CN117065027A - Aibp作为靶点在制备治疗急性肝炎药物中的应用 - Google Patents
Aibp作为靶点在制备治疗急性肝炎药物中的应用 Download PDFInfo
- Publication number
- CN117065027A CN117065027A CN202310977273.0A CN202310977273A CN117065027A CN 117065027 A CN117065027 A CN 117065027A CN 202310977273 A CN202310977273 A CN 202310977273A CN 117065027 A CN117065027 A CN 117065027A
- Authority
- CN
- China
- Prior art keywords
- aibp
- cells
- target
- treatment
- acute liver
- 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
- 101000632037 Homo sapiens NAD(P)H-hydrate epimerase Proteins 0.000 title claims abstract description 117
- 102100028167 NAD(P)H-hydrate epimerase Human genes 0.000 title claims abstract description 116
- 239000003814 drug Substances 0.000 title claims abstract description 27
- 231100000354 acute hepatitis Toxicity 0.000 title claims abstract description 12
- 208000006454 hepatitis Diseases 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title description 4
- 206010067125 Liver injury Diseases 0.000 claims abstract description 53
- 231100000439 acute liver injury Toxicity 0.000 claims abstract description 44
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 16
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 25
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 14
- 230000002018 overexpression Effects 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 12
- 238000001890 transfection Methods 0.000 claims description 8
- 230000007423 decrease Effects 0.000 claims description 7
- 238000011813 knockout mouse model Methods 0.000 claims description 7
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims description 6
- 150000003384 small molecules Chemical class 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 230000014509 gene expression Effects 0.000 claims description 5
- 230000017854 proteolysis Effects 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 238000011740 C57BL/6 mouse Methods 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 claims description 3
- 238000006731 degradation reaction Methods 0.000 claims description 3
- 239000012091 fetal bovine serum Substances 0.000 claims description 3
- 230000002068 genetic effect Effects 0.000 claims description 3
- 230000003345 hyperglycaemic effect Effects 0.000 claims description 3
- 239000002502 liposome Substances 0.000 claims description 3
- 229950010131 puromycin Drugs 0.000 claims description 3
- 239000012096 transfection reagent Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 230000003247 decreasing effect Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 4
- 230000004913 activation Effects 0.000 abstract description 9
