CN102972345B - Modeling processing method of non-alcoholic fatty liver mouse model - Google Patents
Modeling processing method of non-alcoholic fatty liver mouse model Download PDFInfo
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- CN102972345B CN102972345B CN201210533667.9A CN201210533667A CN102972345B CN 102972345 B CN102972345 B CN 102972345B CN 201210533667 A CN201210533667 A CN 201210533667A CN 102972345 B CN102972345 B CN 102972345B
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Abstract
The invention discloses a modeling method of a non-alcoholic fatty liver mouse model, and the method is characterized by consisting of the following steps that (1) a healthy mouse is caught and first adaptively raised for 3 to 7 days in an animal house, a backing material and clean water in a mouse cage are changed every day, and basic fodder is fed during that period of time; (2) after adaptive raising, composite high-fat fodder is fed every day; (3) 5 percent CC14 liquid is injected into the abdominal cavity of the mouse two weeks later, the liquid carbon tetrachloride is diluted to 5 percent emulsion with deionized water, and the liquid which is injected into the abdominal cavity shall be newly prepared; the abdominal cavity injection state continues for the 28th day, and meanwhile the composite high-fat fodder is fed; and the model is obtained. The modeling method has the advantages of short time, high modeling success rate, extremely low mortality rate and low cost.
Description
Technical field
The present invention relates to a kind of modeling technique of animal model, particularly a kind of NASH mouse model novel process.
Background technology
Along with the raising of people's living standard, dietary structure has had obvious change, and fatty liver disease (also the claiming fatty liver) incidence of disease being called as " rich man's disease " significantly improves, especially in Asia.Become the second largest frequently-occurring hepatopathy being only second to virus hepatitis.Fatty liver is caused by multiple inducement, and pathology main body, at lobuli hepatis, is one of common DHD, with triglycerides in liver cell (TG) accumulation too much and diffusivity hepatic cell fattydegeneration be main pathological characteristics.Clinically according to whether having excessive alcohol to take in, fatty liver is divided into alcoholic fatty liver disease (alcoholic fatty liver disease, and non-alcoholic fatty liver disease (nonalcoholic fatty liver disease, NAFLD) AFLD).Non-alcoholic fatty liver disease is comparatively complicated, if controlled not in time, thus inflammatory reaction and liver fibrosis can be caused to occur.It is a kind of without excessive drinking history, with the pathological syndrome of the features such as hepatic cell fattydegeneration, comprises simple fatty liver, fat hepatitis, fatty fibrillatable and fatty cirrhosis 4 pathologic processes.For this pathologic process, each medical medical research institute, all at the specific drug of research treatment non-alcoholic fatty liver disease, is that the safety evaluatio of new drug or curative effect all need animal model and detect in this context.The efficient modeling method of NASH mouse model just seems especially important.
Existing market does not sell the nonalcoholic fatty liver model mouse with genetic stability, all needs cultivate transformation the day after tomorrow.Be directed to different modeling methods, show that the time of model mice is also just different in size.The Method And Principle generally making nonalcoholic fatty liver model mainly carries out from two aspects simultaneously.Namely the lumbar injection of carbon tetrachloride can cause the acute injury of liver, then is combined with the pathology that compound high lipid food causes liver, and be mainly manifested in liver plasma membrane damaged by carbon tetrachloride, the high heat in high lipid food enters with the form of fat drop.
In above-mentioned research, be all form with high lipid food and the amount of carbon tetrachloride and the accumulation of time.Only demand result and ignore cost and efficiency, cause modeling success rate low, lethality rises and the whole process time of modeling is tediously long.And traditional modeling mode is the heat improving high lipid food is improved by lard component ratio, a small amount of with sucrose, then coordinate other drug to mix with basal feed to suppress slivering.Just start mouse appetite still larger, but cause mouse appetite of losing the appetite obviously to decline due to the too high easy generation satiety of lard content and greasy feeling, thus the time of modeling elongated.Tradition modeling method also has a drawback, and namely carbon tetrachloride per injection dosage is 10%, each 0.2ml.Carbon Tetrachloride Concentration is high can effectively cause hepatocellular damage, but mouse physique lumbar injection that is more weak or this dosage out of order is easy to the sudden death causing animal pattern.
Summary of the invention
The object of the invention is: the modeling method and the application that provide a kind of nonalcoholic fatty liver model mouse, it is while preparing animal model, effectively can shorten modeling saving of time modeling cost.
