CN107693507B - 一种腹腔注射法建立恒河猴肝纤维化模型的方法 - Google Patents
一种腹腔注射法建立恒河猴肝纤维化模型的方法 Download PDFInfo
- Publication number
- CN107693507B CN107693507B CN201710910648.6A CN201710910648A CN107693507B CN 107693507 B CN107693507 B CN 107693507B CN 201710910648 A CN201710910648 A CN 201710910648A CN 107693507 B CN107693507 B CN 107693507B
- Authority
- CN
- China
- Prior art keywords
- rhesus monkey
- injection
- ccl
- liver
- hepatic fibrosis
- 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.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 40
- 241000282560 Macaca mulatta Species 0.000 title claims abstract description 22
- 206010019668 Hepatic fibrosis Diseases 0.000 title claims abstract description 21
- 239000007928 intraperitoneal injection Substances 0.000 title claims abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 48
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000000465 moulding Methods 0.000 claims abstract description 13
- 241001465754 Metazoa Species 0.000 claims abstract description 12
- 230000008569 process Effects 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims abstract description 10
- 239000007924 injection Substances 0.000 claims description 16
- 238000002347 injection Methods 0.000 claims description 16
- VZGDMQKNWNREIO-UHFFFAOYSA-N carbon tetrachloride Substances ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 14
- 210000004185 liver Anatomy 0.000 claims description 13
- 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 claims description 12
- 239000000835 fiber Substances 0.000 claims description 7
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 5
- 235000019750 Crude protein Nutrition 0.000 claims description 5
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 5
- 239000011575 calcium Substances 0.000 claims description 5
- 229910052791 calcium Inorganic materials 0.000 claims description 5
- 235000012000 cholesterol Nutrition 0.000 claims description 5
- 208000019425 cirrhosis of liver Diseases 0.000 claims description 5
- 235000019784 crude fat Nutrition 0.000 claims description 5
- 239000011574 phosphorus Substances 0.000 claims description 5
- 229910052698 phosphorus Inorganic materials 0.000 claims description 5
- 239000004006 olive oil Substances 0.000 claims description 4
- 235000008390 olive oil Nutrition 0.000 claims description 4
- 239000011550 stock solution Substances 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 3
- 235000019197 fats Nutrition 0.000 claims 1