- 230000006378 damage Effects 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 6
- 230000005540 biological transmission Effects 0.000 abstract description 2
- 238000011161 development Methods 0.000 abstract description 2
- 230000008030 elimination Effects 0.000 abstract 1
- 238000003379 elimination reaction Methods 0.000 abstract 1
- 230000003647 oxidation Effects 0.000 abstract 1
- 238000007254 oxidation reaction Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 57
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 55
- 208000029618 autoimmune pulmonary alveolar proteinosis Diseases 0.000 description 50
- 241000699670 Mus sp. Species 0.000 description 23
- 230000002950 deficient Effects 0.000 description 14
- 101001109700 Homo sapiens Nuclear receptor subfamily 4 group A member 1 Proteins 0.000 description 12
- 102000043136 MAP kinase family Human genes 0.000 description 12
- 108091054455 MAP kinase family Proteins 0.000 description 12
- 102100022679 Nuclear receptor subfamily 4 group A member 1 Human genes 0.000 description 12
- 210000002966 serum Anatomy 0.000 description 11
- 210000004185 liver Anatomy 0.000 description 10
- 230000019491 signal transduction Effects 0.000 description 9
- 108020004999 messenger RNA Proteins 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 229940079593 drug Drugs 0.000 description 7
- 231100000753 hepatic injury Toxicity 0.000 description 6
- 230000001338 necrotic effect Effects 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 101000588302 Homo sapiens Nuclear factor erythroid 2-related factor 2 Proteins 0.000 description 5
- 102100031701 Nuclear factor erythroid 2-related factor 2 Human genes 0.000 description 5
- 238000003119 immunoblot Methods 0.000 description 5
- 238000000751 protein extraction Methods 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 4
- 108010082126 Alanine transaminase Proteins 0.000 description 4
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 4
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 4
- 208000027418 Wounds and injury Diseases 0.000 description 4
- 230000001640 apoptogenic effect Effects 0.000 description 4
- 210000003494 hepatocyte Anatomy 0.000 description 4
- 208000014674 injury Diseases 0.000 description 4
- 230000008506 pathogenesis Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 208000007788 Acute Liver Failure Diseases 0.000 description 3
- 206010000804 Acute hepatic failure Diseases 0.000 description 3
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 108020002144 NR4 subfamily Proteins 0.000 description 3
- 108091008638 NR4A Proteins 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 229960004308 acetylcysteine Drugs 0.000 description 3
