CN110150222A - The method for constructing mouse nonalcoholic fatty liver disease model - Google Patents
The method for constructing mouse nonalcoholic fatty liver disease model Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/02—Breeding vertebrates
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- 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
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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
Abstract
The invention belongs to fields of biomedicine, are related to the construction method of animal model, and in particular to a kind of nonalcoholic fatty liver disease (NASH) model optimization construction method.The present invention is based on the change of diet style and breeding cycle, on the basis of comparing three kinds of feeding manners formed aspect of model, determines and obtain and the most similar mouse model of NASH clinical indication.Efficacy endpoint is assessed before the quantization observation index for the model that the present invention establishes is suitable for clinical drug, provides a standardization mouse NASH model further to compare different pharmaceutical superiority and inferiority for research.
Description
Technical field
The invention belongs to fields of biomedicine, are related to the construction method of animal model, and in particular to a kind of non-alcoholic rouge
Fat hepatitis (NASH) model optimization construction method.Before the quantization observation index for the model that the present invention establishes is suitable for clinical drug
Efficacy endpoint assessment provides a standardization mouse NASH model further to compare different pharmaceutical superiority and inferiority for research.
Background technique
Prior art discloses nonalcoholic fatty liver disease (NASH) cause of disease complexity, diverse clinical manifestations, and are in chronic
Progressive is non-alcohol fatty liver (NALFD) progressive stage, as obesity, hyperlipidemia, metabolic syndrome, diabetes
Equal systemic diseases liver performance or complication, disease incidence have become the second largest hepatopathy of China.It shows according to investigations, NAFLD
Crowd's overall incidence is 15%, wherein about 20% simple nonalcoholic fatty liver (NAFL) is often accompanied by dysfunction of liver, is entered
NASH stage, the latter further progress to liver fibrosis, cirrhosis or liver cancer.Clinical studies show, NASH's is mainly shown as
Liver function test abnormal (transaminase level raising), blood lipid level exception, insulin resistance (impaired glucose tolerance and empty stomach pancreas islet
Plain horizontal raising)；Liver has fat-free change and vacuolar degeneration and liver fibrosis to need, and needs liver biopsy that could determine.Clinic is ground
Study carefully display, common Imaging Technology such as ultrasonic scanning, Fibroscan cannot effectively judge that the degree of Fat Accumulation and liver are fine
Dimensionization progress, often needs MRI-PDFF to judge intrahepatic fat content, and magnetic resonance elastic force scanning (MRE) judges liver fibrosis
Degree and growth and decline.
Currently, there is no the drug for passing through special treatment NASH through Bureau of Drugs Supervision's examination & approval.The intervention hand that clinical practice is suggested first
Section is living-pattern preservation, increases activity, loses weight；Common drug is that vitamin E is taken June, or uses pancreas islet
Plain receptor sensitizer Pioglitazone (PGZ).It is reported that the drug of research and development treatment NASH, wherein several verified by clinical I phase
Candidate medicine be working on clinical II, III phase and verify, the NASH efficacy endpoint of these clinical verifications concern is concentrated mainly on fat
The degree of change, ballooning degeneration of liver cells, inflammatory infiltration, transaminase level, Fasting insulin level, sugar tolerance singularity in experiment, liver
Fibrosis progression and recession etc.；Therefore, the preclinical study done when assessing NASH drug effect also requires to make referring to These parameters
Obtaining those clinical endpoints has controllability on NASH model and improves space；Accordingly, with respect to can be in a standardized model
Upper assessment different pharmaceutical judges the journey that different pharmaceutical improves all endpoints of NASH or part index number to NASH therapeutic effect
The model of degree becomes the focus of the art.
