CN114717195A - Cell model for screening exogenous compounds mediated by CYP3A7 and metabolized to toxicity, construction method and application thereof - Google Patents
Cell model for screening exogenous compounds mediated by CYP3A7 and metabolized to toxicity, construction method and application thereof Download PDFInfo
- Publication number
- CN114717195A CN114717195A CN202210472652.XA CN202210472652A CN114717195A CN 114717195 A CN114717195 A CN 114717195A CN 202210472652 A CN202210472652 A CN 202210472652A CN 114717195 A CN114717195 A CN 114717195A
- Authority
- CN
- China
- Prior art keywords
- cyp3a7
- screening
- mediated
- cell
- toxicity
- 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
- 150000001875 compounds Chemical class 0.000 title claims abstract description 60
- 101000745715 Homo sapiens Cytochrome P450 3A7 Proteins 0.000 title claims abstract description 49
- 231100000419 toxicity Toxicity 0.000 title claims abstract description 47
- 230000001988 toxicity Effects 0.000 title claims abstract description 47
- 238000012216 screening Methods 0.000 title claims abstract description 44
- 102100039203 Cytochrome P450 3A7 Human genes 0.000 title claims abstract description 42
- 230000001404 mediated effect Effects 0.000 title claims abstract description 31
- 238000010276 construction Methods 0.000 title claims abstract description 12
- 230000002503 metabolic effect Effects 0.000 claims abstract description 47
- 230000001605 fetal effect Effects 0.000 claims abstract description 18
- 231100000331 toxic Toxicity 0.000 claims abstract description 14
- 230000002588 toxic effect Effects 0.000 claims abstract description 14
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 13
- 230000000694 effects Effects 0.000 claims description 14
- 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 14
- 239000013612 plasmid Substances 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 9
- 239000012091 fetal bovine serum Substances 0.000 claims description 8
- 230000004060 metabolic process Effects 0.000 claims description 7
- 229950010131 puromycin Drugs 0.000 claims description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 6
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 claims description 5
- 230000003833 cell viability Effects 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 108091026890 Coding region Proteins 0.000 claims description 4
- 239000007640 basal medium Substances 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 3
- 229960005322 streptomycin Drugs 0.000 claims description 3
- 229930182555 Penicillin Natural products 0.000 claims description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 229940049954 penicillin Drugs 0.000 claims description 2
- 230000001413 cellular effect Effects 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 108010081668 Cytochrome P-450 CYP3A Proteins 0.000 abstract description 5
- 102000004328 Cytochrome P-450 CYP3A Human genes 0.000 abstract description 5
- 231100000041 toxicology testing Toxicity 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 78
- QVCMHGGNRFRMAD-XFGHUUIASA-N monocrotaline Chemical compound C1OC(=O)[C@](C)(O)[C@@](O)(C)[C@@H](C)C(=O)O[C@@H]2CCN3[C@@H]2C1=CC3 QVCMHGGNRFRMAD-XFGHUUIASA-N 0.000 description 31
- QPNKYNYIKKVVQB-UHFFFAOYSA-N crotaleschenine Natural products O1C(=O)C(C)C(C)C(C)(O)C(=O)OCC2=CCN3C2C1CC3 QPNKYNYIKKVVQB-UHFFFAOYSA-N 0.000 description 29
- QVCMHGGNRFRMAD-UHFFFAOYSA-N monocrotaline Natural products C1OC(=O)C(C)(O)C(O)(C)C(C)C(=O)OC2CCN3C2C1=CC3 QVCMHGGNRFRMAD-UHFFFAOYSA-N 0.000 description 29
- 241000700159 Rattus Species 0.000 description 28
- 102100023897 NADPH-cytochrome P450 reductase Human genes 0.000 description 20
- 101710197581 Ketoisovalerate oxidoreductase subunit VorC Proteins 0.000 description 19
- 101710147185 Light-dependent protochlorophyllide reductase Proteins 0.000 description 19
- 101710193909 Protochlorophyllide reductase, chloroplastic Proteins 0.000 description 19
- 101710109491 Pyruvate synthase subunit PorA Proteins 0.000 description 19
- 101710109487 Pyruvate synthase subunit PorB Proteins 0.000 description 19
- 101710109489 Pyruvate synthase subunit PorC Proteins 0.000 description 19
- 101710109484 Pyruvate synthase subunit PorD Proteins 0.000 description 19
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 101001112118 Homo sapiens NADPH-cytochrome P450 reductase Proteins 0.000 description 10
- 238000001514 detection method Methods 0.000 description 10
- 102000047583 human POR Human genes 0.000 description 10
- 229940079593 drug Drugs 0.000 description 9
- 239000003814 drug Substances 0.000 description 9
- 230000006651 lactation Effects 0.000 description 8
- 210000004185 liver Anatomy 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 230000001919 adrenal effect Effects 0.000 description 6
- 210000004100 adrenal gland Anatomy 0.000 description 6
- 238000011156 evaluation Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 229930002356 pyrrolizidine alkaloid Natural products 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 230000002440 hepatic effect Effects 0.000 description 5
- 102000055069 human CYP3A7 Human genes 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 241000700157 Rattus norvegicus Species 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 description 4
- 229940097277 hygromycin b Drugs 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 241000699802 Cricetulus griseus Species 0.000 description 3
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 3
- 206010070863 Toxicity to various agents Diseases 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000001493 electron microscopy Methods 0.000 description 3
- 210000003754 fetus Anatomy 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 210000005228 liver tissue Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000035935 pregnancy Effects 0.000 description 3
- ADRDEXBBJTUCND-UHFFFAOYSA-N pyrrolizidine Chemical class C1CCN2CCCC21 ADRDEXBBJTUCND-UHFFFAOYSA-N 0.000 description 3
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 2
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 2
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 2
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 2
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 2
- 231100000070 MTS assay Toxicity 0.000 description 2