- 231100000836 acute liver failure Toxicity 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 210000005228 liver tissue Anatomy 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 229960005489 paracetamol Drugs 0.000 description 3
- 239000003642 reactive oxygen metabolite Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102100033305 Glutathione S-transferase A3 Human genes 0.000 description 2
- 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 2
- 101000870590 Homo sapiens Glutathione S-transferase A3 Proteins 0.000 description 2
- 101000973778 Homo sapiens NAD(P)H dehydrogenase [quinone] 1 Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102100022365 NAD(P)H dehydrogenase [quinone] 1 Human genes 0.000 description 2
- 101710191474 NAD(P)H-hydrate epimerase Proteins 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 101000779377 Synechocystis sp. (strain PCC 6803 / Kazusa) Aldo/keto reductase slr0942 Proteins 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 206010019692 hepatic necrosis Diseases 0.000 description 2
- 231100000334 hepatotoxic Toxicity 0.000 description 2
- 230000003082 hepatotoxic effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 229940043355 kinase inhibitor Drugs 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 231100000149 liver necrosis Toxicity 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000017074 necrotic cell death Effects 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- -1 oxygen free radical Chemical class 0.000 description 2
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 2
- 239000003757 phosphotransferase inhibitor Substances 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108050008874 Annexin Proteins 0.000 description 1
- 102000000412 Annexin Human genes 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 208000008964 Chemical and Drug Induced Liver Injury Diseases 0.000 description 1
- 206010008635 Cholestasis Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 206010072268 Drug-induced liver injury Diseases 0.000 description 1
- 108010040476 FITC-annexin A5 Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229940122907 Phosphatase inhibitor Drugs 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000012984 biological imaging Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 230000001964 calcium overload Effects 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000010001 cellular homeostasis Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 231100000359 cholestasis Toxicity 0.000 description 1
- 230000007870 cholestasis Effects 0.000 description 1