- 238000007912 intraperitoneal administration Methods 0.000 claims 1
- 238000010171 animal model Methods 0.000 abstract description 11
- 208000019423 liver disease Diseases 0.000 abstract description 5
- 230000007246 mechanism Effects 0.000 abstract description 3
- 230000001717 pathogenic effect Effects 0.000 abstract description 2
- 235000019441 ethanol Nutrition 0.000 description 28
- 210000001519 tissue Anatomy 0.000 description 20
- 238000010186 staining Methods 0.000 description 12
- 206010016654 Fibrosis Diseases 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- 230000004761 fibrosis Effects 0.000 description 9
- 239000001993 wax Substances 0.000 description 9
- 239000008096 xylene Substances 0.000 description 9
- 238000004043 dyeing Methods 0.000 description 8
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 6
- 241000282414 Homo sapiens Species 0.000 description 6
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 238000007789 sealing Methods 0.000 description 6
- 239000008399 tap water Substances 0.000 description 6
- 235000020679 tap water Nutrition 0.000 description 6
- 206010061218 Inflammation Diseases 0.000 description 5
- 230000004054 inflammatory process Effects 0.000 description 5
- 239000012188 paraffin wax Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 210000000683 abdominal cavity Anatomy 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 210000005229 liver cell Anatomy 0.000 description 4
- 210000005228 liver tissue Anatomy 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 230000001476 alcoholic effect Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 210000003855 cell nucleus Anatomy 0.000 description 3
- 235000020188 drinking water Nutrition 0.000 description 3
- 239000003651 drinking water Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 238000013424 sirius red staining Methods 0.000 description 3
- 238000002604 ultrasonography Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 206010057573 Chronic hepatic failure Diseases 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 208000010334 End Stage Liver Disease Diseases 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003150 biochemical marker Substances 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 208000011444 chronic liver failure Diseases 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 230000017074 necrotic cell death Effects 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- XOSXWYQMOYSSKB-LDKJGXKFSA-L water blue Chemical compound CC1=CC(/C(\C(C=C2)=CC=C2NC(C=C2)=CC=C2S([O-])(=O)=O)=C(\C=C2)/C=C/C\2=N\C(C=C2)=CC=C2S([O-])(=O)=O)=CC(S(O)(=O)=O)=C1N.[Na+].[Na+] XOSXWYQMOYSSKB-LDKJGXKFSA-L 0.000 description 2