- 238000000719 MTS assay Methods 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- UCTWMZQNUQWSLP-UHFFFAOYSA-N adrenaline Chemical compound CNCC(O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-UHFFFAOYSA-N 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012761 co-transfection Methods 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 230000006806 disease prevention Effects 0.000 description 2
- 238000000635 electron micrograph Methods 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 238000013537 high throughput screening Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 229960002725 isoflurane Drugs 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 230000003908 liver function Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 229930195730 Aflatoxin Natural products 0.000 description 1
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 1
- 101100441875 Arabidopsis thaliana CYP37 gene Proteins 0.000 description 1
- FMMWHPNWAFZXNH-UHFFFAOYSA-N Benz[a]pyrene Chemical compound C1=C2C3=CC=CC=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 FMMWHPNWAFZXNH-UHFFFAOYSA-N 0.000 description 1
- 101150061952 CYP3A7 gene Proteins 0.000 description 1
- 206010073513 Exposure during pregnancy Diseases 0.000 description 1
- 101000577129 Homo sapiens Monocarboxylate transporter 5 Proteins 0.000 description 1
- 101000577131 Homo sapiens Monocarboxylate transporter 6 Proteins 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 102100025273 Monocarboxylate transporter 5 Human genes 0.000 description 1
- 108010045510 NADPH-Ferrihemoprotein Reductase Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 241000519995 Stachys sylvatica Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000005409 aflatoxin Substances 0.000 description 1
- 210000001557 animal structure Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000007673 developmental toxicity Effects 0.000 description 1
- 231100000415 developmental toxicity Toxicity 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000036267 drug metabolism Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229960005489 paracetamol Drugs 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000013513 substance screening Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
- 239000002676 xenobiotic agent Substances 0.000 description 1
Images
Classifications
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle 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
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
- C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
- C12N9/0042—NADPH-cytochrome P450 reductase (1.6.2.4)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
- C12Y106/02—Oxidoreductases acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
- C12Y106/02004—NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
- C12Y114/14001—Unspecific monooxygenase (1.14.14.1)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
-
- 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
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- 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
- C12N2510/00—Genetically modified 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Reproductive Health (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a cell model for screening CYP3A7 mediated metabolic toxic exogenous compounds, and a construction method and application thereof. The cell model for screening the exogenous compound mediated by CYP3A7 for metabolic toxicity is characterized in that: using Flp-InTMCHO as target cell, and transfecting the target cell with the DNA sequences containing CYP3A7 and POR to obtain the cell model for screening the fetal metabolic toxic exogenous compound. The model established by the invention is novel, reliable, simple and convenient, and can greatly improve the sensitivity of the exogenous compound metabolic toxicity screening cell model mediated by CYP3A 7.
Description
Technical Field
The invention relates to the technical field of drug toxicology, in particular to a cell model for screening exogenous compounds mediated by CYP3A7 metabolic toxicity, and a construction method and application thereof.
Background
The metabolic toxicity of the exogenous compound refers to that the exogenous compound has no toxicity or low toxicity, and generates intermediate metabolites or byproducts with stronger toxicity to damage the organism after metabolism of the organism, which is called metabolic toxicity (or metabolic damage), and exogenous compounds such as aflatoxin, benzopyrene, acetaminophen and the like generate toxic and even carcinogenic metabolites through the metabolism of Cytochrome P450(Cytochrome P450, CYP450) super-family to play a toxic role. CYP is one of the main metabolic enzymes mediating the metabolic toxicity of exogenous compounds, CYP3A4 is the CYP subtype which has the highest expression level in adults and is also the most involved in the metabolism of exogenous compounds, and CYP3A7 is mainly expressed in fetal tissues. CYP3a7 is expressed predominantly in fetal tissues, and postnatal expression begins to decrease but remains the predominant expressed enzyme within 1 year of age. Determining drug doses in neonatal, infancy and childhood patients is complicated by the fact that there is no correlation between visceral organ size, enzymatic activity and body weight, which reflects age-related differences in drug metabolism.
At present, most of evaluation means for metabolic toxicity of the whole animal organ are used as measurement indexes of the developmental toxicity of the exogenous compound, but the evaluation means have the defects of large animal dosage, high requirements on a detection method, low sensitivity and the like, and an existing in-vitro evaluation system for the metabolic toxicity of the exogenous compound, such as a HepaRG or hepatoma cell line, is expensive in price and cannot be passed too high in passage cost, and the later evaluation means influence the detection repeatability of different laboratories at different periods due to the fact that the metabolic enzyme is low in basic expression and needs to transfect DNA sequences randomly inserted into CYP. In summary, the exogenous compound metabolic toxicity in vitro evaluation system has a large limitation, and has high sensitivity, low cost and good repeatability.
