CYP2E1酶在去甲斑蝥素代谢和环孢素诱导肝损伤中的作用研究
[Abstract]:Cytochrome P450 enzyme 2E1 subtype (CYP2E1 enzyme) can mediate a variety of in vivo, exogenous compound metabolism, and is very important in the research of pharmacology and toxicology. The role of CYP2E1 in the pathogenesis of drug metabolism and drug-induced liver injury was discussed. Normotilin is used in the treatment of tumors for many years, but its in vivo metabolic processes are not complete It is clear that in this study, the metaPrint2D software is used, and based on the characteristics of the atomic fingerprint in the noreptilin and its hydrolysis product, the atomic fingerprint feature in the chemical structure is based on the data mining and the statistical analysis technique, and the atoms are calculated by the calculation of the atoms as the metabolic site. The ratio of CYP1A2, CYP2A6, CYP2B6, CYP3A4, CYP2D6, CYP2C9, CYP2E1, and CYP2C19 to normaconic acid was determined by using Sybil software, then the higher of the score was selected, and the in vitro metabolism of human recombinant CYP450 was carried out. The results showed that both CYP2E1, CYP2C19, and CYP2C9 were involved in the mediation of the metabolism of norepinephrine. in that process, the human recombinant CYP2E1 enzyme mediate the in vitro metabolism of the noreptilin in the m's kinetic process, Km is 23.64. mu. M, the CL is 0. 689ml/ mmol of CYP2E1/ min, and finally, the in-vivo metabolism product is separated and identified, and the result is confirmed that the nomotilin enters the body, and then is first hydrolyzed to the nomottle acid, and then an oxygen bridge is generated. in that hydrolysis and six-membered ring, the open-loop or deionization is broken in a different way, the oxidation and metabolism are further happen, the small molecular compound is converted into a small molecular compound to be discharged in vitro, or in combination with the glucuronic acid, the glycine, the glutathione and the like, the water solubility is increased, In vivo, the results of in vitro metabolism experiment are basically The activity of the CYP2E1 enzyme is small, which is beneficial to the entry of the small molecule compound, but the internal force of the molecule and the Van der Waals force between the molecules are high, so that the enzyme-drug complex is not stable; the metabolism of the exogenous compound in the CYP2E1 enzyme-mediated metabolism of the exogenous compound is easy to lead to oxidative stress, induction, The incidence of hepatic injury induced by ciclosporin was about 30%. The results of the study of CYP2E1 genotype were carried out in 322 patients with long-term cyclosporine anti-rejection or immunosuppression-related treatment, and 13 SNPs were first tested by Hardy-Weinberg equilibrium test and genetic linkage analysis, and 6 of them were selected. The results showed that the concentration of the serum trough of the ciclosporin and the hepatic injury did not appear after the administration of the ciclosporin. Significant correlation; rs3813866 (-1563TA) was a risk-sensitive gene for ciclosporin-induced liver injury (OR: 2.325, 95% CI: 1.491-3. 626). Further from the cellular molecular level, using the point mutation technique, the rs3813866 mutant plasmid vector was constructed and transfected into HepG-2 cells to explore how the susceptibility SNP could control the expression of the CYP2E1 protein or the enzyme activity. The results showed that the rs381366 mutation can enhance the activity of the CYP2E1 promoter by about 2.46 times, the mRNA level up to about 1.64 times up-regulation, leading to the induction of liver injury after taking the ciclosporin in a patient carrying the rs381366 mutant gene; the ciclosporin can enhance the activity of the CYP2E1 promoter and increase the activity ability of the promoter to be in direct proportion to the drug concentration, but the regulation effect of the ciclosporin on the activity of the CYP2E1 promoter is far less than that of the rs381 The effect of the 366 mutation: the effect of the ciclosporin on the expression level of CYP2E1mRNA in the cells was opposite to that of the promoter. After the intervention of the ciclosporin, the wild-type and the mutant CYP2E1mRNA were down-regulated and decreased with the increase of the concentration of the ciclosporin, and the CYP2E1 wild-type mRN after the intervention of 20. m The level of level A was reduced by about 2-fold, and the expression of mutant mRNA could be up to 20-fold. Cyclosporin intervention could increase the expression of wild-type and mutant CYP2E1 protein and the activity of enzyme, the higher the concentration of ciclosporin, the higher the expression of protein and the stronger the ability of the enzyme, and the expression of CYP2E1 protein after the intervention of 20. m about 1.72-fold increase in enzyme activity, 27-28% enhancement of enzyme activity, and presence of other feedback regulators in the regulation of the transcription of the CYP2E1 enzyme by the cyclosporine
【学位授予单位】:山东大学
【学位级别】:博士
【学位授予年份】:2014
【分类号】:R575.3
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