It, at present still can be with without single animal model however, due to the diversity of NASH Pathological Physiology and clinical manifestation
Simulate all features of NASH patient.Existing NASH model can only represent specific performance and metabolic disorder in clinic.Usually
NASH is mostly used Rodent Models, can be divided into nutrition induction and heredity is intervened, heredity be intervened, most models are used for specific base
The Function Identification of cause, they are very important in terms of inquiring into the pathologic, physiologic function of specific gene and related pathways, however,
It is little with the cause of disease and clinical manifestation relationship that betide previous health NASH patient；In the NASH model of nutrition dietary induction,
Methionine/choline lack diet (MCD) and high-fat/high caloric diet be it is most common, although the rouge of MCD diet induced
The fat hepatitis duration is relatively short, but weight loss and shortage insulin resistance are modeling NASH clinical manifestations
Major defect.Studies have shown that is high-fat/and High cholesterol diet can lead to weight gain, obesity, hepatic steatosis in two months
Property, light inflammation, insulin resistance, cannot but cause apparent hepatocyte death, inflammation, also have not seen apparent liver fiber
Change；Individually adding high fructose may result in a degree of intrahepatic fat accumulation and inflammatory reaction, however, it hardly causes
Apparent steatohepatitis.Since high-fat/high caloric diet is the most common way of dining of inducing mouse NASH, the model
Western fast food food composition is simulated, however recipe formula, feed combinations and nursing time used in every kind of model are all different, respectively
The comparativity of same endpoint is poor between model；Status based on the prior art, present inventor are quasi- to existing related mould
Type carries out building optimization, establishes a standardization, reliable NASH model, the Preclinical evaluation for curative effect of medication.
Summary of the invention
It is an object of the present invention to it is directed to the status of the prior art, and it is quasi- that building optimization is carried out to existing related model, it establishes
One standardization, reliable NASH model, and in particular to a kind of construction method more particularly to a kind of non-alcoholic of animal model
Steatohepatitis (NASH) model optimization construction method.It is excellent that the model that the present invention establishes can compare different pharmaceutical for further research
One standardization mouse NASH model of bad offer, efficacy endpoint is assessed before quantization observation index is suitable for clinical drug,
The present invention is based on the changes of diet style and breeding cycle, are comparing three kinds of formed aspect of model of feeding manner
On the basis of, it determines and obtains and the most similar mouse model of NASH clinical indication.
The construction method of mouse nonalcoholic fatty liver disease (NASH) model of the invention, including, pass through feeding mouse
High fructose/glucose sugar is added in high-fat/high-calorie feed and drinking water constructs within 16 weeks a kind of and clinic NASH pathological characters
Very much like mouse NASH model, and to its, including Histopathology (liver cell fat become, damage death, inflammatory infiltration,
Liver fibrosis), liver biochemical indicator, lipid-metabolism, inflammatory factor and Adipocyte Factor spectrum variation, insulin resistance change done in detail
Thin measurement and description, determination are built into a standardization, reliable NASH model.
The present invention (was purchased from Nanjing University Nanjing biomedical research institute, and raised using 6-8 weeks male C57BL6J mouse
In Medical Center of Fudan University's experimental animal room, rearing conditions SPF rank, 21~23 DEG C of temperature, free diet) carry out non-alcoholic
The building of steatohepatitis (NASH) model.
Specifically, the construction method of mouse nonalcoholic fatty liver disease (NASH) model of the invention, including,
1) three kinds of Feeding ways, comprising:
1. high fructose/glucose sugar diet；
2. high in fat/high caloric diet；
3. the high syrup diet of high in fat/high heat+4%；
Breeding cycle is 8 weeks and 16 weeks；
2) detection compares three kinds of Feeding way models in Histopathology (change of liver cell fat, liver when feeding 8 weeks and 16 weeks
Cellular damage is dead, liver inflammation infiltration, liver fibrosis), liver biochemical indicator, lipid-metabolism, inflammatory factor and Adipocyte Factor spectrum
Variation, the change of insulin resistance, judge whether these indexs are consistent with the performance of NASH patients clinical；
3) standardization, reliable NASH model are determined；
High in fat/high heat+high fructose/glucose sugar diets 16 weeks can induce mouse and be formed with clinical patient NASH extremely
Similar NASH Pathological Physiology changes, and can be used as mouse NASH master pattern, provides and faces for assessment NASH curative effect of medication
The quantization endpoint that bed verifying matches.