Technical scheme of the present invention: the modeling method of a kind of nonalcoholic fatty liver model mouse, comprises following steps:
(1) get healthy mice, in Animal House, first carry out adaptability raise 3-7 days, change mouse cage bedding and padding and clear water every day, basal feed of feeding during this period;
(2) after adaptability is raised, compound of feeding every day high lipid food;
Lumbar injection concentration 5% CCl is carried out to mouse after (3) two weeks
4liquid, is diluted to liquid carbon tetrachloride the emulsion that concentration is 5% by deionized water, and each lumbar injection all needs Fresh can; Abdominal cavity injection state continues to the 28th day, compound of simultaneously feeding high lipid food; Obtain.
Described compound high lipid food, its formula is: 2% cholesterol+0.5% sodium taurocholate+0.2% propylthiouracil+5% caramel+10% lard+82.3% basal feed.
The described weighing scale by mouse, the injected dose of carbon tetrachloride is 0.1ml/10g.
Described raises in Animal House, and indoor temperature remains on 18 DEG C ~ 29 DEG C, and relative moisture, 40 ~ 70%, ensures clean environment air circulation.
Beneficial effect of the present invention: compared with prior art, the present invention instead of the sucrose in traditional high lipid food after adopting sucrose high temperature to turn to caramel, this method improves the distinctive fragrance of compound high lipid food, thus stimulate the appetite of model mice, solve the problem that traditional high lipid food reduces model mice appetite, and caramel odour masks the cooled fatty taste of lard.The synthesis of all right growing chain fatty acid of caramel, thus the synthesis of triglycerides is increased.The present invention is the 10%CCl of traditional lumbar injection
4liquid changes 5% CCl
4liquid is also divided into double injection, greatly reduces the lethality of modeling, and reaches the perfect condition that modeling lethality is 0%.The inventive method is simple, and easily implement, with low cost, result of use is good, has larger application prospect.
Accompanying drawing explanation
Fig. 1 is nonalcoholic fatty liver model mouse liver even slurry testing result;
Fig. 2 is nonalcoholic fatty liver model mice serum testing result.
Specific embodiment
In order to prove the validity of the modeling method of this kind of nonalcoholic fatty liver model mouse, because this has been following experiment:
The serum TC of model mice and normal mouse, the comparison of TG content:
1, buy the mouse 24 of the Kunming kind cleaning grade with animal number, male and female half and half, body weight is about 18g's.First carry out adaptability after buying laboratory and raise 3 days to about one week, see mouse situation and determine.Adapt to 3 days in order, otherwise 7 days will be extended.When 7 days situation also not good (referring to not vivaciously active) then discardedly do not drop into experiment because Kunming mouse is more bellicose, especially male mice.Change mouse cage bedding and padding and clear water every day, basal feed of feeding during this period.Be divided into two groups at random after animal conforms, one group is Normal group one group is model group.Often organize each 6 of 12 male and female, often organize male and female mouse and separately raise, 6 mouse in each mouse cage.
2, raise in Animal House, indoor temperature remains on 18 DEG C ~ 29 DEG C, and relative moisture, 40 ~ 70%, ensures clean environment air circulation.
3, blank group to be fed basic mouse feed every day, model group to be fed compound high lipid food every day, and its formula is: 2% cholesterol+0.5% sodium taurocholate+0.2% propylthiouracil+5% sucrose (using after being converted into caramel under high temperature)+10% lard+82.3% basal feed.According to said method feed two weeks.
4, carry out lumbar injection concentration 5% CCl to after two weeks model group mouse
4liquid, is diluted to liquid carbon tetrachloride the emulsion (because carbon tetrachloride is water insoluble) that concentration is 5% by deionized water.Each lumbar injection all needs Fresh can.By the lumbar injection dosage of injection every the model mice that how much converts according to the principle of every 10g lumbar injection 0.1ml with its quality after model mice accurate weighing.Abdominal cavity injection state continues to the 28th day, compound of simultaneously feeding high lipid food.
5, after 28 days, stop all operations being stopped eating by two groups of experiment mices not cutting off the water after 24h, 24h accurately weighing and record to two groups of mouse, blood sampling.Put into mouse tail vein injection fixator after mouse weights, take a blood sample from its afterbody.After the alcohol wipe afterbody of 75%, syringe needle is flat lunges blood vessel, and every mouse extracts 0.2ml ~ 0.3ml, anticoagulant with 0.9% physiological saline.If there is muddiness, refrigerated centrifuge (3500r/min, 10min), gets supernatant stand-by.
6, after getting blood, mouse is shifted out fixator, mention its afterbody and rotate seven or eight circles, put it in the large plane of frictional force.There is dizzy sense because mouse turns and lie prone inactive on the table, now draw its afterbody and head to put to death with cervical dislocation.Treat that it is motionlessly dissected immediately, now the heart of mouse is also being beaten, and gets fresh liver sucks surface after physiological saline rinsing moisture with filter paper, record of weighing immediately.
7, get the liver middle period, clip 100mg adds the isopropyl alcohol of 9 times of volumes, grinds to form 10% LH on ice, and 4 DEG C are extracted 24h, centrifugal (3000r/min), extract supernatant stand-by.
Serum and the LH of two groups of experiment mices is detected respectively with Triglyceride Reagent box, Cholesterol Kit.Both all measure absorbance value (OD value) at determined wavelength 500nm place.Shown in following Fig. 1 and Fig. 2 of statistical analysis process data.
Can learn according to Fig. 1, the mouse of model group the 29th day time, liver coefficient, content of triglyceride and the equal severe overweight of cholesterol level compared with normal control group mice.The whole modeling time has only used 28 days, wherein none dead mouse.
Claims (3)
1. a modeling method for nonalcoholic fatty liver model mouse, is characterized in that: comprise following steps:
(1) get healthy mice, in Animal House, first carry out adaptability raise 3-7 days, change mouse cage bedding and padding and clear water every day, basal feed of feeding during this period;
(2) after adaptability is raised, compound of feeding every day high lipid food, compound high lipid food, its formula is: 2% cholesterol+0.5% sodium taurocholate+0.2% propylthiouracil+5% caramel+10% lard+82.3% basal feed;
Lumbar injection concentration 5% CCl is carried out to mouse after (3) two weeks
4liquid, is diluted to liquid carbon tetrachloride the emulsion that concentration is 5% by deionized water, and each lumbar injection all needs Fresh can; Abdominal cavity injection state continues to the 28th day, compound of simultaneously feeding high lipid food; Obtain.
2. the modeling method of a kind of nonalcoholic fatty liver model mouse according to claim 1, is characterized in that: by the weighing scale of mouse, and the injected dose of carbon tetrachloride is 0.1ml/10g.
3. the modeling method of a kind of nonalcoholic fatty liver model mouse according to claim 1, is characterized in that: raise in Animal House, and indoor temperature remains on 18 DEG C ~ 29 DEG C, and relative moisture, 40 ~ 70%, ensures clean environment air circulation.
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104509705B (en) * | 2013-09-29 | 2018-03-30 | 中国人民解放军南京军区南京总医院 | A kind of application of compound high lipid food in rats with nonalcoholic fatty liver disease model is built |
| CN103782955A (en) * | 2013-12-25 | 2014-05-14 | 湖南师范大学 | Method for building SD rat alimentary obesity model |
| CN105850867A (en) * | 2016-03-28 | 2016-08-17 | 大连大学 | Method for modeling mouse models with non-alcoholic fatty liver disease (NAFLD) |
| CN105850868A (en) * | 2016-03-28 | 2016-08-17 | 大连大学 | Method for establishment of non-alcoholic fatty liver disease model by utilizing ApoE-/-mice |
| US11020494B2 (en) * | 2017-06-22 | 2021-06-01 | Crown Bioscience Inc. (Taicang) | Animal models for nonalcoholic fatty liver disease |
| CN107912366B (en) * | 2017-12-06 | 2020-12-11 | 江苏珂玛麒生物科技有限公司 | Non-alcoholic chronic steatohepatitis non-human primate model and construction method and application thereof |
| CN110278914A (en) * | 2019-06-10 | 2019-09-27 | 漳州片仔癀药业股份有限公司 | A kind of alcoholic fibrosis and inflammatory animal model and its construction method and purposes |
| US20220330529A1 (en) * | 2019-09-03 | 2022-10-20 | Innoland Biosciences (Zhejiang) Co., Ltd. | Methods for generating animal models for nonalcoholic fatty liver disease |
| CN112616775B (en) * | 2020-12-30 | 2023-02-10 | 昕慕（上海）科技发展有限公司 | Method for establishing hepatic fibrosis of healthy SD male rat by adopting compound factor method |
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