- YYYARFHFWYKNLF-UHFFFAOYSA-N 4-[(2,4-dimethylphenyl)diazenyl]-3-hydroxynaphthalene-2,7-disulfonic acid Chemical compound CC1=CC(C)=CC=C1N=NC1=C(O)C(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=C12 YYYARFHFWYKNLF-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 1
- 108010082126 Alanine transaminase Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 102000004266 Collagen Type IV Human genes 0.000 description 1
- 108010042086 Collagen Type IV Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010001202 Cytochrome P-450 CYP2E1 Proteins 0.000 description 1
- 102100024889 Cytochrome P450 2E1 Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 208000004930 Fatty Liver Diseases 0.000 description 1
- 206010019708 Hepatic steatosis Diseases 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- RZUBARUFLYGOGC-MTHOTQAESA-L acid fuchsin Chemical compound [Na+].[Na+].[O-]S(=O)(=O)C1=C(N)C(C)=CC(C(=C\2C=C(C(=[NH2+])C=C/2)S([O-])(=O)=O)\C=2C=C(C(N)=CC=2)S([O-])(=O)=O)=C1 RZUBARUFLYGOGC-MTHOTQAESA-L 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 210000000013 bile duct Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 208000030172 endocrine system disease Diseases 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 208000010706 fatty liver disease Diseases 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000007489 histopathology method Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 230000013190 lipid storage Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 210000001087 myotubule Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000414 obstructive effect Effects 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical class OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000037195 reproductive physiology Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 210000000352 storage cell Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 102000014898 transaminase activity proteins Human genes 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/02—Halogenated hydrocarbons
-
- 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
-
- 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
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- 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