In the present invention, high fructose/glucose sugar diet include [the high syrup of common cubed feed+4%, 23.1 grams of L- fructose/
It rises, 18.9 grams of L- glucose/liter]；
In the present invention, high in fat/high caloric diet, including [(Research Diet, D12492 contain 60% rouge to high lipid food
Fat)+common drinking water]；
In the present invention, the high syrup diet of high in fat/high heat+4%, including high lipid food (Research Diet,
D12492)+4% high syrup: 23.1 grams of L- fructose/liter, 18.9 grams of L- glucose/liter；
In the present invention, three kinds of diet styles are compared, the results show that high in fat/high heat+high fructose/glucose sugar at 8 weeks
Diet mouse may occur in which obvious fat, hepatic cell fattydegeneration, while with liver tg, cholesterol, free
Fatty acid, lipoprotein levels increase.In addition, serum GPT levels are significantly raised, there is apoptosis in liver cell and inflammation is anti-
It answers, and insulin resistance and slight liver fibrosis occurs, and high in fat/high caloric diet and high fructose/glucose sugar diet only occur
Slight steatosis, is maintained within normal range without obvious liver inflammation, serum insulin level, and liver is fine without liver liver
Dimensionization feature；
There is severe obesity (weight: 52.4vs. in high in fat/high heat+high fructose/glucose sugar diet mouse at 16 weeks
31.1g) and hepatic cell fattydegeneration (change of size mixed type balloon sample), liver tg level (305.6 vs.44.6mol/
G, 6.9 times), cholesterol levels (64.7vs.18.9mol/g, 3.4 times), free fatty acid levels (184.6vs.30.1mol/g,
6.1 times) extremely rise, and serum glutamic pyruvic transminase (296.9vs.33.6IU/L, 8.8 times) and glutamic-oxalacetic transaminease
(301.2vs.138.4IU/L, 2.2 times) level is also apparently higher than control group mice, in addition, the apparent inflammation of liver appearance is anti-
With Apoptosis (related gene should be damaged and protein level is significantly raised), while apparent insulin resistance (empty stomach pancreas occur
Island element is horizontal: 1.24vs.0.29ng/ml；Insulin resistance index: 8.3vs.2.0) and liver fibrosis (pathological score
1.4vs.0；Hydroxyproline: 0.26vs 0.11g/mg), hepatic lipoprotein is horizontal, inflammatory factor spectrum, Adipocyte Factor is composed and rouge generation
It thanks key enzyme and nuclear factor level and obvious exception also occurs；There is apparent fat change and pancreas in high in fat/high caloric diet group
Insulin resistance, but degree is not as good as high in fat/high heat+high fructose/glucose sugar diet mouse, liver inflammation reaction, cellular damage and
Liver fibrosis is all relatively slight；And the rarely seen hepatic cholesterol of high fructose/glucose sugar diet mouse and Bile Acid in Serum level are increased,
With the change of lipid-metabolism molecular level, but have not seen that significant liver cell fat change, inflammatory reaction, insulin resistance and liver are fine
Dimensionization；Therefore, present invention determine that high in fat/high heat+high fructose/glucose sugar diets 16 weeks can induce mouse formation and clinic
The very much like NASH Pathological Physiology of patient NASH changes, and can be used as mouse NASH master pattern.
The present invention, which constructs determining mouse NASH master pattern, can be used for assessing NASH curative effect of medication, provide and clinical verification
The quantization endpoint to match.
Detailed description of the invention
The distribution and changes of weight of Fig. 1 each group adipose tissue, wherein
A-C. at 8 and 16 weeks, the weight of each group and liver weight；D.16 subcutaneous, internal organ and brown adipose tissue distribution when all
MicroCT 3D rendering；E. the various adipose tissue contents handled with software；*, * * is compared with Control, p < 0.05
With 0.01.A, aa are compared with HF/G, p < 0.05 and 0.01.B, bb are compared with HFCD, p < 0.05 and 0.01.Subcu=
Subcutaneous (subcutaneous fat), Viscera=Visceral (interior fat), Brown=brown fat.
The murine liver tissue of Fig. 2 feeding difference diet changes, wherein
A. each group 8 (a-d) and when 16 weeks (e-h) Hematoxylin-eosin (H&E) dye in liver histological representativeness it is micro-
Picture, amplification factor be 200 ×, scale bar be 100 microns；B. each group 8 (a-d) and oil red O (ORO) is dyed when 16 weeks (e-h)
Representative displaing micro picture, amplification factor be 200 ×, scale bar be 100 microns；C&D. the liver group of each group 8 (C) and 16 (D) weeks
Pathology sxemiquantitative scoring is knitted, *, * * are compared with Control, p < 0.05 and 0.01.A, aa are compared with HF/G, the He of p < 0.05
0.01, bb compared with HFCD, p < 0.01, CNTL=Control.
The hepar damnification index of the mouse of Fig. 3 feeding difference diet, wherein
A. 8 (a-d) and 16 weeks (e-h) TUNEL (apoptosis) dye representative displaing micro picture, amplification factor be 200 ×,
Scale bar is 100 microns；B. the positive apoptosis cells in the liver slice of every group of three mouse count；C&D. the blood at 8 and 16 weeks
The level of clear ALT (C) and AST (D)；E&F. cell factor and Adipocyte Factor are with respect to mRNA level in-site；*, * * is compared with Control, p
< 0.05 and 0.01.A, aa are compared with HF/G, p < 0.05 and 0.01, b, and bb is compared with HFCD, p < 0.05 and 0.01.ALT=third
Histidine amino group transferase；AST=aspartate transaminase；TNF-=tumor necrosis factor；MCP-1=monocyte chemotactic egg
White -1；CCR-2=C-C chemokine receptor-2；Adipo=Adiponectin (adiponectin).
Fig. 4 liver biochemistry and blood lipids index, wherein
A&B. in 8 (A) and 16 weeks (B), total cholesterol of liver (TC), triglycerides (TG) and free fatty acid (FFA) contain
Amount；C&D. in 8 (C) and 16 weeks (D) serum TG, TC, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) water
It is flat；E&F. at 8 weeks and 16 weeks serum total bilirubin (T-BIL) (E) and total cholic acid (TBA) (F) level, *, * * with
Control is compared, p < 0.05 and 0.01, a, and aa is compared with HF/G, p < 0.05 and 0.01.B, bb are compared with HFCD, p < 0.05
With 0.01.
Fig. 5 each group insulin resistance situation, wherein
Intraperitoneal Glucose tolerance test when A&B.8 (A) and 16 (B) is all；Serum islet when C&D.8 (C) and 16 (D) is all
Plain horizontal and Homeostasis model assessment-insulin resistance (HOMA-IR) index；E&F.8 (E) and 16 (F) week INSR, IRS-1 and
IRS-2 is with respect to mRNA level in-site；G.8 horizontal with pIRS-1 at 16 weeks and pAkt protein versus；*, * * is compared with Control, and p <
0.05 and 0.01；A, aa are compared with HF/G, p < 0.05 and 0.01, b, and bb is compared with HFCD, p < 0.05 and 0.01；INSR=pancreas
Island element receptor；IRS-1=insulin receptor substrate-1；IRS-2=Insulin receptor substrate-2；Akt=PKB (protein kinase B)；
The phosphorylation site of IRS1 is serine307, and the phosphorylation site of Akt is serine 473.Fig. 6 liver fibrosis and collagen deposition,
Wherein,
A. in 8 (a-d) and 16 weeks (e-h) liver Masson trichrome stain representative displaing micro picture, amplification factor is
200 ×, scale bar is 100 microns；B.8 the sxemiquantitative scoring of the hepatic fibrosis in mice of week and 16 weeks；C.8 week and 16 weeks liver hydroxyl dried meat
Propylhomoserin (hydroxyproline) content；D. at 8 and 16 weeks in each group mouse liver-SMA protein level；E&F difference drink
Eat the mouse liver I procollagen type (Col-I α 1, Col-I 2) fed, type III precollagen (Col-III 1) and metalloproteinases
The mRNA level in-site of Tissue Inhibitor -1 (TIMP-1)；*, * * is compared with Control, p < 0.05 and 0.01.A, aa and HF/G phase
Than p < 0.05 and 0.01.B, bb are compared with HFCD, p < 0.05 and 0.01, SMA=smooth muscle-actin；Col-I 1=I
Procollagen type α 1；Col-I=I procollagen type α 2；Col-III
1=III procollagen type 1；TIMP-1=Timp -1.
Specific embodiment
Embodiment 1
Animal and diet: 6-8 weeks male C57BL6J mouse is bought from Nanjing biomedical research institute, Nanjing University and is raised
In Medical Center of Fudan University's experimental animal room, rearing conditions SPF rank, 21~23 DEG C of temperature, free diet is suitable by 2 weeks
Mouse is randomly divided into four groups (every group of n=10) by Ying Hou: (1) common cubed feed+common drinking water (Control)；(2) general
23.1 grams of L- fructose and 18.9 grams of L- glucose (4%) (HF/G) are added in logical cubed feed+every liter of drinking water；(3) high in fat
Fat/high-calorie feed (U.S. Research Diet, article No. D12492)+common drinking water；(4) high-fat/high-calorie feed
23.1 grams of L- fructose and 18.9 grams of L- glucose are added in (U.S. Research Diet, article No. D12492)+every liter of drinking water
(4%) (HFCD-HF/G) replaces 2 drinking water weekly and monitors the weight and diet consumption of mouse；Zoopery operation is through multiple
The approval of the animal welfare committee, preclinical medicine institute, denier university, and carried out according to the processing of NIH experimental animal and guide for use；Raising 8
Week puts to death mouse after 16 weeks, collect liver and serum, and it is raw to be stored in -80 DEG C of progress histopathologies, biochemistry and molecule
Object credit analysis；
Whole body quantifies CT scan: showing body fat group by carrying out MicroCT scanning to mouse at feeding 16 weeks
The accumulation and distribution knitted, including subcutaneous, internal organ and brown fat.It is scanned by high-resolution X-ray MicroCT and obtains mouse
Internal detailed three-dimensional structure image, concrete operations are as follows: mouse being anaesthetized with 2.5-3% isoflurane and is placed in scanning platform
On, X-ray power supply is set as 100 μ A of electric current and voltage 80kVp, and every mouse CT scan continues 4 minutes, with 70mm × 40mm
Visual field (FOV) and 144 μm of pixel shoot to form CT image, carry out image segmentation using Volume Edit tool, and make
The quantization for carrying out volume to area-of-interest (ROI) module with software, calculates the content of various fat with software later；
Intraperitoneal glucose toleance test: laggard at nursing mouse 8 weeks and 16 weeks in order to assess insulin-resistant states
Row Intraperitoneal Glucose tolerance test (IGTT).Mouse injectable dextrose monohydrate (2.5g/kg weight) in 15 hours posterior peritoneums of fasting,
0 after injection, 30,60,90 and 120 minutes with blood glucose meter test glucose level；
Serological analysis: taking blood through eye socket and passes through (4 DEG C, 2000g, 10 minutes) separation serum of centrifugation, by giving birth to automatically
Change analysis-e/or determining serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein
(HDL), total bilirubin (TBIL), total bile acid (TBA), alanine aminotransferase (ALT) and aspartate transaminase (AST) water
It is flat, serum insulin level is measured by mouse islets element ELISA kit, and pass through Homeostasis model assessment insulin resistance
(HOMA-IR) calculation formula of index: HOMA-IR=(Diagnostic Value of Fasting Serum insulin (mU/l) × fasting blood-glucose (mmol/l)/22.5
Acquire insulin resistance index；
Liver biochemical analysis: after fresh liver is homogenized by automatic Syrup-homogenizing instrument, using in particular agent box measurement mouse liver
Triglycerides (TG), total cholesterol (TC), free fatty acid (FFA) level.Liver hydroxyl dried meat ammonia is measured by acid-hydrolysis method
The content of sour (hydroxyproline)；
Liver histopathology inspection: fresh mouse liver is fixed in 10% neutral formalin, by paraffin packet
It buries and is cut into 4 μm of slabs, the dyeing in h and E (H&E) by liver section, and via the virologist of profession
H&E is sliced based on histologic characteristics and carries out NAFLD activity scores (NAS), scoring is divided into three classes: steatosis (0-3),
Inflammation (0-2) and ballooning degeneration of liver cells (0-2), if NAS >=5 of liver specimens, which is defined as " NASH ", such as
Fruit NAS is less than 3, then it represents that is " non-NASH ", the degree of liver fibrosis is assessed using Masson trichrome stain, is withered by cell
It dies (TUNEL) dyeing (counting positive cell number in 10 randomly selected regions) and shows mouse liver cell apoptosis situation, by liver
It is dirty to be cut into the frozen sections of 7 μ m thicks through fixed in 4% neutral paraformaldehyde through OTC embedding, and with oil red O stain to show liver
Fat drop deposition in cell, the slice of all dyeing are taken pictures under HPF 20 × object lens optical microscopy；
Quantitative real-time PCR: total serum IgE is isolated from murine liver tissue according to standard operation using TRIZOL reagent, and is made
It is cDNA with Primer-Script RT kit reverse transcription.Using Eppendorf realplex cycle operating system and
Power SYBY green PCR Master Mix carries out quantitative RT-PCR to cDNA, regard house-keeping gene-actin as internal reference
And use the relative level of 2- Δ Δ Ct method analysis target gene；
Western blot analysis: hepatic protein is extracted with RIPA lysate and carries out protein concentration using BCA kit
Measurement, by the albumen of SDS-PAGE electrophoretic separation denaturation and by protein delivery to poly- third difluoroethylene (PVDF) film, room
Temperature is lower to be incubated overnight after close membrane 2 hours with 4 DEG C of primary antibody with 5% skimmed milk power (1X TBST configuration), is then added at room temperature
The secondary antibody of HRP coupling is incubated for 1 hour, using the protein band of ECL luminescence reagent box and the chemiluminescence imaging systematic perspective observation of eyes, and
Gray analysis is carried out to band using Image J software；
All data indicate that statistical analysis is carried out using 17.0 software of SPSS with average value ± SEM, and variance analysis is used
Multiple range test between comparison among groups, two groups has been inspected by LSD, and P < 0.05 is considered to have significant statistical significance；
The results show that at 8 weeks high in fat/high heat+high fructose/glucose sugar diet mouse may occur in which it is obvious it is fat,
Hepatic cell fattydegeneration, while being increased with liver tg, cholesterol, free fatty acid, lipoprotein levels.In addition, blood
Clear gpt level is significantly raised, and apoptosis and inflammatory reaction occurs in liver cell, and insulin resistance and slight liver fibre occurs
Dimensionization, and only there is slight steatosis in high in fat/high caloric diet and high fructose/glucose sugar diet, without obvious liver
Inflammation, serum insulin level are maintained within normal range, and liver is without liver liver fibrosis feature；
There is severe obesity (weight: 52.4vs. in high in fat/high heat+high fructose/glucose sugar diet mouse at 16 weeks
31.1g) and hepatic cell fattydegeneration (change of size mixed type balloon sample), liver tg level (305.6 vs.44.6mol/
G, 6.9 times), cholesterol levels (64.7vs.18.9mol/g, 3.4 times), free fatty acid levels (184.6vs.30.1mol/g,
6.1 times) extremely rise, and serum glutamic pyruvic transminase (296.9vs.33.6IU/L, 8.8 times) and glutamic-oxalacetic transaminease
(301.2vs.138.4IU/L, 2.2 times) level is also apparently higher than control group mice, in addition, the apparent inflammation of liver appearance is anti-
With Apoptosis (related gene should be damaged and protein level is significantly raised), while apparent insulin resistance (empty stomach pancreas occur
Island element is horizontal: 1.24vs.0.29ng/ml；Insulin resistance index: 8.3vs.2.0) and liver fibrosis (pathological score
1.4vs.0；Hydroxyproline: 0.26vs 0.11g/mg), hepatic lipoprotein is horizontal, inflammatory factor spectrum, Adipocyte Factor is composed and rouge generation
It thanks key enzyme and nuclear factor level and obvious exception also occurs；There is apparent fat change and pancreas in high in fat/high caloric diet group
Insulin resistance, but degree is not as good as high in fat/high heat+high fructose/glucose sugar diet mouse, liver inflammation reaction, cellular damage and
Liver fibrosis is all relatively slight；And the rarely seen hepatic cholesterol of high fructose/glucose sugar diet mouse and Bile Acid in Serum level are increased,
With the change of lipid-metabolism molecular level, but have not seen that significant liver cell fat change, inflammatory reaction, insulin resistance and liver are fine
Dimensionization；Therefore, present invention determine that high in fat/high heat+high fructose/glucose sugar diets 16 weeks can induce mouse formation and clinic
The very much like NASH Pathological Physiology of patient NASH changes, and can be used as mouse NASH master pattern.
Claims (6)
1. a kind of nonalcoholic fatty liver disease NASH model optimization construction method, which is characterized in that it includes, small by feeding
Mouse is high-fat/high-calorie feed and drinking water in add fructose/glucose sugar 16 weeks high, construct a kind of and clinic NASH pathological characters
Very much like mouse NASH model, and measure and description its Histopathology, liver biochemical indicator, lipid-metabolism, inflammatory factor
And the variation of Adipocyte Factor spectrum, the change of insulin resistance, determine building standardization, reliable NASH model.
2. method according to claim 1, which is characterized in that itself comprising steps of
1) three kinds of Feeding ways, comprising:
1. high fructose/glucose sugar diet；
2. high in fat/high caloric diet；
3. the high syrup diet of high in fat/high heat+4%；
2) breeding cycle is 8 weeks, 16 weeks；
3) Histopathology, the liver biochemical indicator, lipid of three kinds of Feeding way models are compared in detection respectively when feeding 8 weeks, 16 weeks
Metabolism, inflammatory factor and Adipocyte Factor spectrum variation, insulin resistance change, judge detection index whether with NASH clinic table
Now it is consistent, determines building standardization, reliable NASH model.
3. method as described in claim 2, which is characterized in that the high fructose/glucose sugar diet are as follows: common cubed feed
+ 4% high syrup, 23.1 grams of L- fructose/liter, 18.9 grams of L- glucose/liter；
High in fat/the high caloric diet are as follows: high lipid food Research Diet, D12492, containing 60% fat+commonly drink
Water；
The high syrup diet of the high in fat/high heat+4% is high lipid food Research Diet, D12492+4% high syrup
Its containing 23.1 grams of L- fructose/liter, 18.9 grams of L- glucose/liter.
4. method as described in claim 2, which is characterized in that the Histopathology index include: liver cell fat become,
Hepatocellular injury is dead, liver inflammation infiltrates, liver fibrosis.
5. method as described in claim 1 or 2, which is characterized in that determine that the standardization of building, reliable NASH model are height
Rouge/high heat+high fructose/glucose sugar diets 16 weeks mouse NASH master pattern.
6. method described in claim 5, which is characterized in that the high in fat/high heat+high fructose/glucose sugar diets
16 weeks mouse NASH master patterns provide the quantization terminal to match with clinical verification and refer to for assessing NASH curative effect of medication
Mark.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111727935A (en) * | 2020-07-27 | 2020-10-02 | 中美冠科生物技术（太仓）有限公司 | Method for preparing fatty liver disease animal model by using MS-NASH mice |
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