Flp-InTMThe CHO cell line, from Saimer Feishel scientific Inc. (Cat: R75807), contains a stably integrated FRT site at a transcriptionally active genomic locus. Construction of Flp-In containing the DNA sequence encoding CYP3A7TMExpression vector and Flp-In recombinase vector pOG44 Co-transfect Flp-InTMCHO, targeted integration of CYP3A7 expression vector into the same site in each cellAnd the cell model with uniform CYP3A7 expression level can be obtained at different periods or in different laboratories. It is not clear whether other similar metabolic toxicity evaluation cell model systems reported in the literature express POR.
Disclosure of Invention
One of the purposes of the invention is to provide a cell model for screening exogenous compounds mediated by CYP3A7 metabolic toxicity, and the used cells are Flp-InTMThe CHO cell line is from Saimer Feishel scientific Co. (Cat: R75807), is from Chinese hamster ovary, does not involve complicated ethical problems and can be passaged for many times, and a stably integrated FRT locus is contained at a transcriptionally active genomic locus, so that the CYP3A7 expression vector can be targeted and integrated into the same locus in each cell. After the POR is cotransfected, the sensitivity of the POR to exogenous compounds with metabolism toxicity mediated by CYP3A7 can be greatly improved. The model takes the cell activity as a detection index, and is a detection model with simple method, low cost, sensitivity and rapidness.
The second purpose of the invention is to provide a construction method of a cell model for screening exogenous compounds mediated by CYP3A7 metabolic toxicity, and the modeling method is simple and has strong repeatability.
The invention also aims to provide the application of the cell model for screening the exogenous compound with the metabolic toxicity mediated by CYP3A7 in the detection of the exogenous compound with the metabolic toxicity, the model detection process is rapid, the application range is wide, and the cell model can be used for high-throughput screening.
One of the purposes of the invention adopts the following technical scheme:
a cell model for screening exogenous compounds for CYP3a7 mediated metabolic toxicity, comprising: using Flp-InTMCHO as target cell, and transfecting the target cell with the DNA sequences containing CYP3A7 and POR to obtain the cell model for screening the fetal metabolic toxic exogenous compound.
Preferably, the cell model for screening exogenous compounds with CYP3A 7-mediated metabolic toxicity has two indexes, cell viability and half Inhibitory Concentration (IC)50)。
The second purpose of the invention is realized by adopting the following technical scheme:
a cell model construction method for screening exogenous compounds mediated by CYP3a7 for metabolic toxicity as described above, comprising: the method comprises the following steps:
s1: resuscitating Flp-In basal MediumTM-CHO cells, said basal medium containing 10% fetal bovine serum and 1% F12 of penicillin and streptomycin;
s2: constructing pcDNA5 plasmid containing CYP3A7 coding DNA sequence;
s3: constructing pCMV plasmid containing POR coding DNA sequence;
s4: stably transfecting the pcDNA5 plasmid containing the DNA sequence encoding CYP3A7 constructed In the step S2 into Flp-In after passage of the step S1TM-CHO, cultured in selective DMEM/F12-1 medium containing 10% fetal bovine serum and 500 μ g/ml hygromycin in F12;
s5: the pCMV plasmid containing the POR-encoding DNA sequence constructed In step S3 was stably transfected into Flp-In obtained In step S4TMCulturing in a screening DMEM/F12-2 culture medium in a CHO-CYP3A7 cell, wherein the screening DMEM/F12-2 culture medium contains 10% fetal bovine serum, 500 mu g/ml hygromycin and 50 mu g/ml puromycin F12, and obtaining the cell model of the screening CYP3A7 mediated metabolism toxic exogenous compounds.
The third technical scheme adopted by the invention for realizing the purpose is as follows:
use of a cell model for screening exogenous compounds for metabolic toxicity mediated by CYP3a7 as described above for screening exogenous compounds for metabolic toxicity.
Preferably, when the cell viability is reduced, the exogenous compound to be screened is suggested to have metabolic toxicity mediated by CYP3a 7.
Further, the IC of the foreign compound to be screened is determined according to the difference50The value can be compared with the metabolic toxicity of different exogenous compounds.
Compared with the prior art, the invention has the following beneficial technical benefits:
the invention discovers Flp-In for the first timeTMCHO deficient human cytochrome P450 Oxidoreductase (EC 1.6.2.4; NADPH-Cytochrome P450 Oxidoreductase; POR), which is the only coenzyme that transfers electrons to the CYP enzyme system to help perform CYP oxidation functions. The present invention proposes to stably transfect POR into Flp-In expressing CYP3A7TMIn CHO, the sensitivity of screening cell models through CYP3A7 mediated exogenous compound metabolic toxicity can be greatly improved. The method has the following specific advantages:
1. compared with other metabolic toxicity In-vitro screening systems, the cell model for screening the CYP3A7 mediated metabolic toxicity exogenous compound has the following advantages that: the cells are derived from Chinese hamster ovary, and the problem of complex ethics is not involved; no CYP3A7 basic expression and induction expression, CYP3A7 needs to be expressed in a recombinant mode, and the interference of the CYP3A7 expression of the cell to a screening result is avoided; thirdly, the CYP3A7 expression vector is integrated into the same site in each cell in a targeted manner, so that a cell model with uniform CYP3A7 expression is obtained, and the repeatability is good; the co-transfection POR greatly improves the sensitivity of the screening system; the expression traits of CYP3A7 and POR can be stably maintained under the action of hygromycin and puromycin, and the screening method is sensitive to the reaction of compounds mediated by CYP3A7 and metabolic toxicity and can be used for high-throughput screening.