- A23K20/20—Inorganic substances, e.g. oligoelements
-
- 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
- A23K20/24—Compounds of alkaline earth metals, e.g. magnesium
-
- 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
- A23K20/26—Compounds containing phosphorus
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Inorganic Chemistry (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Environmental Sciences (AREA)
- Dermatology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Birds (AREA)
- Medicinal Preparation (AREA)
- Fodder In General (AREA)
Abstract
本发明提供了一种腹腔注射法建立恒河猴肝纤维化模型的方法，包括以下步骤：选取2‑3岁，体重为5‑9kg的雄性恒河猴作为模型动物，造模过程中喂养高脂饲料，饮用体积浓度为9％‑11％的酒精水溶液，并且在造模过程中向每只恒河猴注射CCl4注射液，每周注射2次，每次注射量为0.8‑1.5mL/kg，注射方式为腹腔注射，持续20‑30周，造模成功。本发明动物模型的建立，其方法简单，且安全可靠，建模时间短，成功率高，重复性好，动物耐受性好，死亡率低，可以用来研究慢性肝病的发生机制、致病因素和治疗手段。
Description
技术领域
本发明属于动物模型构建技术领域，具体涉及一种腹腔注射法建立恒河猴肝纤维化模型的方法。
背景技术
慢性肝病是危害人群健康的重大疾病。我国有上亿人口携带肝炎病毒，在此基础上，环境污染、粮食霉变、泛用食品添加剂以及酒精性和非酒精性过度饮食等因素相互叠加，导致炎症反应、脂肪肝、肝纤维化、其中每年有数百万人演变成肝硬化和肝癌，造成巨大的社会和家庭负担。目前传统的临床药物治疗不能改善终末期肝病的预后，而全球性供体器官的严重短缺，供需矛盾(供:需/1:200)的日益突出，使大多数患者只能在等待的过程中死亡。因此在供体器官严重短缺与终末期肝病病人治疗需求矛盾日益突出的今天，如何拓展新的器官源、或开辟新的替代疗法是我们当前面临的挑战和亟待解决的问题。因此探讨慢性肝病的发病机制、致病因素、治疗手段是根本解决慢性肝病的有效途径，然而理想的动物模型又是其中的关键。
目前应用比较广泛的肝纤维化动物模型有化学性肝纤维化模型、酒精性肝纤维化模型、免疫性肝纤维化模型、胆管阻塞性肝纤维化模型和复合因素性肝纤维化模型，而鼠类及其他常用小动物模型是很成熟和方便的动物模型，但是由于鼠类及其它小动物与人类之间存在很大的遗传背景和生理差距，其构建的模型很难直接与人类的疾病相对应。目前，有实验证实利用一些小鼠模型得到关于疾病机理的研究和人类同样的疾病机理并不相同。灵长类动物的应用始自20世纪初，直至1960年后才用于普通研究机构。这是因为，灵长类动物在亲缘关系上和人类最接近,在组织结构、免疫、生理和代谢等方面与人类高度近似，与人类的遗传基因有75％～98.5％的同源性，是研究人类健康和疾病的理想动物模型，其应用价值远超过其他种属的动物。近年来实验猴动物模型研究广泛被应用于传染病、内分泌疾病、心血管疾病、老年性疾病、生殖生理等病理研究，为更清楚的揭示人类疾病致病机理以及筛选合适的治疗手段和药物提供了很好的研究平台。选择动物模型是关键，大动物实验是嫁接转化医学(from the bench to bedside)之间的桥梁。因此，寻找一种方法简单，成功率高，重复性好且动物耐受性好，死亡率低的建模方法是亟待解决的问题。
发明内容
针对现有技术中存在的上述问题，本发明提供一种腹腔注射法建立恒河猴肝纤维化模型的方法，其造模时间短、造模成功率和造模效率高，且动物耐受性好、死亡率低。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
一种腹腔注射法建立恒河猴肝纤维化模型的方法，包括以下步骤：选取2-3岁，体重为5-9kg的雄性恒河猴作为模型动物，造模过程中所用饲料为高脂饲料，体积浓度为9％-11％的酒精水溶液作为唯一饮用水，并且在造模过程中向每只恒河猴注射CCl4注射液，每周注射2次，每次注射量为0.8-1.5mL/kg，注射方式为腹腔注射，持续20-30周，造模成功。
进一步地，高脂饲料包括以下重量百分数的组分：粗蛋白16％-18％、粗脂肪21％-23％、水9.5％-11.5％、灰分6.5％-7.0％、粗纤维3％-4％、钙0.9％-1.0％、磷0.7％-0.8％、胆固醇0.3％-0.4％。
进一步地，高脂饲料包括以下重量百分数的组分：粗蛋白16.78％、粗脂肪21.35％、水10％、灰分6.8％、粗纤维3.2％、钙0.92％、磷0.71％、胆固醇0.32％。
进一步地，酒精水溶液中酒精的体积浓度为10％。
进一步地，CCl4注射液通过以下方法制备得到：将CCl4原液加入橄榄油中，使CCl4浓度为400mL/L，用紫外线照射灭菌30分钟，制得。
进一步地，每次注射CCl4注射液的量为1.0mL/kg。
本发明提供的一种腹腔注射法建立恒河猴肝纤维化模型的方法，具有以下有益效果：本发明动物模型的建立，其方法简单，且安全可靠，建模时间短，成功率高，可以用来研究肝病的发生机制。
