CN111727935A - Method for preparing fatty liver disease animal model by using MS-NASH mice - Google Patents
Method for preparing fatty liver disease animal model by using MS-NASH mice Download PDFInfo
- Publication number
- CN111727935A CN111727935A CN202010730891.1A CN202010730891A CN111727935A CN 111727935 A CN111727935 A CN 111727935A CN 202010730891 A CN202010730891 A CN 202010730891A CN 111727935 A CN111727935 A CN 111727935A
- Authority
- CN
- China
- Prior art keywords
- preparing
- fatty liver
- animal model
- nash
- liver disease
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010053219 non-alcoholic steatohepatitis Diseases 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 42
- 238000011713 fatty liver animal model Methods 0.000 title claims abstract description 13
- 241000699670 Mus sp. Species 0.000 title claims description 24
- 210000004185 liver Anatomy 0.000 claims abstract description 19
- 208000021907 Central cloudy dystrophy of François Diseases 0.000 claims abstract description 18
- 238000010171 animal model Methods 0.000 claims abstract description 18
- 238000012360 testing method Methods 0.000 claims abstract description 11
- 210000004369 blood Anatomy 0.000 claims description 34
- 239000008280 blood Substances 0.000 claims description 34
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 23
- 241001465754 Metazoa Species 0.000 claims description 17
- 208000010706 fatty liver disease Diseases 0.000 claims description 15
- 210000002966 serum Anatomy 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 238000005303 weighing Methods 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 6
- 150000001720 carbohydrates Chemical class 0.000 claims description 6
- 235000014633 carbohydrates Nutrition 0.000 claims description 6
- 230000037396 body weight Effects 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- 230000034994 death Effects 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 210000003462 vein Anatomy 0.000 claims description 4
- 206010015548 Euthanasia Diseases 0.000 claims description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 3
- 239000001569 carbon dioxide Substances 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 238000007619 statistical method Methods 0.000 claims description 3
- 230000015271 coagulation Effects 0.000 claims description 2
- 238000005345 coagulation Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 claims description 2
- 238000001543 one-way ANOVA Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 238000003672 processing method Methods 0.000 claims description 2
- 238000007492 two-way ANOVA Methods 0.000 claims description 2
- 206010061218 Inflammation Diseases 0.000 abstract description 11
- 230000006698 induction Effects 0.000 abstract description 11
- 230000004054 inflammatory process Effects 0.000 abstract description 11
- 230000008859 change Effects 0.000 abstract description 5
- 238000002474 experimental method Methods 0.000 abstract description 5
- 230000001575 pathological effect Effects 0.000 abstract description 5
- 230000007170 pathology Effects 0.000 abstract description 4
- 230000003908 liver function Effects 0.000 abstract description 2
- 230000036285 pathological change Effects 0.000 abstract 1
- 231100000915 pathological change Toxicity 0.000 abstract 1
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 7
- 231100000240 steatosis hepatitis Toxicity 0.000 description 7
- 241000282414 Homo sapiens Species 0.000 description 6
- 235000005911 diet Nutrition 0.000 description 6
- 206010019708 Hepatic steatosis Diseases 0.000 description 5
- 230000037213 diet Effects 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000007863 steatosis Effects 0.000 description 5
- 206010016654 Fibrosis Diseases 0.000 description 4
- 208000008589 Obesity Diseases 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 230000004761 fibrosis Effects 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 238000010172 mouse model Methods 0.000 description 4
- 235000020824 obesity Nutrition 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 4
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 4
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 4
- 206010022489 Insulin Resistance Diseases 0.000 description 3
- 208000019425 cirrhosis of liver Diseases 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 230000028709 inflammatory response Effects 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 208000032928 Dyslipidaemia Diseases 0.000 description 2
- 208000004930 Fatty Liver Diseases 0.000 description 2
- 208000017170 Lipid metabolism disease Diseases 0.000 description 2
- 238000013231 NASH rodent model Methods 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 238000010562 histological examination Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000004584 weight gain Effects 0.000 description 2
- 235000019786 weight gain Nutrition 0.000 description 2
- 102100031251 1-acylglycerol-3-phosphate O-acyltransferase PNPLA3 Human genes 0.000 description 1
- 102000014777 Adipokines Human genes 0.000 description 1
- 108010078606 Adipokines Proteins 0.000 description 1
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 1
- 108010082126 Alanine transaminase Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 102000017011 Glycated Hemoglobin A Human genes 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001129184 Homo sapiens 1-acylglycerol-3-phosphate O-acyltransferase PNPLA3 Proteins 0.000 description 1
- 101000597918 Homo sapiens Transmembrane 6 superfamily member 2 Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000016267 Leptin Human genes 0.000 description 1