2. The invention provides a cell model for screening CYP3A7 mediated metabolism toxic exogenous compounds to obtain cell activity and IC50The value is used as a detection index, and when the cell activity is reduced, the exogenous compound to be screened is prompted to have metabolic toxicity mediated by CYP3A7, and the method is simple, high in sensitivity and wide in application range. IC of exogenous compound to be screened according to different50The value can be compared with the metabolic toxicity of different exogenous compounds, and has important significance for rapidly screening the exogenous compounds mediated by CYP3A 7.
Drawings
FIG. 1 shows the construction of the DNA sequence Flp-In containing CYP3A7 code and POR code In the present inventionTMSchematic representation of the method of the CHO model.
FIG. 2 is a basic skeleton diagram of a tool vector used in the construction of a DNA sequence encoding CYP3A7 in example 1 of the present invention.
FIG. 3 is a diagram of the basic skeleton of the tool carrier used in the construction of DNA sequence containing POR code in example 1 of the present invention.
FIG. 4 is an immunoblot of protein-identified recombinant clones in example 1 of the present invention;
in fig. 4: vector is protein extracted from CHO cell expressing empty vector (vector), POR-3A7 is protein extracted from CHO cell transfected with human POR and CYP3A7 genome, POR is protein extracted from CHO cell transfected with human POR genome, and 3A7 is protein extracted from CHO cell transfected with human CYP3A7 genome.
FIG. 5 is a diagram showing the MTS assay cell viability of Rekaline (RTS) in example 1 of the present invention;
in fig. 5: after administration of the drug RTS, the inhibition was stronger on CHO-POR-CYP3A7 cells compared to CHO cells and CHO-CYP3A7 cells. The IC50 for drug RTS is lowest in CHO-POR-CYP3A7 cells as shown.
FIG. 6 is a MTS assay cytoactive map of Monocrotaline (MCT) in example 1 of the present invention;
in FIG. 6: after administration of the drug MCT, the inhibition was stronger on CHO-POR-CYP3A7 cells compared to CHO cells and CHO-CYP3A7 cells. As shown, the IC50 for drug MCT was lowest in CHO-POR-CYP3A7 cells.
FIG. 7 is a graph of the adrenal pathology in rats after intrauterine Pyrrolizidine Alkaloids (PAs) exposure in example 2 of the present invention;
in fig. 7: panel A shows a control of fetal Adrenaline hematoxylin-eosin staining in rats. Panel B is a pathological picture of fetal adrenohematoxylin-eosin staining in rats after RTS exposure. Panel C is a pathological picture of rat fetal hematoxylin-eosin staining after MCT exposure. Panel D and G are electron micrographs of fetal adrenal glands from rats. Panels E and H are electron micrographs of fetal adrenal glands after RTS exposure. Panels F and I are electron microscopy pathology of rat fetal adrenal glands after MCT exposure.
FIG. 8 is a pathological diagram of liver threo-xylogen-eosin staining of rats three weeks after exposure to Retroricine (RTS) in lactation in example 3 of the present invention.
FIG. 9 is a graph showing the relative indices of liver function in rats of three weeks after exposure to Retrocrine (RTS) in lactation in example 3 of the present invention;
in fig. 9: ALT activity in serum of rats three weeks after RTS exposure in lactation.
FIG. 10 is a pathological graph showing staining of hepatic soja-eosin in three-week-old rats after exposure to Monocrotaline (MCT) in example 3 of the present invention.
FIG. 11 is a graph showing the relative indices of liver function in rats at three weeks of age after exposure to Monocrotaline (MCT) in lactation in example 3 of the present invention;
in fig. 11: ALT activity in serum of rats three weeks after MCT exposure in lactation.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the accompanying drawings and specific embodiments.
Example 1: construction of CHO-POR-CYP3A7 cell model for in vitro fetal drug toxicity screening
1. Test cell
Flp-In used In this experimentTMCHO cells were purchased from seemer feishel scientific.
2. Experimental procedure
2.1 Co-transfection of Flp-In with the constructed pcDNA5 recombinant plasmid containing the full-length cDNA of human CYP3A7 and the helper plasmid pOG44TMCHO cells. The specific transfection steps are as follows:
1) CHO cells were maintained in F12 (Ham's) medium containing 10% FBS and 1% streptomycin after recovery and passage 3.
2) 24 hours before transfection, CHO cells were seeded in six-well plates, so that on the day of transfection CHO cells grew to 80-90% confluency.
3) Mu.g of POR-pcDNA5 plasmid (simultaneously, pcDNA5 empty vector without CYP3A7 gene cDNA was used in parallel as a negative control) and 5. mu.g of helper plasmid pOG44 were added to 250. mu.L of serum-free medium and mixed with 10. mu.L of P3000 transfection reagent, and the mixture was left at room temperature for 5 min.