本发明中提供的特定的高脂饲料可为恒河猴提供营养，再饮用特定浓度的酒精水溶液，酒精水溶液浓度不需要逐次增加，其过程简单，然后再注射CCl4注射液，在给药20次后肝脏基础结构出现异常，炎症反应严重，给药40次后可见窦周及汇管区纤维化，肝内不完全纤维隔形成，给药60次后已为中度肝纤维化，造模时间短，效率高。
在制备CCl4注射液时采用橄榄油作为CCl4原液的溶剂，其相容性好，利于注射，同时还有利于CCl4损伤质膜，破坏肝细胞的膜性结构和功能，造成肝细胞变性坏死，同时促使肝纤维化；此外，橄榄油也不会对恒河猴造成伤害。
附图说明
图1为腹腔给药组肝组织穿刺样本染色结果图。
具体实施方式
实验原理：CCl4是最广泛使用的化学性肝毒物，CCl4在肝细胞内质网中经肝内的细胞色素2E1(CYP2E1)激活后代谢产生的自由基可直接损伤质膜，启动脂质过氧化作用，破坏肝细胞的膜性结构和功能，造成肝细胞变性坏死，同时静止的贮脂细胞活化，释放Ⅳ型胶原酶，降解Ⅳ型胶原，合成分泌Ⅰ型胶原，促使肝纤维化，整个过程炎症和坏死常先于纤维化。
实施例1
选取2-3岁，体重为5-9kg的雄性恒河猴10只作为模型动物，在造模过程中，起初是用常规饲料喂养15天，饮用水为正常的自来水，15天后将常规饲料改为高脂饲料，饮用水为体积浓度为10％的酒精水溶液(无水乙醇兑纯净水制得)，每周给每只恒河猴注射两次CCl4注射液，每次注射量为1.0mL/kg，注射方式为腹腔注射，在注射20次(10周)后，病理活检提示脂肪样变；持续30周，恒河猴表现为中度纤维化症状。
其中，高脂饲料包括以下重量百分数的组分：粗蛋白16.78％、粗脂肪21.35％、水10％、灰分6.8％、粗纤维3.2％、钙0.92％、磷0.71％、胆固醇0.32％。
10％的酒精水溶液是将无水乙醇与纯净水按体积比为1:9混合，制得。
CCl4注射液通过以下方法制备得到：将CCl4原液加入橄榄油中，使CCl4浓度为400mL/L，用紫外线照射灭菌30分钟，制得。
需注意的是，在造模过程中，若模型动物体重在一周内下降1kg左右，或者有任何注射药物后的不良反应或其他可能威胁实验动物安全性的情况时，需停止实验。
模型动物每20次给药后，行腹部彩色多普勒超声判断肝纤维化情况，并在彩色多普勒超声下行穿刺取样，所得样本行HE、Masson、天狼星红染色判断肝纤维化情况。
实验例 恒河猴肝损伤诊断模式评价方法和结果
1、血清生化标志物检测
血清生化标志物主要包括谷丙转氨酶(ALT)、谷草转氨酶(AST)、白蛋白(ALB)、胆红素(BIL)、血清总胆固醇(TCH)、甘油三脂(TG)、凝血时间(PT和肌酐(Cr)。
造模在10周和20周后，实验恒河猴禁食12h，然后注射舒泰0.5mL进行肌肉麻醉，在后肢静脉采血5mL，3000r/min离心15min，分离血清，测试血清中生化标志物含量，发现各标志物含量与对照组差异显著(P＜0.05)；对照组为在整个过程中喂养常规饲料，饮用自来水。
2、病理组织学分析过程：
注射舒泰0.5mL进行肌肉麻醉，取仰卧位，在在Supersonic彩色多普勒超声诊断仪引导下，采用BARD全自动活检穿刺枪(MG15-22，美国)获取肝组织样本，然后进行如下试验：
(1)组织石蜡包埋切片实验
取材：新鲜组织固定于4％多聚甲醛24h以上，将组织从固定液取出在通风橱内用手术刀将目的部位组织修平整，将修切好的组织和对应的标签放于脱水盒内。
脱水：将脱水盒放进吊篮里，置于脱水机内依次按照以下梯度酒精及程序进行脱水：75％酒精4h-85％酒精2h-90％酒精2h-95％酒精1h-无水乙醇30min-无水乙醇30min-醇苯5-10min-二甲苯5-10min-二甲苯5-10min-蜡1h-蜡1h-蜡1h。
包埋：将浸好蜡的组织于包埋机内进行包埋。先将融化的蜡放入包埋框，待蜡凝固之前将组织从脱水盒内取出按照包埋面的要求放入包埋框并贴上对应的标签，于-20°冻台冷却，蜡凝固后将蜡取出按照包埋面的要求放入包埋框并贴上对应的标签，于-20°冻台冷却，蜡凝固后将蜡块从包埋框中取出并修整蜡块。
切片：将修整好的蜡块置于石蜡切片机上切片，片厚4μm，切片漂浮于摊片机40℃温水上将组织展平，用载玻片将组织捞起，并放进60℃烘箱内烤片，待水烤干蜡烤化后取出常温保存备用。
(2)HE染色
石蜡切片脱蜡至水洗：依次将切片放入二甲苯20min-二甲苯20min-无水乙醇10min-无水乙醇10min-95％酒精5min-90％酒精5min-80％酒精5min-70％酒精5min-蒸馏水洗。
苏木素染细胞核：切片入Harris苏木素染3-8min，自来水洗，1％的盐酸酒精分化数秒，自来水冲洗，0.6％氨水返蓝，流水冲洗。
伊红染细胞质：切片入伊红染液中染色1-3min。
脱水封片：将切片依次放入95％酒精5min-95％酒精5min-无水乙醇5min-无水乙醇5min-二甲苯5min-二甲苯5min中脱水透明，将切片从二甲苯拿出来稍晾干，中性树胶封片。显微镜镜检，图像采集分析。
染色结果：细胞核蓝色，细胞质红色。
(3)Masson染色
石蜡切片脱蜡至水洗：依次将切片放入二甲苯20min-二甲苯20min-无水乙醇10min-无水乙醇10min-95％酒精5min-90％酒精5min-80％酒精5min-70％酒精5min-蒸馏水洗。
苏木素染细胞核：masson染色试剂盒内Weigert氏铁苏木素染5min，自来水洗，1％的盐酸酒精分化数秒，自来水冲洗，流水冲洗数分钟返蓝。
丽春红染色：masson染色试剂盒内丽春红酸性品红液染5-10min，蒸馏水快速漂洗。
磷钼酸处理：masson染色试剂盒内磷钼酸水溶液处理约3-5min。
苯胺蓝染色：不用水洗，直接用masson染色试剂盒内苯胺蓝液复染5min。
分化：1％冰醋酸处理1min。