- 108010092277 Leptin Proteins 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 102100035330 Transmembrane 6 superfamily member 2 Human genes 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 239000000478 adipokine Substances 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000013237 diet-induced animal model Methods 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 208000016097 disease of metabolism Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 108091005995 glycated hemoglobin Proteins 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 235000009200 high fat diet Nutrition 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 1
- 229940039781 leptin Drugs 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 229940118019 malondialdehyde Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000006371 metabolic abnormality Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 230000003234 polygenic effect Effects 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 208000018655 severe necrosis Diseases 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000011820 transgenic animal model Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Images
Classifications
-
- 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
-
- 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
-
- 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
- A01K2207/00—Modified animals
- A01K2207/25—Animals on a special diet
-
- 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
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- 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
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Endocrinology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Diabetes (AREA)
- Animal Husbandry (AREA)
- Gastroenterology & Hepatology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Rheumatology (AREA)
- Toxicology (AREA)
- Urology & Nephrology (AREA)
- Epidemiology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the field of fatty liver animal models, in particular to a method for preparing a fatty liver animal model by utilizing an MS-NASH mouse; the invention takes MS-NASH as an experimental animal, and tests the pathological change of the liver caused by the change of the liver function after being induced by CCDF and CDAHFD feeds; compared with the traditional feed induction result, the invention needs shorter pathological result reaching the same level, has more obvious adiposity, inflammatory reaction, balloon deformation and higher NAS score, and can obtain a more definite model of pathology in shorter induction time to enter an experiment.
Description
Technical Field
The invention relates to the field of fatty liver animal models, in particular to a method for preparing a fatty liver animal model by using an MS-NASH mouse.
Technical Field
Histological examination results of non-alcoholic fatty liver disease (NAFLD) are characterized by steatosis, hepatocyte injury and inflammatory cell infiltration, but a clinical disease in which patients have no history of excessive alcohol consumption. According to a recent study report, the number of NAFLD cases in different regions of the world will increase by 18.3-29.3% by 2030. Chinese NAFLD is associated with a rapid rise in the incidence of obesity and metabolic disease, estimated to increase from 2.463 billion in 2016 to 3.145 billion in 2030. Another study reported that approximately 20% of NAFLD progresses to NASH (nonalcoholic steatohepatitis), with approximately 25% of patients likely to have advanced liver fibrosis (fibrosis at stage 2 or later) at the time of diagnosis.
However, to date, the FDA has not approved therapeutic drugs for NASH liver fibrosis. Not only because NASH was named and gradually known and studied only in 1980, but also because it is a complex multifactorial disease, no clear genetic etiology has been found in humans, making it difficult for researchers to model in the laboratory for systematic studies. The NASH model as a study and validation of efficacy should reflect not only the characteristics of NASH liver histopathology, but also the overall metabolic disturbance of human NASH patients. This means that the animal model chosen should replicate not only hepatic steatosis, lobular inflammation, hepatocyte swelling and fibrosis, but also metabolic abnormalities such as obesity (weight gain and fat mass), body fat distribution, insulin resistance (blood glucose and insulin levels), fasting hyperglycemia, dyslipidemia and abnormal adipokines. However, the existing NASH model based on gene modification and specific diet induction cannot faithfully simulate the process and characteristics of the occurrence and development of the similar diseases of human beings.
The genetically modified animal model plays a very important role in elucidating certain specific pathways in the NASH development process. For example, T cell knockout mouse lines were used to demonstrate that adaptive immunity has a key role in NASH and its progression to hepatocellular carcinoma. In addition, transgenic animal models can also be used to elucidate the effect of genetic background on NASH. It is well known that different single nucleotide polymorphisms are associated with NASH, most notably variants of PNPLA3 and TM6SF2, while specific monogenic conditions lead to severe NAFLD symptoms. However, most mouse models of genetic NASH contain mutations in genes not normally found in patients (e.g., ob/ob, db/db, foz/foz mice, etc.). In this case, the value of these models lies in being able to study the consequences of isolated pathways and their dysfunctions related to metabolic homeostasis. Although it is also possible to mimic advanced NASH by inducing the development of inflammation and fibrosis in the genetic model mice by applying additional stimuli (by feeding an abnormal diet), the animal model itself is clearly deficient.