4) mu.L of Lipo3000 transfection reagent was added to 250. mu.L of serum-free medium, mixed well, and then left at room temperature for 5 min.
5) Mixing the reagents of steps S2) and S3)Homogenizing, standing at room temperature for 10-15min, and mixing with Flp-In cultured In 1mL serum-free Ham' sF12 mediumTMCHO cells were co-incubated for 24 hours, after which the reaction was stopped by changing with 2mL of complete Ham's F12 medium containing 10% fetal bovine serum.
6) After 12 hours of recovery from complete medium, transfected cells were transferred from 6-well plates to 100mm diameter petri dishes.
7) After 12 hours of the transfer, hygromycin B (hygromycin B) was added to the medium to a final concentration of 500. mu.g/mL to screen positive clones.
8) After positive clones were screened out, single clones were picked and maintained in hygromycin B (hygromycin B) at a final concentration of 500. mu.g/mL.
2.2 the packaged lentivirus containing the full-length human POR cDNA was infected into CHO-CYP3A7 cells.
The specific infection steps are as follows:
1) CHO-CYP3A7 cells were seeded in six-well plates 24 hours prior to infection, allowing cells to grow to 80-90% confluency on the day of infection.
2) 500uL of virus fluid packed with human POR full-length cDNA (also run in parallel with no load of virus without POR full-length cDNA as negative control) was mixed with 2mL of Ham's F12 medium and polybrene (8mg/mL) was added to a final concentration of 8ug/mL (2mL +0.5 mL-2.5 mL, i.e., 2.5uL polybrene was added). After being mixed evenly, the mixture is centrifuged at 3000rpm for 1.5h at normal temperature (the protection and the pollution prevention are realized during centrifugation).
3) After 24h of infection, the infection was repeated once more.
4) After 48h of infection, the cells in each well of the six-well plate were transferred partially to a 100mm diameter culture dish, and the remaining cells were used to extract proteins and tested by western to determine whether the transfection was successful. After 12h of attachment, puromycin (puromycin) was added to the medium to a final concentration of 50. mu.g/mL to screen for positive clones.
5) After the cells in the dish have grown into single colonies (small white spots can be seen visually after aspirating most of the medium), the single colonies can be picked, optionally with filter paper or a small gun, into a 24-well plate and maintained on puromycin (puromycin) at a final concentration of 50. mu.g/mL.
6) After the cells in the 24-well plate grow up, the cells are transferred to a 6-well plate, and after the cells in the 6-well plate grow up, a part of the cells are collected to detect the expression of the POR protein and carry out activity detection. Cells with high expression and high activity are frozen.
Western blot detection of protein expression of CHO-POR-CYP3A7 cells
1) Extracting the constructed CHO-POR-CYP3A7 cell protein:
a) washing the cells in the six-well plate twice with pre-cooled PBS to remove residual culture medium; mu.L of cell lysate (containing 1% PMSF) was added to each well, the cells were scraped off with a cell scraper and then transferred to a 1.5mL EP tube, blown 5 times with a pipette to sufficiently lyse the cells, and left on ice for 5 minutes.
b) Centrifuge at 12000rpm for 5 minutes at 4 ℃.
c) The centrifuged supernatant was transferred to a new 0.5mL Eppendorf tube and stored at-80 ℃ until use.
2) BCA assay for sample protein concentration:
a) taking 0, 1, 2, 4, 8, 12, 16 and 20 mu L of Bovine Serum Albumin (BSA) standard substance of 0.5mg/mL respectively to a 96-well plate, and adding double distilled water to complement to 20 mu L;
b) adding 200 mu L of BCA working solution, and standing at 37 ℃ for 30 minutes;
c) the absorbance (A) at a wavelength of 570nm was measured by an ultraviolet spectrophotometer, and the protein concentration was calculated from the standard curve.
3) Polyacrylamide gel electrophoresis of proteins.
Toxic effects of RTS and MCT on Flp-In CHO cell lines expressing CYP3A7 and POR and CYP3A7
Successfully screened CHO-CYP3A7 and CHO-POR-CYP3A7 cells were expanded and cryopreserved, and then CHO-CYP3A7 cells were cultured and cryopreserved&CHO-CYP3A7&CHO-POR-CYP3A7 at 5-9X 103Density per well 96 well plates (n ═ 4), treated 24h later with RTS or MCT at 0, 0.000001, 0.00001, 0.0001, 0.001, 0.01 and 0.1mM, respectively, 20 μ L of MTS was added to each well at the end of the treatment, incubated at 37 ℃ for 30min until the color turned yellow-brown, and the absorbance value was measured at OD 490nm in a microplate reader. After the formal experiment is completedDrug inhibition curves for RTS and MCT were generated by calculating and plotting the OD values of the sample and control wells in prism software.
5. Results of the experiment
5.1 protein expression of CHO-POR-CYP3A7 cells constructed
POR and CYP37 protein expression of CHO-POR-CYP3A7 cells constructed are shown in FIG. 4. Compared with CHO cells expressing an empty vector (vector), POR protein expression is detected by transfecting a human POR genome, CYP3A7 protein expression is detected by transfecting a human CYP3A7 genome, and POR and CYP3A7 protein expression is detected by transfecting human POR and CYP3A7 genomes. The experimental results show that the human POR and CYP3A7 genes have been stably transfected into CHO cells and successfully expressed.