- 208000035850 clinical syndrome Diseases 0.000 description 1
- 230000002498 deadly effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 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 1
- 230000005713 exacerbation Effects 0.000 description 1
- 230000003090 exacerbative effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 230000010224 hepatic metabolism Effects 0.000 description 1
- 230000028974 hepatocyte apoptotic process Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000001865 kupffer cell Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000004769 mitochondrial stress Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000000955 prescription drug Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000012342 propidium iodide staining Methods 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- 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
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- 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
- 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; AVICULTURE; APICULTURE; PISCICULTURE; 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
- A01K2267/035—Animal model for multifactorial diseases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Environmental Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pain & Pain Management (AREA)
- Epidemiology (AREA)
- Rheumatology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明提供AIBP作为靶点在制备治疗急性肝炎药物中的应用，涉及生物医学技术领域，本申请中提供了一种新的治疗靶点，本发明的治疗靶点是新发现的急性肝损伤相关基因。和目前的治疗药物主要抑制氧化应激不同，AIBP基因过表达后，主要抑制MAPK信号通路的激活，从而抑制损伤信号的传递，本申请提供了急性肝损伤中的重要基因，从而方便开发新的治疗方法，从而从根本上进一步提高急性肝损伤的治疗效果，或者实现根除急性肝损伤的目的。
Description
技术领域
本发明涉及生物医学技术领域，尤其涉及AIBP作为靶点在制备治疗急性肝炎药物中的应用。
背景技术
急性肝损伤是由各种因素造成的肝脏急性炎症性疾病，是一种由多种因素共同参与所引起的致命临床综合征，其特点是死亡率高，肝功能迅速恶化，无潜在的慢性肝病。其损伤的病因包括药物性、胆汁淤积性、病毒性等，其损伤机制可能与钙超载、大量氧自由基生成、细胞因子激活、Kupffer细胞激活及中性粒细胞聚集等相关。急性肝损伤或者急性肝衰竭作为一种临床急症，其治疗方案有限，目前有效的治疗方案仅有肝脏移植，但是因为肝脏供源紧缺，因此，随着急性肝损伤的快速进展，探索其他有效的治疗方法来提高急性肝损伤或者肝衰竭的总生存率已成为临床医生面临的挑战。
在急性肝损伤的病因中，由药物诱导的肝损伤在临床中占据最大比例。在我国，各种处方药和中药以及含有药用成分的膳食等均是引起急性肝损伤的病因。在药理上，多数的急性肝损伤由个体敏感性引起，个体差异大。据统计，由对乙酰氨基酚(APAP)过量引起的急性肝损伤是西方国家急性肝衰竭的主要病因。APAP诱导的急性肝损伤在动物模型中已经得以复现且具有剂量依赖性，使得对于急性肝损伤机制的研究有迹可循。
对于急性肝损伤的发病机制至今仍没有完全阐明，目前研究发现在肝损伤中发挥作用的主要细胞器为内质网和线粒体。肝毒性药物在经肝代谢后生成的肝毒性活性物质，是造成肝脏损伤的主要元凶。肝毒性物质具有氧化性，会耗竭还原性谷胱甘肽诱发线粒体应激，干扰氧化呼吸并造成细胞氧化损伤。同时肝毒性物质结合蛋白质后会造成蛋白变性，使其失去正常的生物学功能。在损伤下，内质网应激产生蛋白质的错误折叠，加剧了细胞稳态的失衡。
目前临床上唯一经过批准可以使用的治疗APAP导致的急性肝损伤的药物是N乙酰半胱氨酸(NAC)，然而，NAC治疗仅在过量后20小时内有效，对晚期肝损伤患者敏感性较差。针对JNK磷酸化的抑制剂在动物模型中被证实可以抑制急性肝损伤的进展，然而因为其抑制肝脏再生和增殖的副作用，目前仍然停留在实验阶段。其他可能的治疗药物正在被陆续报道，然而这些药物对于肝损伤的作用有限，没有明确的作用靶点。表明，我们对于急性肝损伤的发病机制认识还不足，依然有关键的治疗靶点等待去发现和开发。
