the rest weeks, the injection is performed for 4 weeks, the injection is performed according to the original dose in the first two weeks, the administration dose is adjusted according to the weight of the rat in the injection third week, the amplitude is adjusted, and the following scheme is adopted: the body weight is not changed or reduced by less than 10 g, and the original dose is used for administration; the original dosage of 75 percent is used when the weight is reduced by 10 to 30 grams; the weight is reduced by 31-50 g, and the original dosage of 50% is used; when the weight loss exceeds 50g, the injection is suspended once, and the dosage is adjusted according to the weight change after 1 week.
2. A method for constructing an animal model with ascites due to cirrhosis comprises the following steps: the construction method is characterized by comprising the following steps: after the weight of the rat reaches 220g, the model is started to be molded, 0.5% DMN solution is used for intraperitoneal injection according to 0.2ml/100g of the weight of the rat, three, four and five doses are administered once every week, the rest six, one, two doses are not interfered in the rest weeks, the injection is performed for 4 weeks, the injection is performed according to the original dose in the first two weeks, the administration dose is adjusted according to the weight of the rat in the injection third week, the amplitude is adjusted, and the following scheme is adopted: the body weight is not changed or reduced by less than 10 g, and the original dose is used for administration; the original dosage of 75 percent is used when the weight is reduced by 10 to 30 grams; the weight is reduced by 31-50 g, and the original dosage of 50% is used; when the weight loss exceeds 50g, the injection is suspended once, and the dosage is adjusted according to the weight change after 1 week.
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| JP4879520B2 (en) * | 2004-06-07 | 2012-02-22 | 小林製薬株式会社 | Liver fibrosis inhibitor |
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