5.2 toxicity of Pyrrolizidine Alkaloids (PAs) to CHO-POR-CYP3A7 cells
Pyrrolizidine Alkaloids (PAs), mainly serving as Reburnine (RTS) and Monocrotaline (MCT) pairs CHO&CHO-CYP3A7 cell toxicity As shown in FIGS. 5 and 6, the present inventors have found that RTS and MCT, two drugs, are more potent in inhibiting CHO-POR-CYP3A4 cell viability than CHO-CYP3A7 cells. IC of two drugs RTS and MCT as shown in FIGS. 5 and 650The lowest among CHO-POR-CYP3A7 cells. The data show that CHO-POR-CYP3A7 cells are more sensitive than CHO-CYP3A7 cells when screening drugs that are metabolically detoxified by CYP3A 7.
The results of this example suggest that the present invention demonstrates that it is possible to study fetal drug toxicity screening In vitro by using a method of stably transfecting human POR and CYP3A7 genes In Flp-In CHO cells, respectively, to obtain a cell line stably expressing human POR CHO-CYP3A 7. The establishment of the CHO-POR-CYP3A7 cell model has various applications, including clinical drug and toxicity screening related research of environmental xenobiotics.
Example 2: intrauterine in vivo toxicity verification based on toxic exogenous compounds RTS and MCT of example 1
1. Laboratory animal
SPF grade healthy adult Wistar rats (male body weight: 280. + -.20 g, female body weight: 200. + -.20 g) were purchased from Hubei province disease prevention and control center. License number: SCXK 2020-.
2. Animal treatment
Wistar rats were acclimatized to a 12 hour light cycle at 25. + -. 2 ℃ and 1w prior to acclimatization, and the ratio of female: male-2: 1 coop conception. Pregnant rats were randomly divided into 3 groups: control, RTS, and MCT groups, 3-4 of each group. RTS group: the mother rats are administered 20mg/kg RTS or MCT by intragastric administration once a day from the ninth day of pregnancy; control group: the mice were given an equal volume of vehicle for 12 days. At 20 days after pregnancy, the mice were sacrificed 2h on the morning after drug administration, and the mice and fetuses were anesthetized with isoflurane and adrenal specimens were collected and fixed in 4% neutral paraformaldehyde and electron microscopy fixative for HE staining and electron microscopy pictures. The remaining adrenal tissue specimens were stored at-80 ℃.
3. Morphological detection of fetal rat adrenal gland
The fetal rat adrenal tissue is fixed for 48 hours, dehydrated, waxed, embedded and sliced, then stained with hematoxylin-eosin stain, and finally observed and photographed under a microscope.
The preparation of block samples was carried out after the fetal rat adrenal gland tissue electron microscope was fixed for 24 hours and observed under a transmission electron microscope.
4. Results of the experiment
4.1 Effect of RTS and MCT Exposure during pregnancy on fetal adrenal morphology
As shown in FIG. 7, the cells in the cortical region of the fetal rat adrenal tissue of the control group are clearly arranged, regularly and orderly and have normal structure. And the cortical cells of the RTS20 and MCT20 administration groups were swollen, disorganized, indistinct in boundaries and fuzzy in structure.
Example 3: lactation in vivo toxicity test based on toxic exogenous compounds RTS and MCT of example 1
1. Laboratory animal
SPF grade healthy adult Wistar rats (male body weight: 280. + -.20 g, female body weight: 200. + -.20 g) were purchased from Hubei province disease prevention and control center. License number: SCXK 2020-.
2. Animal treatment
Wistar rats were acclimatized to a 12 hour light cycle at 25. + -. 2 ℃ and 1w prior to acclimatization, and the ratio of female: male-2: 1 coop conception. Pregnant rats were randomly divided into 5 groups after production: the control group, RTS5 and RTS20 groups, and MCT5 and MCT20 groups each had 3-4 litters, each with 4 males and females. RTS group: the mother rat starts intragastric administration from the first day after production and is administered RTS with the concentration of 5 mg/kg or 20mg/kg once a day; MCT group: the mother rat starts intragastric administration with 5 or 20mg/kg MCT once a day from the first day after production; control group: the rats were given an equal volume of vehicle for 21 consecutive days. After 2h of administration on the morning on day 21 after birth, 2 males and 2 females were randomly selected per litter for sacrifice. Offspring mice were anesthetized with isoflurane and blood and liver specimens were collected. Each group randomly selected 3 nests of male and female progeny livers, fixed in 4% neutral paraformaldehyde, for HE staining. Serum and remaining liver tissue specimens were stored at-80 ℃.
3. Three-week rat liver morphology assay
After the liver tissues of three-week rats are fixed for 48 hours, the liver tissues are dehydrated, waxed, embedded and sliced, then stained by hematoxylin-eosin staining solution, and finally observed and photographed under a microscope.
4. Results of the experiment
4.1 Effect of RTS and MCT Exposure on liver morphology in three-week rats
As shown in FIG. 8, the contour of the central vein and the boundaries between hepatic cells in the hepatic tissue of the control group are clear, hepatic cords are radially arranged along the central vein, the hepatic plate is regularly and orderly arranged, the hepatic sinus structure is normal, and no lipid drop is found. The liver cells of the RTS20 administration group are disorderly arranged, the boundary is not clear, the liver cord structure is fuzzy, and obvious vacuolar degeneration appears in the liver cells. As shown in fig. 10, significant vacuolar degeneration occurred in hepatocytes for the administered group of MCT20, compared to the control group.
4.2 Damage to the liver of three-week-old rats by RTS and MCT Exposure
As shown in figure 9, RTS exposure resulted in elevated ALT activity in serum of rats for three weeks compared to the control group. As shown in figure 11, MCT exposure also resulted in elevated ALT activity in serum of three weeks rats, resulting in liver damage, compared to controls.
In summary, the present invention is based on Flp-InTMThe CHO cell line is obtained by transfecting the target cells with the CYP3A7 and POR-containing coding DNA sequences respectivelyThe cell model for screening the fetal metabolic toxicity exogenous compound is disclosed. The toxicity of two exogenous compounds RTS and MCT on a CHO-POR-CYP3A7 model is detected, which indicates that the cell model is more stable, sensitive and convenient compared with the prior cell model. Then, RTS and MCT exposure are given to the female mouse during pregnancy on the whole animal model to confirm the adverse effect of the two toxic exogenous compounds on the function of adrenal gland in the uterus, and RTS and MCT exposure are given to the female mouse during lactation to confirm the adverse effect of the two toxic exogenous compounds on the function of offspring liver in lactation, which is consistent with the cell model result of CHO-POR-CYP3A7 screened by the in vitro fetal drug toxicity constructed by the invention. The exogenous substance screening system established by the invention has the characteristics of high sensitivity, high stability and the like, can be used for screening exogenous compounds which are metabolized and detoxified by CYP3A7, and has important significance for screening the toxicity of exogenous substances during the development period.
Claims (6)
1. A cell model for screening exogenous compounds for CYP3a7 mediated metabolic toxicity, comprising: using Flp-InTMCHO as target cell, and transfecting the target cell with the DNA sequences containing CYP3A7 and POR to obtain the cell model for screening the fetal metabolic toxic exogenous compound.
2. The cellular model for screening exogenous compounds for CYP3a7 mediated metabolic toxicity according to claim 1, wherein: the cell model for screening exogenous compounds with CYP3A7 mediated metabolic toxicity has two indexes, cell activity and half Inhibitory Concentration (IC)50)。
3. A cell model construction method for screening exogenous compounds with metabolic toxicity mediated by CYP3a7 according to claim 1, wherein: the method comprises the following steps:
s1: reviving Flp-In a basal mediumTM-CHO cells, said basal medium containing 10% fetal bovine serum and 1% F12 of penicillin and streptomycin;
s2: constructing pcDNA5 plasmid containing CYP3A7 coding DNA sequence;
s3: constructing pCMV plasmid containing POR coding DNA sequence;
s4: stably transfecting the pcDNA5 plasmid containing the DNA sequence encoding CYP3A7 constructed In the step S2 into Flp-In after passage of the step S1TM-CHO, cultured in selective DMEM/F12-1 medium containing 10% fetal bovine serum and 500 μ g/ml hygromycin in F12;
s5: the pCMV plasmid containing the POR-encoding DNA sequence constructed In step S3 was stably transfected into Flp-In obtained In step S4TMCulturing in a screening DMEM/F12-2 culture medium in a CHO-CYP3A7 cell, wherein the screening DMEM/F12-2 culture medium contains 10% fetal bovine serum, 500 mu g/ml hygromycin and 50 mu g/ml puromycin F12, and obtaining the cell model of the screening CYP3A7 mediated metabolism toxic exogenous compounds.
4. Use of a cell model for screening exogenous compounds for metabolic toxicity mediated by CYP3a7 according to claim 1 for screening exogenous compounds for metabolic toxicity.
5. Use of a cell model for screening exogenous compounds for metabolic toxicity mediated by CYP3a7 according to claim 4 for screening exogenous compounds for metabolic toxicity, wherein: when the cell viability is reduced, it is suggested that the exogenous compound to be screened has metabolic toxicity mediated by CYP3a 7.