急性肝损伤的治疗药物较少，并且有很大的局限性，而且在目前的治疗方法下，依然有较大一部分病人反应不佳或无反应。表明我们对于急性肝损伤的发病机制有待深入研究，寻找急性肝损伤中的未知重要基因，进而开发新的治疗方法，能从根本上进一步提高急性肝损伤的治疗效果，或实现根除急性肝损伤的目的。
发明内容
本发明的目的是为了解决现有技术中急性肝损伤的治疗药物较少，并且有很大的局限性，而且在目前的治疗方法下，依然有较大一部分病人反应不佳或无反应。表明我们对于急性肝损伤的发病机制有待深入研究，寻找急性肝损伤中的未知重要基因，进而开发新的治疗方法，能从根本上进一步提高急性肝损伤的治疗效果，或实现根除急性肝损伤的目的。
为了实现上述目的，本发明采用了如下技术方案：
AIBP作为靶点在制备治疗急性肝炎药物中的应用。
优选的，所述药物包括抑制AIBP表达下降或抑制AIBP蛋白降解的小分子。
优选的，所述药物包括过表达AIBP或重组AIBP蛋白的小分子。
优选的，所述药物通过抑制AIBP下降或过表达及重组蛋白，而实现抑制急性肝损伤的进展或治疗急性肝损伤。
优选的，所述抑制AIBP下降包括抑制AIBP的蛋白降解。
本申请还提供了一种AIBP过表达细胞系的建立方法，包含以下步骤：用于转染的细胞先种植于六孔板中，当细胞生长至70％密度时，将培养基更换为无血清基础培养基DMEM，用脂质体2000转染试剂转染细胞。转染六小时后，将DMEM跟换为仅含10％FBS的DMEM完全培养基，48小时后加入嘌呤霉素筛选细胞。
优选的，所述用于转染的细胞为使用DMEM高血糖培养基(含10％胎牛血清和1％抗生素)在37℃和5％CO2条件下培养。
本申请还提供了一种AIBP敲除小鼠模型的建立方法，包含以下步骤：
S1：设计了特异性识别第二外显子两端的两个sgRNA；
S2：将AIBP+/-小鼠杂交产生AIBP-/-小鼠，并在C57BL/6遗传背景上回交10代以上。
本申请中提供AIBP作为靶点在制备治疗急性肝炎药物中的应用，和目前的治疗药物主要抑制氧化应激不同，AIBP基因过表达后，主要抑制MAPK信号通路的激活，从而抑制损伤信号的传递，本申请提供了急性肝损伤中的重要基因，从而方便开发新的治疗方法，从而从根本上进一步提高急性肝损伤的治疗效果，或者实现根除急性肝损伤的目的。
附图说明
图1为本发明一实施方式中验证AIBP在急性肝损伤时下调对比图。(A)APAP诱导小鼠急性肝损伤时，小鼠肝脏中AIBP的蛋白质水平。(B)在不同的肝细胞系中，使用不同浓度的APAP诱导肝细胞损伤，不同细胞中的AIBP蛋白质水平。(C)健康体检者和急性肝损伤患者血清中AIBP的蛋白分泌量；
图2为本发明一实施方式中验证AIBP缺陷小鼠，在APAP诱导下的肝脏损伤加重的对比图。(A)AIBP野生型和AIBP缺陷小鼠，在APAP处理后谷丙转氨酶和谷草转氨酶的血清浓度。(B)AIBP野生型和AIBP缺陷小鼠，在APAP处理后的肝脏坏死面积。(C)AIBP野生型和AIBP缺陷小鼠，在APAP处理后肝脏凋亡和坏死标志物的蛋白质水平。(D)AIBP野生型和AIBP缺陷小鼠，在APAP处理后的生存率；
图3为本发明一实施方式中验证过表达AIBP缓解APAP诱导的细胞死亡的对比图。(A)过表达对照和AIBP的HepG2细胞，在APAP处理后，坏死细胞的荧光染色图片。(B)过表达对照和AIBP的HepG2细胞，在APAP处理后，凋亡细胞的百分比。(C)过表达对照和AIBP的HepG2细胞，对不同剂量APAP处理的敏感性；
图4为本发明一实施方式中验证AIBP维持NR4A1的基因表达。(A)在过表达对照和AIBP的HepG2细胞中，NR4A1的蛋白质水平。(B)在过表达对照和AIBP的HepG2细胞中，NR4A1的mRNA水平。(C)在过表达对照和AIBP的HepG2细胞中，APAP处理之后，NR4A1的蛋白质水平。(D)在过表达对照和AIBP的HepG2细胞中，APAP处理之后，NR4A1的mRNA水平；
图5为本发明一实施方式中AIBP抑制MAPK信号通路活化，抑制NR4A1的蛋白质降解。(A)在过表达对照和AIBP的HepG2细胞中，APAP处理后，MAPK信号通路的活化水平。(B)AIBP野生型和AIBP缺陷小鼠，在APAP处理后肝脏NR4A1的蛋白质和MAPK信号通路的活化水平。(C)HepG2细胞在使用MAPK信号通路的激酶抑制剂后，APAP处理后，NR4A1的蛋白质水平；
图6为本发明一实施方式中AIBP抑制APAP诱导的氧化应激。(A)在过表达对照和AIBP的HepG2细胞中，APAP处理后，转录因子NRF2的蛋白质水平。(B)在过表达对照和AIBP的HepG2细胞中，APAP处理后，NRF2的下游抗氧化靶基因的mRNA转录水平。(C)HepG2细胞在使用MAPK信号通路的激酶抑制剂后，APAP处理后，细胞产生的活性氧水平。
具体实施方式
以下结合具体实施例，对本发明作进一步地详细说明。
AIBP作为靶点在制备治疗急性肝炎药物中的应用。
所述AIBP的官方名NAD(P)HX epimerase(NAXE)，又称AIBP；基因编码的蛋白号：
NP_658985：1msrlrallgl gllvagsrvp riksqtiacr sgptwwgpqr lnsggrwdsevmastvvkyl
61sqeeaqavdq elfneyqfsv dqlmelagls cataiakayp ptsmsrsppt vlvicgpgnn
121ggdglvcarh lklfgyepti yypkrpnkpl ftalvtqcqk mdipflgemp aepmtidely
181elvvdaifgfsfkgdvrepfhsilsvlkgl tvpiasidip sgwdvekgna ggiqpdllis
241ltapkksatq ftgryhylgg rfvppalekk yqlnlppypd tecvyrlq
所述药物包括抑制AIBP表达下降或抑制AIBP蛋白降解的小分子；
在另一实施方式中，所述药物包括过表达AIBP或重组AIBP蛋白的小分子。
所述药物通过抑制AIBP下降(包括抑制AIBP的蛋白降解等)或过表达及重组蛋白，而实现抑制急性肝损伤的进展或治疗急性肝损伤。
以下结合具体实施例对本申请进行阐述：
准备实验：
A、AIBP过表达细胞系和AIBP敲除小鼠模型的建立