The specific food induction model mainly takes high-fat, high-sugar and other diets as main materials. After the mice are fed with the high-fat feed for about 6 weeks, the liver function indexes, triglyceride and total cholesterol of the mice are obviously increased. Histological examination shows that the liver cells are obviously swollen, and fat drops are dispersedly distributed in the liver. After 24 weeks, inflammatory reactions of the liver tissue occurred. The model has the advantages of low cost and simple operation, and has the disadvantages of long induction time and unobvious hepatic fibrosis. After the mice are fed with the high fructose feed, the weight and triglyceride of the mice are obviously increased, and the mice have obvious steatosis. Although the model is simple in modeling and obvious in steatosis, the inflammatory reaction is slight, and insulin resistance does not exist, so that the model cannot faithfully reflect the disease occurrence background of a patient, and can only be used for primary screening of single functions of the medicine. After 4 weeks of choline-proteinate deficient diet (MCD) feeding C57BL/6 mice, weight gain, steatosis, marked elevation of triglyceride, alanine aminotransferase and malondialdehyde, and mild inflammatory injury. The method can obtain moderate inflammatory reaction after feeding for 8 weeks, and has the disadvantages of high feed price, no compliance of MCD diet with human dietary structure, large difference between modeling cause and NASH patient, and unsuitability for comprehensive and accurate evaluation of drug effect. The obvious triglyceride rise is contrary to the pathological characteristics of NASH patients.
Carbon tetrachloride, tetracycline and other toxic agents that cause liver damage can be administered alone or in combination with high fat diets to induce fatty liver or liver fibrosis. For example, carbon tetrachloride is mainly used to induce oxidative stress in the liver, which results in the continuous production and accumulation of harmful lipid and protein peroxidation products and severe necrosis, thereby causing the structural and functional destruction of liver cells. This approach is currently thought to be used in conjunction with genetically modified or diet-induced models to promote the progression of fibrosis. The method has short modeling time, but the pathogenesis, the course change and the histological morphology of the liver are greatly different from those of human fatty liver, the toxicity of the medicine is strong, the death of the animal is easy to be actuated, and the method is still in the research stage in academia and the groove and tongue industry.
Disclosure of Invention
The MS-NASH polygene mouse model is a new generation model of type 2 diabetes and obesity. Has the characteristic of complete functional leptin pathway, and the model can spontaneously form obesity, dyslipidemia and insulin resistance after being fed with standard feed (ND, protein 23.5%, fat 6.5%, Purina), and is very similar to the pathogenesis basis of human beings. The invention discloses a method for preparing a fatty liver disease animal model by using an MS-NASH mouse on the basis of an MS-NASH polygenic mouse model, which is shorter than the conventional feed induction result in order to achieve the same level of pathological result, and has more obvious adiposity, inflammatory reaction, ballooning change and higher NAS score. A more definite model of the pathology can be obtained in a shorter induction time to enter an experiment.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for preparing a fatty liver disease animal model by using an MS-NASH mouse is characterized by comprising the following steps:
(1) selecting male MS-NASH mice of 6-8 weeks old, feeding with CCDF or CDAHFD feed, and measuring body weight once a week;
(2) collecting blood before, during and after feeding high fat feed to measure blood sugar and Hba1 c; preparing serum by using the collected blood and detecting blood biochemical indexes;
(3) after the last blood collection, all animals were euthanized, the livers were dissected and weighed, and data processing was performed.
Further, in the method for preparing the animal model with fatty liver disease by using the MS-NASH mouse, the CCDF feed in the step 1 contains 18% of protein, 21% of carbohydrate and 61% of fat.
Further, in the method for preparing the animal model with fatty liver disease by using the MS-NASH mice, the CDAHFD feed in the step 1 contains 18% of protein, 36% of carbohydrate and 46% of fat.