6. Use of a cell model for screening exogenous compounds for metabolic toxicity mediated by CYP3a7 according to claim 4 for screening exogenous compounds for metabolic toxicity, wherein: IC of exogenous compound to be screened according to difference50The value can be compared with the metabolic toxicity of different exogenous compounds.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210472652.XA CN114717195A (en) | 2022-04-29 | 2022-04-29 | Cell model for screening exogenous compounds mediated by CYP3A7 and metabolized to toxicity, construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210472652.XA CN114717195A (en) | 2022-04-29 | 2022-04-29 | Cell model for screening exogenous compounds mediated by CYP3A7 and metabolized to toxicity, construction method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114717195A true CN114717195A (en) | 2022-07-08 |
Family
ID=82245576
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210472652.XA Pending CN114717195A (en) | 2022-04-29 | 2022-04-29 | Cell model for screening exogenous compounds mediated by CYP3A7 and metabolized to toxicity, construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114717195A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024160863A1 (en) * | 2023-01-31 | 2024-08-08 | Brandenburgische Technische Universität Cottbus-Senftenberg | Test system comprising stably cyp-transfected or transduced mammalian cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888382A (en) * | 2012-10-17 | 2013-01-23 | 吉瑞泰（武汉）生物科技有限公司 | Cell model, construction method and application thereof for screening nervous disorder resistant medicines |
| CN103468722A (en) * | 2013-09-08 | 2013-12-25 | 浙江大学 | Construction expression and application of miniature pig cytochrome P450 recombinase |
| CN108251437A (en) * | 2017-11-30 | 2018-07-06 | 中山珐玛斯医药科技有限公司 | A kind of method for improving cyp3A5 expression enzyme metabolic activities |
| CN109688810A (en) * | 2016-06-27 | 2019-04-26 | 贝勒医学院 | People liver with defective P450 oxidoreducing enzyme is fitted into non-human animal and its application method |
-
2022
- 2022-04-29 CN CN202210472652.XA patent/CN114717195A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888382A (en) * | 2012-10-17 | 2013-01-23 | 吉瑞泰（武汉）生物科技有限公司 | Cell model, construction method and application thereof for screening nervous disorder resistant medicines |
| CN103468722A (en) * | 2013-09-08 | 2013-12-25 | 浙江大学 | Construction expression and application of miniature pig cytochrome P450 recombinase |
| CN109688810A (en) * | 2016-06-27 | 2019-04-26 | 贝勒医学院 | People liver with defective P450 oxidoreducing enzyme is fitted into non-human animal and its application method |
| CN108251437A (en) * | 2017-11-30 | 2018-07-06 | 中山珐玛斯医药科技有限公司 | A kind of method for improving cyp3A5 expression enzyme metabolic activities |
Non-Patent Citations (2)
| Title |
|---|
| 孙莉 等: "稳定表达CYP2A13 和CYP2A13 /POR 的Flp-In CHO 重组细胞系的构建", pages 795 - 799, Retrieved from the Internet <URL:https: //kns.cnki.net/kcms/detail/34.1086.R.20220426.1321.048.html> * |
| 王守林 等: "细胞色素P450 2A13及其自然变异体的功能研究", 中国毒理学第二届中青年学者科技论坛会议论文集, vol. 1, pages 24 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024160863A1 (en) * | 2023-01-31 | 2024-08-08 | Brandenburgische Technische Universität Cottbus-Senftenberg | Test system comprising stably cyp-transfected or transduced mammalian cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bonapace et al. | Np95 is regulated by E1A during mitotic reactivation of terminally differentiated cells and is essential for S phase entry | |
| Kojima et al. | Combined expression of pancreatic duodenal homeobox 1 and islet factor 1 induces immature enterocytes to produce insulin | |
| Doetsch et al. | Subventricular zone astrocytes are neural stem cells in the adult mammalian brain | |
| US5928943A (en) | Embryonal cardiac muscle cells, their preparation and their use | |
| Liang et al. | Clinical implications of HBV PreS/S mutations and the effects of PreS2 deletion on mitochondria, liver fibrosis, and cancer development | |
| Sheth et al. | Decoupling the function of Hox and Shh in developing limb reveals multiple inputs of Hox genes on limb growth | |
| Ramezankhani et al. | Organoid and microfluidics-based platforms for drug screening in COVID-19 | |
| CN114717195A (en) | Cell model for screening exogenous compounds mediated by CYP3A7 and metabolized to toxicity, construction method and application thereof | |
| US10100284B2 (en) | Method for inducing differentiation of pluripotent stem cells into endodermal cells | |
| Albright et al. | Characterization of cultured microglia that can be infected by HIV-1 | |
| CN107417772B (en) | Polypeptide HIP-20 capable of antagonizing RNA binding activity of hnRNPU protein and application thereof | |
| Dorison et al. | What can we learn from kidney organoids? | |
| WO2012047037A2 (en) | Embryonic stem cell-derived cardiomyocytes and cell therapy product using same as an active ingredient | |
| EP1498483A1 (en) | Endocrine cell lines and method of using the same | |
| US20100260731A1 (en) | Propagation of primary cells and the use thereof | |
| US11825817B2 (en) | Rat model of down syndrome and method for producing same | |
| CN108795874B (en) | Method for reprogramming fibroblast into Sertoli cell in vitro and application thereof | |
| JPWO2016002937A1 (en) | Pancreatic endocrine cells, method for producing the same, and transdifferentiation agent | |
| Rulli Jr et al. | Mutant murine leukemia virus Gag proteins lacking proline at the N-terminus of the capsid domain block infectivity in virions containing wild-type Gag | |
| CN114891750A (en) | Cell model for screening exogenous compounds mediated by CYP3A4 and metabolized to toxicity, construction method and application thereof | |
| CN114478706B (en) | Polypeptide for three-dimensional culture of fiber network structure and application thereof | |
| Catanuto et al. | Mouse retinal pigmented epithelial cell lines retain their phenotypic characteristics after transfection with human papilloma virus: a new tool to further the study of RPE biology | |
| Lim et al. | ARL6IP1 gene delivery reduces neuroinflammation and neurodegenerative pathology in hereditary spastic paraplegia model | |
| Sorel et al. | Restoration of T and B Cell Differentiation after RAG1 Gene Transfer in Human RAG1 Defective Hematopoietic Stem Cells | |
| Du et al. | Generation and application of immortalized sertoli cell line from sheep testis |
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 |