脱水封片：将切片依次放入95％酒精5min-95％酒精5min-无水乙醇5min-无水乙醇5min-二甲苯5min-二甲苯5min中脱水透明，将切片从二甲苯拿出来稍晾干，中性树胶封片。显微镜镜检，图像采集分析。
染色结果：胶原纤维、粘液、软骨呈蓝色；肌纤维、纤维素和红细胞呈红色；细胞核呈蓝黑色。
(4)天狼猩红染色
石蜡切片脱蜡至水洗：依次将切片放入二甲苯20min-二甲苯20min-无水乙醇10min-无水乙醇10min-95％酒精5min-90％酒精5min-80％酒精5min-70％酒精5min-蒸馏水洗。
天狼猩红染色液染色：切片入饱和苦味酸天狼猩红染色液内染色8min。
漂洗：切片入无水酒精漂洗数分钟，显微镜下观察满意为止。
封片：切片于60℃烤箱烤干后于二甲苯透明5min，中性树胶封片。显微镜镜检，图像采集分析。
染色结果：光学显微镜下胶原纤维红色，背景黄色。
腹腔给药组肝组织穿刺样本染色结果见图1。
由图1可知，给药前，恒河猴肝脏组织，结构正常，细胞排列整齐，狄氏间隙明显。给药20次后，腹腔给药组肝脏基础结构出现异常，炎症反应严重，肝细胞内可见大小不一的空泡，部分细胞核偏向细胞一侧，肝脏呈脂肪样变趋势。给药40次后，肝脏细胞排列紊乱，中央静脉缺如，肝小叶消失，炎症反应严重，可见窦周及汇管区纤维化，肝内不完全纤维隔形成；Laennec肝穿刺组织纤维化分级评分2分，提示已有肝纤维化形成；给药60次后，可见肝内纤维隔长度增加，间隔增粗；腹腔给药组Laennec评分3分提示中度肝纤维化。Laennec肝穿刺组织纤维化分级评分标准见表1。
表1 Laennec肝穿刺组织纤维化分级评分标准
Claims (4)
1.一种腹腔注射法建立恒河猴肝纤维化模型的方法，其特征在于，包括以下步骤：选取2-3岁，体重为5-9kg的雄性恒河猴作为模型动物，造模过程中喂养高脂饲料，饮用体积浓度为9％-11％的酒精水溶液，并且在造模过程中向每只恒河猴注射CCl4注射液，每周注射2次，每次注射量为1.0mL/kg，注射方式为腹腔注射，持续10周后，肝脏呈脂肪样变趋势，持续20-30周，表现为中度肝纤维化，造模成功；高脂饲料包括以下重量百分数的组分：粗蛋白16％-18％、粗脂肪21％-23％、水9.5％-11.5％、灰分6.5％-7.0％、粗纤维3％-4％、钙0.9％-1.0％、磷0.7％-0.8％、胆固醇0.3％-0.4％。
2.根据权利要求1所述的腹腔注射法建立恒河猴肝纤维化模型的方法，其特征在于，高脂饲料包括以下重量百分数的组分：粗蛋白16.78％、粗脂肪21.35％、水10％、灰分6.8％、粗纤维3.2％、钙0.92％、磷0.71％、胆固醇0.32％。
3.根据权利要求1所述的腹腔注射法建立恒河猴肝纤维化模型的方法，其特征在于，酒精水溶液中酒精的体积浓度为10％。
4.根据权利要求1-3任一项所述的腹腔注射法建立恒河猴肝纤维化模型的方法，其特征在于，CCl4注射液通过以下方法制备得到：将CCl4原液加入橄榄油中，使CCl4浓度为400mL/L，用紫外线照射灭菌30分钟，制得。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710910648.6A CN107693507B (zh) | 2017-09-29 | 2017-09-29 | 一种腹腔注射法建立恒河猴肝纤维化模型的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710910648.6A CN107693507B (zh) | 2017-09-29 | 2017-09-29 | 一种腹腔注射法建立恒河猴肝纤维化模型的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107693507A CN107693507A (zh) | 2018-02-16 |
| CN107693507B true CN107693507B (zh) | 2020-07-31 |
Family
ID=61175749
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710910648.6A Active CN107693507B (zh) | 2017-09-29 | 2017-09-29 | 一种腹腔注射法建立恒河猴肝纤维化模型的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107693507B (zh) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111631187B (zh) * | 2019-03-01 | 2022-04-22 | 广西中医药大学 | 快速诱导肝纤维化动物模型的方法 |
| CN110278914A (zh) * | 2019-06-10 | 2019-09-27 | 漳州片仔癀药业股份有限公司 | 一种酒精性肝纤维化和炎症动物模型及其构建方法和用途 |
| CN110926911A (zh) * | 2019-12-23 | 2020-03-27 | 苏州堪赛尔生物技术有限公司 | 马松染色试剂盒及其染色方法 |
| CN111812314B (zh) * | 2020-07-09 | 2023-03-10 | 嘉兴市第二医院 | 一种完全弗氏佐剂诱导的冻结肩动物模型建立方法 |
| CN112056275B (zh) * | 2020-09-27 | 2022-05-10 | 澎立生物医药技术（上海）有限公司 | 一种肠粘膜炎动物模型及其构建方法和应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103006690A (zh) * | 2012-12-25 | 2013-04-03 | 广西玮美生物科技有限公司 | 食蟹猴肝损伤造模方法 |