Further, in the method for preparing a fatty liver disease animal model by using an MS-NASH mouse, the method for weighing body weight in the step 1 is: and opening the balance, placing a mouse weighing bowl, resetting the balance, then placing the mouse, and reading the reading of the balance to obtain the weight of the animal.
Further, in the method for preparing the animal model with fatty liver disease by using the MS-NASH mouse, the method for detecting blood sugar and HbA1c in the step 2 comprises: the test started fasting animals for about 6 hours in the morning, and whole blood was collected in the afternoon for blood glucose and HbA1c testing, at the mouse orbital venous plexus, tail vein or heart.
Further, in the method for preparing a fatty liver animal model by using an MS-NASH mouse, the biochemical blood indicators in the step 2 comprise ALT, AST, ALP, TG, TC, LDL, HDL and n-HDL; the preparation method of the serum comprises the following steps: collecting blood, standing at room temperature for at least 30 min for coagulation, centrifuging at 4 deg.C for 10 min at 10000 rpm, collecting serum, subpackaging into EP tubes, and storing in-80 deg.C refrigerator before serum sample detection.
Further, in the method for preparing the animal model with fatty liver disease by using the MS-NASH mouse, the euthanasia method in the step 3 is: death was ensured by neck removal or chest opening using 99% excess carbon dioxide inhalation.
Further, in the method for preparing the animal model with fatty liver disease by using the MS-NASH mouse, the method for dissecting and weighing the liver in the step 3 comprises: collecting and weighing the wet weight of the liver, dividing the left leaf of the liver into 2 parts, immediately fixing one part in 10% formalin, and quickly freezing and storing the other part by using liquid nitrogen.
Further, in the method for preparing a fatty liver disease animal model by using an MS-NASH mouse, the data processing method in step 3 is: all data will be compared by statistical analysis, one-way or two-way analysis of variance, with P < 0.05 indicating statistical significance.
The invention has the following beneficial effects:
1. the invention reserves the characteristics of the MS-NASH mouse, better conforms to the characteristics of NASH disease occurrence of human patients compared with other traditional known NASH animal models, and is more suitable to be used as a strict medicine research model animal.
2. Compared with the traditional feed induction result, the invention needs shorter time to achieve the same level of pathological result, and has more obvious adiposity, inflammatory reaction, ballooning change and higher NAS score. A more definite model of the pathology can be obtained in a shorter induction time to enter an experiment.
3. The key inducing substance is added into the solid feed, so that each mouse takes the inducing substance more evenly, and the difference between experimental control groups of each batch in the group is smaller.
Drawings
FIG. 1 is a comparison of the differences in hepatic steatosis among the control, CCDF and CDAHFD groups in accordance with the present invention;
FIG. 2 is a comparison of the differences in liver inflammatory responses between the control, CCDF and CDAHFD groups, in accordance with the present invention:
FIG. 3 is a comparison of differences between liver ballooning between the control, CCDF and CDAHFD groups in accordance with an embodiment of the present invention;
FIG. 4 is a comparison of the difference between NAS scores in the control, CCDF and CDAHFD groups, in accordance with an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Examples
The experimental process comprises the following steps: male mice (6-8 weeks old) were randomly assigned to 3 groups, which were ND group (control group), CCDF group and CDAHFD group, respectively; the animals were fed with ND, CCDF and CDAHFD feeds, respectively, and 3 feeds were fed for 12 weeks, and the body weights were weighed 1 time per week. Before, during and after feeding high fat feed, blood was collected for blood glucose measurement and Hba1c, ALT, AST, ALP, TG, TC, LDL, HDL, n-HDL were collected. After blood collection, all animals were euthanized, and livers were dissected and weighed.
During the experiment, all animals can freely eat drinking water without special condition, and the animals are respectively fed with ND feed (23.5% of protein, 6.5% of fat and Purina) from 6 to 8 weeks of age; CDAHFD feed: protein (18%) carbohydrate (36%) fat (46%) (name: A16092003 source: Changzhou mouse-mouse two Biotech limited); CCDF is a feed: protein (18%) carbohydrate (21%) fat (61%) feed (name: A06071302 source: Changzhou mouse-mouse two Biotech Co., Ltd.).