在一实施方式中，所用细胞使用DMEM高血糖培养基(含10％胎牛血清和1％抗生素)在37℃和5％CO2条件下培养。
用于转染的细胞事先种植于六孔板中，当细胞生长至70％密度时，将培养基更换为无血清基础培养基DMEM，用脂质体2000转染试剂转染细胞。转染六小时后，将DMEM跟换为仅含10％FBS的DMEM完全培养基，48小时后加入嘌呤霉素筛选细胞。
为了获得AIBP敲除小鼠，在一实施方式中，设计了特异性识别第二外显子两端的两个sgRNA,AIBP基因敲除小鼠的制备程序由上海模式生物中心有限公司完成。将AIBP+/-小鼠杂交产生AIBP-/-小鼠，并在C57BL/6遗传背景上回交10代以上。这些老鼠被饲养在南通大学实验动物科学研究所，所有研究和程序均经南通大学动物研究委员会批准。
B、急性肝损伤小鼠模型的建立
在一实施方式中，将APAP(对乙酰氨基酚)溶解在温磷酸盐缓冲盐水中，待充分溶解后冷却至37℃以250mg/kg的剂量腹腔注射。在APAP处理后的6h、12h、24h眼球取血备用，腹腔注射戊巴比妥深度麻醉后颈椎脱臼处死小鼠，取肝备用。
实施例1：AIBP在急性肝损伤时下调
步骤一：蛋白质提取和免疫印迹验证：
APAP诱导小鼠肝组织和细胞使用含有1％磷酸酶抑制剂和2％蛋白酶抑制剂的RIPA裂解缓冲液裂解匀浆，收集裂解液于4℃以12000rpm离心10分钟后收集上清。加入5x装载缓冲液，混匀后100℃煮沸5分钟获得蛋白质样品。蛋白质于SDS-PAGE分离后，电转到PVDF膜上。然后将PVDF膜用含5％脱脂牛奶的TBST室温封闭2h，TBS清洗5min后与稀释后的一抗孵育过夜。TBST洗涤10分钟三次后，与特异性二抗室温孵育2h，TBST洗涤三次后使用增强发光剂以生物成像系统观察条带。GAPDH作为蛋白质内参被使用。
请参阅图1中A和B，其中图1A为APAP诱导小鼠急性肝损伤时，小鼠肝脏中AIBP的蛋白质水平；图1B为在不同的肝细胞系中，使用不同浓度的APAP诱导肝细胞损伤，不同细胞中的AIBP蛋白质水平。可以看出与对比组相比，AIBP在急性肝损伤时下调明显。
步骤二：血清Elisa测定
首先用碳酸氢盐缓冲液稀释抗体，在反应孔中加入100ul，4℃过夜。次日弃去孔内溶液，用洗涤液冲洗三次。取100ul待测样品(健康体检者和急性肝损伤患者的血清)置于反应孔中，37℃孵育1小时，然后洗涤。于各反应孔中加入新鲜稀释的酶标抗体100ul，37℃孵育1小时，然后洗涤。于各反应孔中加入临时配置的TMB底物溶液100ul，37℃孵育30分钟。最后，于各反应孔中加入2M硫酸50ul终止反应。终止反应后，于酶标仪检测450nm处的OD值，换算血清样本中的抗原浓度。
请参阅图1C，图1C为健康体检者和急性肝损伤患者血清中AIBP的蛋白分泌量。可以看出，急性肝损伤患者血清中AIBP蛋白分泌量明显小于健康体检者。
实施例2：AIBP缺陷小鼠，在APAP诱导下的肝脏损伤加重
步骤一：血清Elisa测定
本实施例中步骤一与实施例1中步骤二相似，其不同之处在于：本申请中的样品为AIBP野生型和AIBP缺陷小鼠，在APAP处理后谷丙转氨酶和谷草转氨酶的血清；
请参阅图2A，图2A为AIBP野生型和AIBP缺陷小鼠在APAP处理后，不同时间节点血清中谷丙转氨酶和谷草转氨酶的分泌量。可以看出，AIBP缺陷小鼠血清中谷丙转氨酶和谷草转氨酶的分泌量明显高于野生型。
步骤二：苏木素-伊红染色：
将肝脏组织切块，浸在4％的多聚甲醛中24小时，第二天取出，进行酒精梯度脱水，石蜡包埋。组织块切成5um厚的切片，切片脱蜡、水化，苏木精染色30min，伊红染色5s，将染好的切片酒精脱水、二甲苯透明、树脂封片后镜下观察。使用ImageJ对坏死面积进行定量。
结果请参阅图2B，在APAP处理后，AIBP野生型小鼠的肝脏坏死的面积明显小于AIBP缺失小鼠。
步骤三：蛋白质提取和免疫印迹
本实施例中步骤三与实施例1中步骤一基本相同，其不同之处在于：本实施例中所使用的样品为AIBP野生型和AIBP缺陷小鼠在APAP处理后的组织和细胞。
请参阅图2C，可以看出，在APAP处理后，AIBP缺陷小鼠与AIBP野生型小鼠相比，肝细胞凋亡和坏死水平加重。
步骤四：生存曲线对比图：
将对乙酰氨基酚(APAP)溶解在温磷酸盐缓冲盐水中，待充分溶解后冷却至37℃以500mg/kg的剂量分别腹腔注射于野生型和AIBP基因敲除鼠。药物注射后每日观察，记录各组小鼠死亡数量。使用GraphPad进行生存曲线绘制。
请参阅图2D，可以看出，本步骤中，AIBP基因敲除鼠的生存率明显低于野生型小鼠。
实施例3：验证过表达AIBP缓解APAP诱导的细胞死亡：
步骤一：坏死细胞染色
碘化丙啶可以穿透坏死细胞破损的细胞膜，嵌入双链DNA后释放红色荧光。细胞被诱导损伤后，用PBS温和清洗细胞，重新加入含1ug/ml碘化丙啶的细胞培养液。37℃避光孵育30分钟，PBS温和清洗后用荧光显微镜观察细胞。Hoechst33342(Beyotime,China)作为活细胞对照。
请参阅图3A，可以看出，过表达AIBP的HepG2细胞在APAP处理后，坏死细胞明显少于对照组。
步骤二：细胞凋亡检测
收集待测细胞样本制备单细胞悬液，加入5ul Annexin V-FITC和10ul碘化丙啶染色液，混匀后室温避光孵育20分钟，每5分钟重悬细胞以充分染色，PBS重悬后用流式细胞仪检测。使用FlowJo-10.6.2定量实验结果，Annexin V阳性细胞定义为早期凋亡细胞，Annexin V-PI双阳性细胞定义为晚期凋亡或坏死细胞。
请参阅图3B，过表达AIBP的HepG2细胞中在APAP处理后，凋亡细胞的百分比少于对照组。
步骤三：细胞活力测定
将处于对数生长期的AIBP过表达和对照HepG2细胞消化后制备单细胞悬液，以5000个每孔接种于96孔板中。待细胞贴壁后，将不同浓度的APAP加入培养基中处理细胞。24小时后，于450nm处检测OD值。最后根据与空白对照组的OD值比例，计算细胞活力百分比。
请参阅图3C，过表达AIBP抑制了随着APAP药物剂量增加所诱导的细胞死亡。
实施例4：AIBP维持NR4A1的基因表达
步骤一：蛋白质提取和免疫印迹
本步骤与实施例1中步骤一基本类似，其不同之处在于：本步骤中用于验证NR4A1的蛋白质。
