CN102138928A - Establishing method of hepatocyte apoptosis animal model and application thereof - Google Patents
Establishing method of hepatocyte apoptosis animal model and application thereof Download PDFInfo
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- CN102138928A CN102138928A CN2010105238234A CN201010523823A CN102138928A CN 102138928 A CN102138928 A CN 102138928A CN 2010105238234 A CN2010105238234 A CN 2010105238234A CN 201010523823 A CN201010523823 A CN 201010523823A CN 102138928 A CN102138928 A CN 102138928A
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Abstract
The invention relates to a preparation method of a hepatocyte apoptosis animal disease model by coinduction of D-galactosamine and lipopolysaccharide, in particular to a simple inducer formula and a simple operation process which can generate a hepatocyte apoptosis rate above 80% and reproduce a stable and reliable hepatocyte apoptosis animal model. The invention also relates to the application of the hepatocyte apoptosis animal model to the research on the pathogenesis of hepatitis and evaluation of novel anti-hepatitis medicaments.
Description
One, technical field
The present invention relates to a kind of preparation method of animal disease model, particularly a kind of simple derivant prescription and shirtsleeve operation program, produce the hepatocellular apoptosis rate more than 80%, copy stable, reliable hepatocellular apoptosis animal model and the application in incidence of hepatitis Study on Mechanism and the evaluation of novel anti hepatitis medicament thereof.
Two, background technology
Hepatitis is the general name of liver generation inflammatory lesion, can be divided into viral, ethanol, medicine source property and autoimmune hepatitis etc. according to the cause of disease, and for seeing, wherein based on hepatitis B, hepatitis C is taken second place more with viral hepatitis.Hepatitis B is a kind of very harmful worldwide popular infectious disease that is caused by hepatitis B virus (HBV), the whole world has 3.5 hundred million HBV carriers approximately at present according to statistics, and the China high infected zone that is HBV, the HBV carrier accounts for 10%～15% of total population, wherein about 3,000 ten thousand examples of chronic viral hepatitis B patient have 350,000 people to die from the chronic viral hepatitis B relevant disease every year approximately.Though playing the neonate vaccination that China carries out, nineteen ninety significantly reduced child's chronic viral hepatitis B infection rate, the ascendant trend of not checking the hepatitis B sickness rate, and hepatitis B and relevant disease thereof are still the important public health problem of China.Because the pathogeny (comprising viral infection and hepatocyte death) of hepatitis B is still not fully aware of, causes treating hepatitis B still not have safe and effective medicine and radical cure way so far.Therefore, the morbidity of further investigation hepatitis and lapse to mechanism, continuing to develop safely and effectively, anti-hepatitis B original new drug and Therapeutic Method are still the task of top priority.Existing treating hepatitis strategy mainly is by suppressing virus replication, human body immunity improving function and improve the hepatocyte metabolism to alleviate hepar damnification.Recent study is found excessive apoptosis of hepatocyte and various hepatitis, and especially the viral hepatitis relation is very close, plays an important role in viral hepatitis chronicity and canceration of hepatic cell.The regulation and control hepatocellular apoptosis alleviates liver inflammatory reaction and pathology damage degree, is considered to have the new way of the treating hepatitis of prospect.And, depend on the foundation and the application of the inside and outside model of disease association to a great extent to the discussion of incidence of hepatitis mechanism and the screening and the evaluation of potential medicine.Can use but still lack stable, reliable hepatocellular apoptosis model so far, cause great difficulty for the expansion for the treatment of hepatitis strategy and the exploitation of novel anti hepatitis medicament.
Three, summary of the invention
The technical problem to be solved in the present invention provides a kind ofly induces with the hepatotoxicant prescription, be applicable to the normal experiment animal, simple, the hepatocellular apoptosis animal model that produce hepatocellular apoptosis rate 80% or more, stablize, sensitivity is high, and the labor intensive financial resources are few, experimental period is short, become mould rate height, be applicable to the incidence of hepatitis Study on Mechanism and suppress the evaluation of hepatocellular apoptosis medicine, for the exploitation of the expansion for the treatment of hepatitis strategy and novel anti hepatitis medicament provides a kind of stable, hepatocellular apoptosis model reliably.For this reason, the present invention takes following technical scheme:
A kind of simple, can produce the hepatocellular apoptosis rate more than 80%, stable, reliable, and also the labor intensive financial resources are few, and experimental period, weak point can obtain the method for building up of hepatocellular apoptosis animal model.The foundation of hepatocellular apoptosis animal model need not special device, is easy to carry out.The hepatocellular apoptosis model of setting up can well be simulated the especially hepatocyte injury apoptosis mechanism of viral hepatitis of various hepatitis.Utilize the pathogenesis of this model further investigation hepatitis and the medicine that exploitation suppresses hepatocellular apoptosis,, can significantly alleviate liver inflammatory reaction and pathology damage degree, new direction will be provided the treatment of hepatitis in early days apoptosis being regulated and control of disease.
Existing hepatocellular apoptosis model instability, become the mould rate low, do not have clear and definite evaluation index and standard, do not carry out comprehensive, systematically estimate, immature.And can reach more than 80% with the hepatocellular apoptosis animal model hepatocellular apoptosis rate that the present invention obtains, and the disease model that obtains is reliable and stable, simple and convenient, the cycle is short, cost is low.
A kind of with D-Gal amine and lipopolysaccharide-induced, simple, can produce the hepatocellular apoptosis rate more than 80%, reliable and stable, and also the labor intensive financial resources are few, the method for building up of the hepatocellular apoptosis animal model that experimental period is short.Concrete preparation method is as follows:
Get the ICR mice of 18～22 grams,, injected back 8 hours, promptly can be used for the exploitation of incidence of hepatitis Mechanism Study and antiapoptotic drug by lumbar injection D-Gal amine (750mg/kg) and lipopolysaccharide (8mg/kg).
The present invention compared with prior art has following advantage: (1) is with D-Gal amine and lipopolysaccharide-induced, and is simple, can produce the hepatocellular apoptosis rate more than 80%, reliable and stable hepatocellular apoptosis animal model; (2) the labor intensive financial resources are few, and experimental period is short, become mould rate height; (3) be suitable for incidence of hepatitis Mechanism Study and inhibition apoptosis drug evaluation; (4) has good dependency with hepatitis especially viral hepatitis hepatocyte injury apoptosis.
Four, description of drawings
Fig. 1-3 is breach end-labelling (TUNEL) analysis result that DNA electrophoretic analysis, histopathological analysis, the deoxyribonucleotide terminal transferase of embodiment 1 resulting hepatocellular apoptosis mediates.
Fig. 1 is embodiment 1 resulting mouse liver cell apoptosis liver histopathological analysis result
Fig. 2 is embodiment 1 resulting mouse liver cell apoptosis DNA electrophoretic analysis result
Fig. 3 is embodiment 1 resulting mouse liver cell apoptosis TUNEL staining analysis result
Five, the specific embodiment
Embodiment 1: the preparation of hepatocellular apoptosis mouse model and evaluation
1 prepares following material:
1.1 laboratory animal: the ICR mice is provided by Zhejiang Province's Experimental Animal Center.
1.2D-aminogalactose amine: the permanent outstanding biological engineering company limited product in Jiaxing.
1.3 lipopolysaccharide: U.S. SIGMA company product.
1.4 apoptosis DNALadder detection kit: Nanjing KaiJi Biology Science Development Co., Ltd.
1.5 the two transfection reagent boxes of apoptosis fluorescence Hoechst33342/PI: Nanjing KaiJi Biology Science Development Co., Ltd.
1.6?In?Situ?Cell?Death?Detection?Kit，POD：Roche?Kit
2 preparation methoies are as follows:
2.1 mice elder generation adaptability was raised 3 days before the experiment.
2.2 prepare inducer of apoptosis D-Gal amine and lipopolysaccharide solution with the formula proportion that normal saline is determined by screening.
2.3 weighing, animal after the numbering, gives the lumbar injection inducer of apoptosis by prescription dosage.
2.4 derivant was injected back 8 hours, can form typical hepatocellular apoptosis model.
2.5 the DNA electrophoretic analysis of hepatocellular apoptosis is estimated: take off cervical vertebra and put to death mice, get hepatic tissue PBS buffer flush away blood stains; Take by weighing tissue and be put into homogenizer homogenate on a small quantity; Homogenate back is well washed and is poured in the centrifuge tube of a 1.5ml with PBS buffer, presses test kit operating routine extracting DNA then; Extracting DNA is trapezoid-shaped strips with 1.5% agarose gel electrophoresis.
2.6 the comprehensive verification of hepatocellular apoptosis: adopt TUNEL method, liver histopathological analysis, flow cytometry and electronic microscope photos overall merit and checking.
Embodiment 2: the antiapoptotic drug evaluation
1 prepares following material:
1.1 laboratory animal: the ICR mice is provided by Zhejiang Province's Experimental Animal Center.
1.2D-aminogalactose amine: the permanent outstanding biological engineering company limited product in Jiaxing.
1.3 test medicinal liquid (diluting) with normal saline
1.4 lipopolysaccharide: U.S. SIGMA company product.
1.5 apoptosis DNA Ladder detection kit: Nanjing KaiJi Biology Science Development Co., Ltd.
2 evaluation methodologys are as follows:
2.1ICR the mice animal is divided into large, medium and small three dosage groups and solvent control group at random, every group of 10 mices.
2.2 modeling first three day prevention administration, every day 1 time; Administration 1 time again after the modeling, after the last administration 4 hours, put to death mice, get serum and survey the Caspase-3 activity with the ELISA method, get hepatic tissue and do DNA electrophoresis detection or the analysis of TUNEL method.
2.3 according to trial test and actual needs, the Concentraton gradient of design experiment sample.
2.4 do qualitative analysis according to the DNA electrophoresis, detect by Caspase-3 activity and TUNEL method and do quantitative analysis, judge inhibition degree to hepatocellular apoptosis.
Claims (10)
1. the method for building up of a hepatocellular apoptosis animal model, comprise the screening of apoptosis induction agent prescription, the selection of apoptosis evaluation index, the method and the steps such as definite and model evaluation establishment of standard of the best induction time of apoptosis, it is characterized in that adopting a kind of simple derivant prescription and shirtsleeve operation program, produce the hepatocellular apoptosis rate more than 80%, copy stable, reliable hepatocellular apoptosis animal model and the application in incidence of hepatitis Study on Mechanism and the evaluation of novel anti hepatitis medicament thereof.
2. the method for building up of hepatocellular apoptosis animal model according to claim 1 is characterized in that described animal is a mice, especially the ICR mice.
3. the method for building up of hepatocellular apoptosis animal model according to claim 1, the optimal proportion that it is characterized in that described apoptosis induction agent prescription are D-Gal amine (750mg/kg) and lipopolysaccharide (8mg/kg).
4. the method for building up of hepatocellular apoptosis animal model according to claim 1 and 2, the route of administration that it is characterized in that described inducer of apoptosis is a lumbar injection.
5. according to the method for building up of the described hepatocellular apoptosis animal model of claim 1-5, it is characterized in that the best induction time of described apoptosis is that inducer of apoptosis was injected back 8 hours.
6. a kind of according to claim 1 simple derivant prescription of the method for building up of a hepatocellular apoptosis animal model and shirtsleeve operation program produce the hepatocellular apoptosis rate more than 80%, copy stable, reliable hepatocellular apoptosis animal model.It is characterized in that described hepatocellular apoptosis Preparation of model method comprises that the selection of the screening of apoptosis induction agent prescription, apoptosis evaluation index, the method steps such as definite and apoptosis model evaluation establishment of standard of apoptosis induction time carry out in the following order:
A. mice elder generation adaptability was raised 3 days before the experiment.
B. press formulated inducer of apoptosis D-Gal amine and the lipopolysaccharide that screening is determined with normal saline.
C. after animal is weighed numbering, give the lumbar injection inducer of apoptosis by prescription dosage.
D. derivant was injected back 8 hours, can form typical hepatocellular apoptosis model.
7. the preparation method of hepatocellular apoptosis animal model as claimed in claim 6 is characterized in that described apoptosis model can be the fragmentation trapezoid-shaped strips with hepatic tissue DNA electrophoresis and estimate as follows and carry out:
A. take off cervical vertebra and put to death mice, get hepatic tissue PBS buffer flush away blood stains.
B. take by weighing tissue and be put into homogenizer homogenate on a small quantity.
C. homogenate is washed and is poured in the centrifuge tube of a 1.5ml with PBS buffer in the back well, presses test kit operating routine extracting DNA then.
D. extracting DNA is trapezoid-shaped strips with 1.5% agarose gel electrophoresis.
8. as the preparation method of the described hepatocellular apoptosis animal model of claim 6-7, it is characterized in that breach end-labelling (TUNEL), flow cytometry and the electronic microscope photos overall merit of the also available deoxyribonucleotide terminal transferase mediation of described apoptosis model.
9. hepatocellular apoptosis animal model according to claim 1 be based upon application in the incidence of hepatitis Mechanism Study, it is characterized in that the especially viral hepatic injury pathological change of described hepatic injury apoptosis and hepatitis has good dependency.
10. hepatocellular apoptosis animal model according to claim 1 set up the application of novel anti hepatitis medicament in estimating, it is characterized in that concrete good susceptiveness, specificity and the stability of described hepatocellular apoptosis animal model, can be used for suppressing the screening and the evaluation of hepatocellular apoptosis medicine.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103920165A (en) * | 2013-01-10 | 2014-07-16 | 中国计量学院 | Establishment method and application of novel immunological hepatitis model |
| CN104706657A (en) * | 2013-12-12 | 2015-06-17 | 中国科学院深圳先进技术研究院 | Method for establishing acute viral hepatitis animal model |
| CN105707004A (en) * | 2016-01-26 | 2016-06-29 | 浙江省中医院 | Building method of mouse liver apoptosis animal model induced by nanometer zinc oxide |
| CN106822159A (en) * | 2017-02-27 | 2017-06-13 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | One kind treats the medicine of lipopolysaccharides/D amine-galactose amine induced liver injuries |
| CN108685939A (en) * | 2018-07-23 | 2018-10-23 | 遵义医学院附属医院 | A kind of acute otherness hepatic injury derivant and its preparation method and application |
| CN111579538A (en) * | 2020-04-22 | 2020-08-25 | 山东第一医科大学(山东省医学科学院) | Apoptosis kit applied to circulating tumor cell detection and detection method thereof |
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2010
- 2010-10-29 CN CN2010105238234A patent/CN102138928A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103920165A (en) * | 2013-01-10 | 2014-07-16 | 中国计量学院 | Establishment method and application of novel immunological hepatitis model |
| CN104706657A (en) * | 2013-12-12 | 2015-06-17 | 中国科学院深圳先进技术研究院 | Method for establishing acute viral hepatitis animal model |
| CN105707004A (en) * | 2016-01-26 | 2016-06-29 | 浙江省中医院 | Building method of mouse liver apoptosis animal model induced by nanometer zinc oxide |
| CN106822159A (en) * | 2017-02-27 | 2017-06-13 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | One kind treats the medicine of lipopolysaccharides/D amine-galactose amine induced liver injuries |
| CN108685939A (en) * | 2018-07-23 | 2018-10-23 | 遵义医学院附属医院 | A kind of acute otherness hepatic injury derivant and its preparation method and application |
| CN111579538A (en) * | 2020-04-22 | 2020-08-25 | 山东第一医科大学(山东省医学科学院) | Apoptosis kit applied to circulating tumor cell detection and detection method thereof |
| CN111579538B (en) * | 2020-04-22 | 2022-12-30 | 山东第一医科大学(山东省医学科学院) | Method for detecting circulating tumor cells by using apoptosis kit |
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Application publication date: 20110803 |