The specific experimental process is as follows
(1) Selecting 30 male MS-NASH mice with the age of 8 weeks, randomly dividing into 3 groups, and feeding 10 mice in each group with ND, CCDF and CDAHFD feeds; weighing for 1 time per week, opening the balance, placing a mouse weighing bowl, resetting the balance, then placing the mouse, and reading the scale to obtain the animal weight.
(2) Before, during and after feeding high-fat feed, blood is respectively collected to measure blood sugar and Hba1c, and the specific method comprises the following steps: on the test day, fasting animals are started for about 6 hours in the morning, animal whole blood is collected in the afternoon for blood sugar and HbA1c test, the blood sugar test instrument is a Roche Diagnostics (Accuchek), and the HbA1c test instrument is a Siemens DCA glycated hemoglobin Analyzer (Siemens DCA variable HbA1c Analyzer). The blood collection site was the orbital venous plexus, tail vein or heart of the mouse.
The method for preparing serum and detecting biochemical indexes of blood by using collected blood comprises the following steps: on the test day, animals were fasted for about 6 hours in the morning, and animal sera were collected in the afternoon for the measurement of biochemical indicators of blood (ALT, AST, ALP, TG, TC, LDL, HDL, n-HDL) at the orbital venous plexus, tail vein or heart of mice. Blood was collected into AXYGEN EP tubes (MCT-150-C), allowed to clot at room temperature for at least 30 minutes, then centrifuged at 10000 rpm for 10 minutes at 4 deg.C, and serum was drawn and dispensed into pre-labeled AXYGEN EP tubes (MCT-150-C) and stored in a-80 deg.C freezer prior to serum sample testing.
(3) At 4, 6, and 12 weeks respectively, a fraction of mice from each group were sacrificed by inhalation with 99% excess carbon dioxide and death was ensured by an additional euthanasia procedure (decapitation or open chest), and livers were dissected and weighed wet. Dividing the left leaf of the liver into 2 parts, immediately fixing one part in 10% formalin, and quickly freezing and preserving the other part by using liquid nitrogen; and (3) carrying out microscopic observation on the fixed liver, analyzing adiposity, inflammatory reaction, ballooning and NAS scoring, comparing all data through statistical analysis one-way or two-way variance analysis, and indicating that P is less than 0.05 and has statistical significance. Wherein hepatic steatosis is shown in FIG. 1, and shows that when fed with CCDF or CDAHFD, the steatosis is obviously increased compared with the control group; inflammatory responses as shown in figure 2, with CCDF or CDAHFD, the inflammatory responses were significantly increased compared to the control (no inflammation); as shown in fig. 3, ballooning was significantly increased over the control group by CDAHFD feeding (histogram in the figure shows control groups at 4 and 6 weeks, CCDF group at 4 weeks, CDAHFD group at 6 and 12 weeks); as shown in fig. 4, NAS (NAFLD activity score) score was significantly higher than normal group fed CCDF or CDAHFD. In the above fig. 1-4, the control group, CCDF group and CDAHFD are sequentially provided from left to right; the bars from left to right in each group were data for 4 weeks, 6 weeks and 12 weeks in sequence.
The experimental data show that the method for preparing the animal model with the fatty liver disease by using the MS-NASH mice is shorter than the conventional feed induction result in order to achieve the pathological result of the same level, and has more obvious adiposity, inflammatory reaction, ballooning change and higher NAS score. A more definite model of the pathology can be obtained in a shorter induction time to enter an experiment.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should also be construed as the protection scope of the present invention.
Claims (9)
1. A method for preparing a fatty liver disease animal model by using an MS-NASH mouse is characterized by comprising the following steps:
(1) selecting male MS-NASH mice of 6-8 weeks old, feeding with CCDF or CDAHFD feed, and measuring body weight once a week;
(2) collecting blood before, during and after feeding high fat feed to measure blood sugar and Hba1 c; preparing serum by using the collected blood and detecting blood biochemical indexes;
(3) after the last blood collection, all animals were euthanized, the livers were dissected and weighed, and data processing was performed.
2. The method for preparing an animal model with fatty liver disease by using MS-NASH mice as claimed in claim 1, wherein the CCDF feed in the step 1 contains 18% of protein, 21% of carbohydrate and 61% of fat.
3. The method for preparing an animal model with fatty liver disease from MS-NASH mice as claimed in claim 1, wherein the CDAHFD feed in step 1 contains 18% protein, 36% carbohydrate and 46% fat.
4. The method for preparing a fatty liver disease animal model by using an MS-NASH mouse according to claim 1, wherein the method for weighing the body weight in the step 1 comprises the following steps: and opening the balance, placing a mouse weighing bowl, resetting the balance, then placing the mouse, and reading the reading of the balance to obtain the weight of the animal.
5. The method for preparing an animal model with fatty liver disease from MS-NASH mice according to claim 1, wherein the method for detecting blood sugar and HbA1c in step 2 comprises: the test started fasting animals for about 6 hours in the morning, and whole blood was collected in the afternoon for blood glucose and HbA1c testing at the mouse orbital venous plexus, tail vein or heart.
6. The method for preparing an animal model with fatty liver disease using MS-NASH mice as claimed in claim 1, wherein the biochemical indices of blood in step 2 include ALT, AST, ALP, TG, TC, LDL, HDL and n-HDL; the preparation method of the serum comprises the following steps: collecting blood, standing at room temperature for at least 30 min for coagulation, centrifuging at 4 deg.C for 10 min at 10000 rpm, collecting serum, subpackaging into EP tubes, and storing in-80 deg.C refrigerator before serum sample detection.
7. The method for preparing an animal model with fatty liver disease from MS-NASH mice as claimed in claim 1, wherein the euthanasia method in step 3 is: death was ensured by neck removal or chest opening using 99% excess carbon dioxide inhalation.
8. The method for preparing an animal model with fatty liver disease from MS-NASH mice as claimed in claim 1, wherein the method for dissecting and weighing liver in step 3 comprises: collecting and weighing the wet weight of the liver, dividing the left leaf of the liver into 2 parts, immediately fixing one part in 10% formalin, and quickly freezing and storing the other part by using liquid nitrogen.
9. The method for preparing an animal model with fatty liver disease from MS-NASH mice as claimed in claim 1, wherein the data processing method in step 3 is: all data will be compared by statistical analysis, one-way or two-way analysis of variance, with P < 0.05 indicating statistical significance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010730891.1A CN111727935A (en) | 2020-07-27 | 2020-07-27 | Method for preparing fatty liver disease animal model by using MS-NASH mice |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010730891.1A CN111727935A (en) | 2020-07-27 | 2020-07-27 | Method for preparing fatty liver disease animal model by using MS-NASH mice |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111727935A true CN111727935A (en) | 2020-10-02 |
Family
ID=72657853
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010730891.1A Pending CN111727935A (en) | 2020-07-27 | 2020-07-27 | Method for preparing fatty liver disease animal model by using MS-NASH mice |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111727935A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104365543A (en) * | 2014-10-28 | 2015-02-25 | 华中科技大学同济医学院附属同济医院 | Method for establishing non-alcoholic fatty liver disease combined with viral hepatitis mouse model |
| CN105510597A (en) * | 2015-12-17 | 2016-04-20 | 泸州医学院附属医院 | Method for regulating and controlling rat stearoyl-coenzyme A desaturase and liver X receptor alpha genes of NAFLD (nonalcoholic fatty liver disease) with quercetin |
| 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 |
| CN109105333A (en) * | 2017-06-22 | 2019-01-01 | 中美冠科生物技术（太仓）有限公司 | Animal model for nonalcoholic fatty liver disease |
| CN110150222A (en) * | 2018-02-10 | 2019-08-23 | 复旦大学 | The method for constructing mouse nonalcoholic fatty liver disease model |
| CN111328920A (en) * | 2020-03-10 | 2020-06-26 | 昆明科灵生物科技有限公司 | Feed for constructing non-alcoholic steatohepatitis model of non-human primate and using method thereof |
-
2020
- 2020-07-27 CN CN202010730891.1A patent/CN111727935A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104365543A (en) * | 2014-10-28 | 2015-02-25 | 华中科技大学同济医学院附属同济医院 | Method for establishing non-alcoholic fatty liver disease combined with viral hepatitis mouse model |
| CN105510597A (en) * | 2015-12-17 | 2016-04-20 | 泸州医学院附属医院 | Method for regulating and controlling rat stearoyl-coenzyme A desaturase and liver X receptor alpha genes of NAFLD (nonalcoholic fatty liver disease) with quercetin |
| 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 |
| CN109105333A (en) * | 2017-06-22 | 2019-01-01 | 中美冠科生物技术（太仓）有限公司 | Animal model for nonalcoholic fatty liver disease |
| CN110150222A (en) * | 2018-02-10 | 2019-08-23 | 复旦大学 | The method for constructing mouse nonalcoholic fatty liver disease model |
| CN111328920A (en) * | 2020-03-10 | 2020-06-26 | 昆明科灵生物科技有限公司 | Feed for constructing non-alcoholic steatohepatitis model of non-human primate and using method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Brumbaugh et al. | Developmental origins of nonalcoholic fatty liver disease | |
| Shepherd et al. | Altered adipocyte properties in the offspring of protein malnourished rats | |
| Zhang et al. | Changes in serum adipocyte fatty acid‐binding protein in women with gestational diabetes mellitus and normal pregnant women during mid‐and late pregnancy | |
| CN107773566B (en) | A kind of construction method of tire source property metabolic syndrome animal model and its application | |
| CN106857406A (en) | A kind of method for building up by diet induced SD rat diabetes animal models | |
| CN103638531A (en) | LGR4 gene for screening of medicines promoting browning of white fat | |
| Maruhama et al. | Hepatic steatosis and the elevated plasma insulin level in patients with endogenous hypertriglyceridemia | |
| Alkan et al. | Hippocampal neural cell loss in high-fat diet-induced obese rats–exploring the protein networks, ultrastructure, biochemical and bioinformatical markers | |
| Balasubramanyam et al. | Non-traditional forms of diabetes worldwide: implications for translational investigation | |
| Li et al. | A mouse model of gestation-specific transient hyperglycemia for translational studies | |
| Toller-Kawahisa et al. | Systemic lupus erythematosus onset in lupus-prone B6. MRL/lpr mice is influenced by weight gain and is preceded by an increase in neutrophil oxidative burst activity | |
| CN111727935A (en) | Method for preparing fatty liver disease animal model by using MS-NASH mice | |
| CN109142419A (en) | A kind of potential marker metabolic pathway of serum and research method of the moringa seeds anti-diabetic based on metabolism group | |
| CN115279411B (en) | Application of EphB4 as target in screening of insulin sensitivity increasing drugs or models | |
| Verbeek et al. | Development of a representative mouse model with nonalcoholic steatohepatitis | |
| Chen et al. | Late-Onset Carnitine–Acylcarnitine Translocase Deficiency with SLC25A20 c. 199-10T> G Variation: Case Report and Pathologic Analysis of Liver Biopsy | |
| CN109730028A (en) | A method of Models of Nonalcoholic Fatty Liver Disease is established using mouse | |
| CN110478485B (en) | Application of ASB3 in preparation of medicine for treating non-alcoholic fatty liver disease | |
| Pratiwi et al. | Alterations in body weight, blood glucose levels, and lipid profiles in high-fat diet-low dose streptozotocin-induced diabetic rats | |
| CN105267954A (en) | Application of hepatocyte nuclear factors HNF-1alpha and HNF-4alpha in preparation of medicine used for preventing or treating intrauterine growth retardation (IUGR) | |
| CN116396965B (en) | Application of AHDC1 in construction of obese animal model, method and drug screening method | |
| CN114557318B (en) | Construction method and application of non-alcoholic steatohepatitis mouse model based on PEDF/LDLR double gene knockout | |
| CN115136928B (en) | Rapid modeling method for tree shrew type II diabetes | |
| CN114596965A (en) | Premature infant and SGA catch-up growth management and CMDs risk early warning system thereof | |
| Simion | THE LIVER FUNCTIONS IN DIABETES MELLITUS |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20211013 Address after: 311215-8, bonded building, west of bonded Road, Hangzhou Airport Economic Zone, Xiaoshan District, Hangzhou City, Zhejiang Province Applicant after: Zhejiang YaoYuan Xindi Biotechnology Co.,Ltd. Address before: 215400 science and technology entrepreneurship Park, No. 6, Beijing West Road, Taicang Economic Development Zone, Suzhou, Jiangsu Applicant before: CROWN BIOSCIENCE Inc. (TAICANG) |
|
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201002 |