结果如图4A和图4C所示：过表达AIBP提高了NR4A1的蛋白质水平；过表达AIBP部分缓解了APAP诱导的NR4A1的蛋白质水平降低。
步骤二：RNA提取与实时荧光定量PCR：
用TRIZOL试剂提取肝组织和细胞总RNA,用生物分光光度计检测RNA含量。cDNA合成试剂盒用于RNA逆转录，然后使用SYBER GREEN试剂盒进行RT-qPCR。18s作为mRNA内参被使用。所用引物序列如下：
Gene Forwardprimer(5’→3’) Reverseprimer(5’→3’)
18s GGGGATTGGTTTTGACGTTTTTGCG AAGCATTAAATAAAGCGAATACATCCTTAT
NR4A1 AAAATCCCTGGCTTCATTGAG TTTAGATCGGTATGCCAGGCG
请参阅图4B和图4D，过表达AIBP促进NR4A1的mRNA转录；过表达AIBP部分缓解了APAP诱导的NR4A1的mRNA转录水平降低。
实施例5：AIBP抑制MAPK信号通路活化，抑制NR4A1的蛋白质降解。
蛋白质提取和免疫印迹：本实施例步骤与实施例1中步骤一步骤基本类似，其不同指出在于：本实施例中所采用的样品为过表达AIBP的HepG2细胞和空白组的HepG2细胞、AIBP野生型和AIBP缺陷小鼠在APAP处理后的组织、不同MAPK抑制剂预处理之后的HepG2细胞。
结果如图5所示：A过表达AIBP抑制APAP处理后诱导的MAPK信号通路的活化；B基因缺陷性AIBP小鼠在APAP处理后MAPK信号通路活化增强、NR4A1蛋白质水平降低；C不同的MAPK抑制剂不同程度的回复了APAP诱导的NR4A1蛋白质水平下调。
实施例6：AIBP抑制APAP诱导的氧化应激
步骤一：蛋白质提取和免疫印迹
本步骤与实施例1步骤基本相同，其不同之处在于：本步骤中用于验证NRF2的蛋白质。
结果如图6A所示：过表达AIBP增加了NRF2的蛋白质水平，并部分缓解了APAP诱导的NRF2的蛋白质水平降低。
步骤二：RNA提取与实时荧光定量PCR
本步骤与实施例4中步骤二基本类似。其不同之处在于：本步骤中所用引物序列如下：
结果如图6B所示：过表达AIBP促进NQO1、GSTA3、AKR1C的mRNA转录；过表达AIBP部分缓解了APAP诱导的NQO1、GSTA3、AKR1C的mRNA转录水平降低。
步骤三：活性氧检测
按照1：1000用DMEM稀释DCFH-DA探针(Beyotime,China),收集样本细胞重悬于稀释后的DCFH-DA探针溶液中，37℃培养箱孵育20分钟。每5分钟颠倒混匀一次，使探针和细胞充分接触。用DMEM洗涤细胞三次，去除未进入细胞的DCFH-DA。PBS重悬后，使用流式细胞仪(FACSCalibur；BD,USA)以488nm激发波长、525nm发射波长检测荧光信号。使用FlowJo-10.6.2定量实验结果。
结果如图6C所示:对MAPK信号通路的抑制，不同程度的抑制了APAP诱导的细胞活性氧生成。
Claims (8)
1.AIBP作为靶点在制备治疗急性肝炎药物中的应用。
2.根据权利要求1所述的AIBP作为靶点在制备治疗急性肝炎药物中的应用，其特征在于：所述药物包括抑制AIBP表达下降或抑制AIBP蛋白降解的小分子。
3.根据权利要求2所述的AIBP作为靶点在制备治疗急性肝炎药物中的应用，其特征在于：所述药物包括过表达AIBP或重组AIBP蛋白的小分子。
4.根据权利要求1所述的AIBP作为靶点在制备治疗急性肝炎药物中的应用，其特征在于：所述药物通过抑制AIBP下降或过表达及重组蛋白，而实现抑制急性肝损伤的进展或治疗急性肝损伤。
5.根据权利要求4所述的AIBP作为靶点在制备治疗急性肝炎药物中的应用，其特征在于：所述抑制AIBP下降包括抑制AIBP的蛋白降解。
6.一种AIBP过表达细胞系的建立方法，其特征在于：包含以下步骤：用于转染的细胞先种植于六孔板中，当细胞生长至70％密度时，将培养基更换为无血清基础培养基DMEM，用脂质体2000转染试剂转染细胞。转染六小时后，将DMEM跟换为仅含10％FBS的DMEM完全培养基，48小时后加入嘌呤霉素筛选细胞。
7.根据权利要求6所述的AIBP过表达细胞系的建立方法，其特征在于：所述用于转染的细胞为使用DMEM高血糖培养基(含10％胎牛血清和1％抗生素)在37℃和5％CO2条件下培养。
8.一种AIBP敲除小鼠模型的建立方法，其特征在于：包含以下步骤：
S1：设计了特异性识别第二外显子两端的两个sgRNA；
S2：将AIBP+/-小鼠杂交产生AIBP-/-小鼠，并在C57BL/6遗传背景上回交10代以上。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310977273.0A CN117065027A (zh) | 2023-08-04 | 2023-08-04 | Aibp作为靶点在制备治疗急性肝炎药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310977273.0A CN117065027A (zh) | 2023-08-04 | 2023-08-04 | Aibp作为靶点在制备治疗急性肝炎药物中的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117065027A true CN117065027A (zh) | 2023-11-17 |
Family
ID=88710663
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310977273.0A Pending CN117065027A (zh) | 2023-08-04 | 2023-08-04 | Aibp作为靶点在制备治疗急性肝炎药物中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117065027A (zh) |
-
2023
- 2023-08-04 CN CN202310977273.0A patent/CN117065027A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shen et al. | Uric acid aggravates myocardial ischemia–reperfusion injury via ROS/NLRP3 pyroptosis pathway | |
| JP6879931B2 (ja) | 併用療法用の医薬組成物 | |
| Dong et al. | Juglanin suppresses fibrosis and inflammation response caused by LPS in acute lung injury | |
| Wei et al. | Blocking mammalian target of rapamycin (mTOR) attenuates HIF-1α pathways engaged-vascular endothelial growth factor (VEGF) in diabetic retinopathy | |
| Zhang et al. | Irisin pretreatment protects kidneys against acute kidney injury induced by ischemia/reperfusion via upregulating the expression of uncoupling protein 2 | |
| Bai et al. | CDK5 promotes renal tubulointerstitial fibrosis in diabetic nephropathy via ERK1/2/PPARγ pathway | |
| Liu et al. | Discovery of a novel rice-derived peptide with significant anti-gout potency | |
| CN111358789A (zh) | Nsc228155在制备防治慢性肾纤维化药物中的用途 | |
| Shi et al. | Intestinal vitamin D receptor signaling ameliorates dextran sulfate sodium‐induced colitis by suppressing necroptosis of intestinal epithelial cells | |
| Hou et al. | Taurine transporter regulates adipogenic differentiation of human adipose-derived stem cells through affecting Wnt/β-catenin signaling pathway | |
| Tang et al. | Acinar cell-derived extracellular vesicle MiRNA-183-5p aggravates acute pancreatitis by promoting M1 macrophage polarization through downregulation of FoxO1 | |
| Jin et al. | 5-aminolevulinate and CHIL3/CHI3L1 treatment amid ischemia aids liver metabolism and reduces ischemia-reperfusion injury | |
| CN111671749A (zh) | 双香豆素在制备HBx蛋白稳定性抑制剂中的用途 | |
| CN117065027A (zh) | Aibp作为靶点在制备治疗急性肝炎药物中的应用 | |
| WO2022166839A1 (zh) | 银杏叶提取物在制备靶向衰老细胞、抑制肿瘤或延长寿命的药物中的应用 | |
| Liu et al. | Salvianolic acid B inhibits hepatic stellate cell activation and liver fibrosis by targeting PDGFRβ | |
| CN109172559B (zh) | 依他尼酸在制备治疗和预防肥胖及相关代谢性疾病药物中的应用 | |
| CN114432302A (zh) | 小分子sr9009在抗衰老以及减轻衰老引起的慢性炎症中的用途 | |
| Xu et al. | Tert-butyl hydroperoxide induces ferroptosis of bone mesenchymal stem cells by repressing the prominin2/BACH1/ROS axis | |
| CN116047066B (zh) | Sgk1作为靶点在制备诊断、预防、治疗冠状病毒所致疾病的产品中的应用 | |
| CN115287359B (zh) | Mcoln2在结直肠癌中的应用 | |
| CN117205225B (zh) | 栀子苷在制备治疗骨髓增殖性肿瘤药物中的应用 | |
| Yang et al. | HIF-1α Pathway Orchestration by LCN2: A Key Player in Hypoxia-Mediated Colitis Exacerbation | |
| Izquierdo-Fernandez | Role of Hypoxia Inducible Factors in Alveolar Macrophage Physiology | |
| Zhou et al. | GYY4137 ameliorates sepsis-induced cardiomyopathy via inhibiting NLRP3 Inflammasome activity and reduction of oxidative stress in the myocardium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |