CN107801691A - A kind of chronic fat hepatitis rat model of non-alcoholic and its construction method and purposes - Google Patents
A kind of chronic fat hepatitis rat model of non-alcoholic and its construction method and purposes Download PDFInfo
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- 238000010171 animal model Methods 0.000 claims abstract description 14
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- 238000011160 research Methods 0.000 claims abstract description 11
- 239000003814 drug Substances 0.000 claims abstract description 9
- 206010019668 Hepatic fibrosis Diseases 0.000 claims abstract description 8
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims abstract description 8
- 229930182817 methionine Natural products 0.000 claims abstract description 8
- 235000013305 food Nutrition 0.000 claims abstract description 7
- 150000002632 lipids Chemical class 0.000 claims abstract description 7
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Classifications
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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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/158—Fatty acids; Fats; Products containing oils or fats
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/50—Feeding-stuffs specially adapted for particular animals for rodents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- Animal Husbandry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
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- Animal Behavior & Ethology (AREA)
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- Inorganic Chemistry (AREA)
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- Biomedical Technology (AREA)
- Biodiversity & Conservation Biology (AREA)
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- Urology & Nephrology (AREA)
- Gastroenterology & Hepatology (AREA)
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Abstract
The present invention relates to biological technical field, discloses a kind of chronic fat hepatitis of non-alcoholic（NASH）Rat model and its construction method and purposes.The special feed that methionine and choline lack is fed to adult rat（MCD feeds）Or the special high lipid food that choline lacks（CDAA feeds）, a certain amount of sodium nitrite solution of animal is given in certain time, structure, which obtains the chronic fat hepatitis rat model of non-alcoholic, has chronic hepatic fibrosis and early-phase hepatocirrhosis feature.Rat model building process provided by the present invention is simple, technological means is easy, construction cost is low, value with the animal model needed for high scientific research and medicament research and development,, with seriousness liver fibrosis and the particular animals model in early-phase hepatocirrhosis stage, belong to domestic initiation especially as in NASH.
Description
Technical field
The present invention relates to biological technical field, the chronic fat hepatitis rat model of more particularly to a kind of non-alcoholic and its
Construction method and purposes.
Background technology
Non-alcohol fatty liver (non-alcoholic fattyliver disease, NAFLD) is to be clinical normal
One of liver diseases seen, wherein nonalcoholic fatty liver disease (non-alcoholic steatohepatitis, NASH)
It is that NASH (non-alcoholic fatty liver, NAFL) is transformed to non-alcoholic fatty liver hardening
A very important link in journey, is one of major reason of cryptogenic cirrhosis.Research shows that NASH prognosis is not
Good, about 50% NASH follow-up of patients can enter after 6 years transforms into liver fibrosis, and 10%-15% can enter to transform into hepatic sclerosis in 10-20,
And 9%-26% is dead in the 4-10 Nei Keyin End-stage liver diseases of follow-up.Therefore, the control strategy for studying NASH is the current country
One of focus of outer hepatopathy circle.
NASH pathogenesis is not fully understood, it is necessary to carry out in-depth study by substantial amounts of zoopery so far
However, NASH animal models both domestic and external differ in a jumble at present, lack unified standard, limit NASH diseases to a certain extent
The further investigation of reason mechanism and medicine.Nonalcoholic fatty liver disease (NASH) animal model is exploring the morbidity machine of disease
The fields such as system, evaluation diagnostic method, screening protective agents play an important role.
In recent years, many scholars replicate nonalcoholic fatty liver disease pathological model using experimental animal and it are carried out
Substantial amounts of research.The nonalcoholic fatty liver disease animal model commonly used both at home and abroad at present, including trophic disturbance, drug poisoning
Property, particular line and complex factors induction Models of Fatty Liver.Trophic disturbance fatty liver model is the most frequently used both at home and abroad
Nonalcoholic fatty liver disease animal model, including high fat diet fatty liver model, high-carbonhydrate diet fatty liver animal mould
Type and choline-methionine deficiency fatty liver model.With genetic model and trophic model), wherein methionine-choline lacks
The NASH models that (methionine-choline-deficient, MCD) diet induces are the animals being widely recognized as in the world
Model, it is easy to operate, it is high into mould rate, turn into the primary animal model of research NASH pathogenesis and protective agents.
As CN200710066801.8 discloses a kind of construction method of non-alcohol fatty liver mouse model.Will
C57BL/6 mouse using choline and methionine deficiency forage feed 1-8 weeks and evaluate corresponding group mouse into mould situation.《Egg
Propylhomoserin-choline lacks the foundation and dynamic monitoring of the nonalcoholic fatty liver disease mouse model of diet induced》（World Chinese disappears
Change magazine on April 18th, 2011; 19(11): 1122-1129）It has studied methionine-choline and lack (MCD) diet induced
Nonalcoholic fatty liver disease (NASH) mouse liver steatosis and inflammation occur, the pathological characteristic of development.However, existing skill
The NASH animal model degree of hepatic fibrosis of existing MCD diet induceds is lighter under art, fails to embody progressivity liver fibrosis
Or the pathogenesis of hepatic sclerosis.It is existing because NASH with liver fibrosis, hepatic sclerosis is the disease research and the core point for the treatment of
Animal model under technology can not very well in simulation actual clinical disease pathology feature, NASH researchs and treatment can not be met
Requirement.Meanwhile the mouse model that MCD is induced under prior art serious the weight of animals occurs and declined, within the test period
Body weight loss reaches or close to 40%, seriously violates Laboratory Animal Welfare principle, animal model has very big defect.Based on life
The exploitation of thing macromolecular drug, medicament research and development market in urgent need energy growing day by day for new nonalcoholic fatty liver disease demand
Enough solves the new experimental animal disease model of above-mentioned model problem.
The content of the invention
The present invention in view of the shortcomings of the prior art, in view of the above existing model the shortcomings that, it is an object of the invention to carry
For a kind of construction method of rat model for simulating mankind NASH and application thereof, and further provide for by described rat model
Rat model that construction method structure obtains and application thereof.The present invention gives specific methionine to adult rat, choline lacks
Special feed, and doses natrium nitrosum induced animal hepatic tissue fat become, inflammatory disorderses and aggravate liver cell
Apoptosis produces NASH basic pathology process, the pathological change of significant liver fibrosis and early-phase hepatocirrhosis particularly occurs.
Concrete technical scheme of the present invention is as follows：
A kind of construction method of the chronic fat hepatitis rat model of non-alcoholic, comprises the following steps：
（1）The special high lipid food that the special feed or choline that methionine and choline lack lack is fed to adult rat；
（2）After feeding special high lipid food certain time（Usually 8-12 weeks）, while rat sodium nitrite solution is given, build
The chronic fat hepatitis rat model of non-alcoholic.
Periodic detection Liver Function is used for the Clinical course for monitoring NASH in above-mentioned feeding period；Periodic detection rats'liver
Popular name for reason judges that NASH different pathological changing period includes, and the fatty liver phase, the fat hepatitis phase, fat hepatitis are fine with liver
Dimensionization phase, pathologic process of the liver fibrosis progression into the early-phase hepatocirrhosis phase.
It is real that rat of the present invention is selected from, but not limited to, the rats such as SD rats or the Waster rats of female or male series
Test animal.
The special feed that methionine and choline of the present invention lack is MCD feeds, the special high fat that the choline lacks
Feed is CDAA feeds.The scale of feeding of the MCD feeds or CDAA feeds is every 20-30 grams daily, feeding time 8-12
Week.
Step of the present invention（2）Sodium nitrite solution gives mode as daily oral or intraperitoneal injection.The nitrous acid
The concentration of sodium solution is 30-100mg/mL, and it is 30-100mg/kg that natrium nitrosum, which gives dosage,.When the sodium nitrate solution is given
Between be 4-6 weeks.
Another object of the present invention is to provide a kind of non-alcoholic chronic fat hepatitis rat model, using claim
The method of the invention is built-up.Model of the present invention is special with the pathology of chronic hepatic fibrosis and early-phase hepatocirrhosis
Sign.
Another object of the present invention is to provide the chronic fat hepatitis rat model of non-alcoholic of the present invention to exist
Screening prevention or the treatment chronic fat hepatitis of non-alcoholic, liver fibrosis, application in the medicine of hepatic sclerosis or liver cancer and
As animal model in chronic fatty liver, fat hepatitis, liver fibrosis, hepatic sclerosis, hepatocellular carcinoma, metabolic disease,
Diabetes, the application in the research of diabetic complication.
The present invention has the beneficial effect that：The method of the invention establishes obtained non-alcoholic fatty type hepatitis model companion
There are conspicuousness liver fibrosis and early-phase hepatocirrhosis pathological characters, the toy NASH models with prior art（Model with and it is light
The fibrosis of degree, its fibrosis and the difference of the intrinsic fibr tissue of liver are very small, are only fiber in fibrillatable pathological classification
Change stage phase, it is impossible to meet the research and development of anti-fibrosis medicine）Compared to significant advantage, with mankind NASH and liver fibrosis disease
Disease is highly similar.Model is used for NASH, liver fibrosis, the teiology of early-phase hepatocirrhosis or even liver cancer diseases, pathology and pharmacy
The model of evaluation.Because its pathologic process and people NASH, liver fibrosis, hepatic sclerosis pathologic process are highly similar, possesses high disease
It is worth because learning with pathological research, it is significant in the evaluation to medicine.
Brief description of the drawings
Fig. 1 is MCD of the embodiment of the present invention group-III and MCD+NaCO2Rat model livers of the group-II after ten weeks.(A：MCD
Group-III, B: MCD+NaCO2Group-II).
Fig. 2 is each test group liver weight change of the embodiment of the present invention.
Fig. 3 is each test group Serum ALT change of the embodiment of the present invention.
Fig. 4 is each test group serum AST change of the embodiment of the present invention.
Fig. 5 is each test group liver NAS scorings of the embodiment of the present invention.
Fig. 6 is that each test group liver pathology of the embodiment of the present invention changes H＆E dyeing.（C：Intact animal, D: MCD-4
Week, E：MCD-8 weeks, F：MCD-10 weeks, G：Intact animal+NaCO2, H：MCD-8 weeks+NaCO2, I：MCD-10 weeks+NaCO2）.
Fig. 7 is each test group liver fibrosis quantitative analysis of the embodiment of the present invention.
Fig. 8 is that each test group liver pathology of the embodiment of the present invention changes sirius red stains result.（C：Intact animal,
D:MCD-4 weeks, E：MCD-8 weeks, F：MCD-10 weeks, G：Intact animal+NaCO2, H：MCD-8 weeks+NaCO2, I：MCD-10 weeks+
NaCO2）.
Embodiment
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described implementation
Example is only the part of the embodiment of the present invention, rather than whole embodiments.It is common based on the embodiment in the present invention, this area
All other embodiment that technical staff is obtained under the premise of creative work is not made, belong to the model that the present invention protects
Enclose.
Embodiment：By taking SD male rats as an example, nonalcoholic fatty liver disease is built（NASH）With chronic hepatic fibrosis,
The animal model of early-phase hepatocirrhosis
From the SD male rats of 35 7-8 week old, body weight is 220-240 grams for this experiment.12 h illumination and dark cycle, temperature
22 DEG C ± 2 DEG C of degree, humidity 50%-60%.
MCD is formulated：(l-amino acid 175.7g/kg, sucrose 441.9g/kg, the g/kg of corn starch 150.0, dextrorotation wheat
Bud sugar 50.0g/kg, cellulose 30.0g/kg, corn oil 100.0g/kg, the g/kg of sodium acid carbonate 7.4, salt mixture 35.0g/
Kg, vitamin mixtures 10.0g/kg), methionine-choline abundance control diet MCS formula be MCD diet formulations, plus courage
Alkali 2g/kg, methionine 3g/kg. MCD and MCS feed are clear by Nantong Te Luofei feed corporation,Ltds processing and fabricating
Clean level feed, 4 DEG C of Cord bloods.
35 rats give normal diet adaptability to be randomly divided into 7 groups after feeding 1 week, wherein：
Blank group 5, feeding MCS diet 10 weeks;
Blank group adds natrium nitrosum group 5, feeding MCS diet 10 weeks, starts within the 5th week natrium nitrosum input, continues 6 weeks;
MCD model groups-I 5, feeding MCD diet 4 weeks, animal euthanasia；
MCD model groups-II 5, feeding MCD diet 8 weeks, animal euthanasia；
MCD model groups-III 5, feeding MCD diet 10 weeks, animal euthanasia；
MCD+ natrium nitrosum groups-I 5, feeds MCD 8 weeks altogether, natrium nitrosum 4 weeks, animal euthanasia since MCD is the 5th week.
MCD+ natrium nitrosum groups-II 5, feeds MCD 10 weeks altogether, the natrium nitrosum 6 weeks since MCD is the 5th week, animal peace
It is happy dead.
Experimental endpoints, animal are euthanized.Peripheral blood is gathered, separates Virus monitory NASH correlation liver function indexs.Liver
Dirty tissue is fixed through 10% neutral formalin, the analysis of row hepatic pathology.
Experimental result：
Fig. 1 is MCD of the embodiment of the present invention group-III and MCD+NaCO2Rat model livers of the group-II after ten weeks.(A：MCD groups-
III, B: MCD+NaCO2Group-II).Fig. 2 is that each test group liver weight of the embodiment of the present invention changes, * p<0.05 vs. is normal
Animal, * * p<0.01 vs. intact animals；##p<0.01 vs. MCD-10wk）As a result after showing natrium nitrosum two-hit
No natrium nitrosum treatment group is compared on animal's liver surface, and to become conspicuousness coarse, illustrates early stage liver after natrium nitrosum two-hit
Denaturation aggravates, and liver weight slightly increases, and the later stage aggravates with stem cell injuries, the increase of liver fibrosis, and liver weight mitigates.
Fig. 3 is each test group Serum ALT change of the embodiment of the present invention（*p<0.05 vs. intact animals, * * p<0.01
Vs. intact animal；***p<0.001 vs. intact animals）.Fig. 4 is each test group serum AST change of the embodiment of the present invention（*
p<0.05 vs. intact animals, * * p<0.01 vs. intact animals；***p<0.001 vs. intact animals）.Fig. 5 is this hair
Bright each test group liver NAS scorings of embodiment.Fig. 6 is that each test group liver pathology of the embodiment of the present invention changes H＆E dyeing knots
Fruit.（C：Intact animal, D:MCD-4 weeks, E：MCD-8 weeks, F：MCD-10 weeks, G：Intact animal+NaCO2, H：MCD-8 weeks+
NaCO2, I：MCD-10 weeks+NaCO2）.As a result show, raisings and nitrous acid of the hepatic injury Enzyme target ALT and AST with MCD
The two-hit Serum ALT and AST of sodium gradually rise, and natrium nitrosum strike group is slightly above non-Asia in same time point
Sodium nitrate strike group（Fig. 3 and Fig. 4）.Liver histological testing result shows that MCD raising surroundings conspicuousness hepatic steatosis occur
And cell infiltration, raised 10 weeks to MCD and reach severe fatty liver and severe fat hepatitis, natrium nitrosum two-hit has no
Conspicuousness increase fat becomes and inflammation（Fig. 5 and Fig. 6）.
Fig. 7 is each test group liver fibrosis quantitative analysis results of the embodiment of the present invention（***p<0.001 vs. is normally moved
Thing, ##p<0.01 vs. MCD-8weeks；$$$p<0.001 vs. MCD-10wk）.Fig. 8 is that the embodiment of the present invention is respectively tested
Group liver pathology change sirius red stains result.（C：Intact animal, D:MCD-4 weeks, E：MCD-8 weeks, F：MCD-10 weeks,
G：Intact animal+NaCO2, H：MCD-8 weeks+NaCO2, I：MCD-10 weeks+NaCO2）.Hepatic fibrosis-renal tubular ectasia syndrome testing result shows MCD
Raising only occurs mild fibrosis for 10 weeks, main centralized system portal area and central vein area, between hepatic cell cords it is rarely seen on a small quantity
Deposition of fibrous tissue, consistent with conventional mouse model, liver cirrhosis pathology scoring is a phase.Natrium nitrosum two-hit surrounding
It can be seen that conspicuousness fibrosis, the fibrosis between particularly visible conspicuousness hepatic cell cords, liver cirrhosis pathology scoring was 3 phases.
The natrium nitrosum two-hit hepatic fibrosis-renal tubular ectasia syndrome pathological score of six weeks is the 3-4 phases, sees the hepatic sclerosis pathological change of early stage, is realized
The final goal of this model.
The present invention is given natrium nitrosum in the regular period and is carried out two-hit to liver using the special feed raisings of MCD
NASH is induced with chronic hepatic fibrosis, early-phase hepatocirrhosis, meets the morbidity general principle of current mankind NASH and liver fibrosis,
Energy real simulation mankind NASH pathologic process.Absolutely, stability is good, and the death rate is low for model success rate；The neck is filled up
The blank of the NASH thing models in domain, for mankind NASH and the research of the research, particularly early-phase hepatocirrhosis of liver fibrosis, Yi Jixin
Medicine research and development provide indispensable animal model.
Claims (10)
1. a kind of construction method of the chronic fat hepatitis rat model of non-alcoholic, it is characterised in that comprise the following steps：
（1）The special high lipid food that the special feed or choline that methionine and choline lack lack is fed to adult rat；
（2）After feeding special high lipid food certain time, while give rat sodium nitrite solution and continue special high lipid food
Raising, build the chronic fat hepatitis rat model of non-alcoholic.
2. construction method as claimed in claim 1, it is characterised in that the rat for female or male SD rats or
Waster rats.
3. construction method as claimed in claim 1, it is characterised in that the special feed that the methionine and choline lack is MCD
Feed, the special high lipid food that the choline lacks is CDAA feeds.
4. construction method as claimed in claim 3, it is characterised in that the scale of feeding of the MCD feeds or CDAA feeds is daily
Every 20-30 grams of animal, feeding time are 8-12 weeks.
5. construction method as claimed in claim 1, it is characterised in that the step（2）It is every that sodium nitrite solution, which gives mode,
Day is oral or is injected intraperitoneally.
6. construction method as claimed in claim 5, it is characterised in that the concentration of the sodium nitrite solution is 30-100mg/
ML, it is 30-100mg/kg that natrium nitrosum, which gives dosage,.
7. construction method as claimed in claim 5, it is characterised in that the sodium nitrate solution gave the time as 4-6 weeks.
8. a kind of chronic fat hepatitis rat model of non-alcoholic, it is characterised in that using any one of claim 1-7 right
It is required that methods described is built-up.
9. the chronic fat hepatitis rat model of non-alcoholic as claimed in claim 8, it is characterised in that the model with
Chronic hepatic fibrosis and/or early-phase hepatocirrhosis pathological characters.
10. the chronic fat hepatitis rat model of non-alcoholic as claimed in claim 8 or 9 is in screening prevention or treats non-wine
Application and conduct animal model in the medicine of the chronic fat hepatitis of essence, liver fibrosis, hepatic sclerosis or liver cancer is chronic
Fatty liver, fat hepatitis, liver fibrosis, hepatic sclerosis, hepatocellular carcinoma, metabolic disease, diabetes, diabetic complication
Research in application.
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