| CN106389394A (zh) * | 2016-08-31 | 2017-02-15 | 广西防城港常春生物技术开发有限公司 | 一种食蟹猴酒精性肝病模型的构建方法 |
-
2017
- 2017-09-29 CN CN201710910648.6A patent/CN107693507B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103006690A (zh) * | 2012-12-25 | 2013-04-03 | 广西玮美生物科技有限公司 | 食蟹猴肝损伤造模方法 |
| CN106389394A (zh) * | 2016-08-31 | 2017-02-15 | 广西防城港常春生物技术开发有限公司 | 一种食蟹猴酒精性肝病模型的构建方法 |
Non-Patent Citations (2)
| Title |
|---|
| Establishment of a liver fibrosis model in cynomolgus monkeys;Ke Ding et al.;《Experimental and Toxicologic Pathology》;20141231;第66卷;257-261 * |
| Ke Ding et al..Establishment of a liver fibrosis model in cynomolgus monkeys.《Experimental and Toxicologic Pathology》.2014,第66卷257-261. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107693507A (zh) | 2018-02-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107693507B (zh) | 一种腹腔注射法建立恒河猴肝纤维化模型的方法 | |
| LU500561B1 (en) | In vitro construction method and use of liver organoids | |
| Yoshida et al. | Interleukin-1 alpha, interleukin-1 beta, and tumor necrosis factor gene expression in endotoxin-induced uveitis. | |
| Needham et al. | Physico-chemical experiments on the amphibian organizer | |
| CN104173365B (zh) | N6-(2-羟乙基)腺苷在抗肾衰竭中的用途 | |
| CN102106476A (zh) | 一种高脂饲料及在构建非酒精性脂肪肝动物模型中的应用 | |
| CN108348543A (zh) | 促进肝再生的方法 | |
| CN107519153B (zh) | 一种灌胃法建立恒河猴肝纤维化模型的方法 | |
| Al-Qudsi et al. | Effect of monosodium glutamate on chick embryo development | |
| Crocker et al. | Developmental defects of the kidney: a review of renal development and experimental studies of maldevelopment | |
| CN108586582B (zh) | 一种抗凝多肽fx18及其应用 | |
| CN108866179A (zh) | lncRNA-SCARNA10在制备肝纤维化检测试剂盒及治疗肝纤维化药物的用途 | |
| US20210308191A1 (en) | Drug Used for Treating Tissue Necrosis or for Improving Cardiac Function | |
| CN112206231A (zh) | 苦参碱衍生物在治疗非酒精性脂肪性肝炎中的用途 | |
| CN105213523B (zh) | 一种从黄连中提取有效成份的方法及该提取物的用途 | |
| CN112402431A (zh) | 帕立骨化醇在制备治疗胆汁淤积性肝损伤药物中的新用途 | |
| WO2021052450A1 (zh) | 反相色谱分离非人动物羊水中的活性成分 | |
| CN108640979B (zh) | 一种抗凝多肽th16及其应用 | |
| CN101554386B (zh) | 治疗肝病的药物组合物及其制备方法 | |
| CN108498546B (zh) | 一种预防或治疗溃疡性结肠炎癌变的中药组合物及应用 | |
| CN108379246A (zh) | 莽草酸在制备预防或治疗脱髓鞘疾病药物中的应用 | |
| CN104001156A (zh) | 真核肽链释放因子3b片段（eRF3b-36）在治疗肝损伤中的应用 | |
| Olariu-Jurca et al. | Study of morphological changes in sheep fluke. | |
| CN102600376B (zh) | 天麻及天麻提取物在制备治疗外周神经损伤再修复药物中的应用 | |
| CN105998101A (zh) | 维药斯米孜·欧提提取物在制备肝保护药物和保健品中的应用 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |