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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

发布时间:2018-11-12 10:43
【摘要】:细胞色素P450酶2E1亚型(CYP2E1酶)可介导多种体内、外源性化合物代谢,在药理学和毒理学研究方面作用十分重要,本研究以去甲斑蝥素和环孢素为切入点,分别对CYP2E1酶在药物代谢和药物性肝损伤发病过程中的作用进行探讨。 去甲斑蝥素用于肿瘤治疗多年,但其体内代谢过程尚未完全清楚。本研究首先采用MetaPrint2D软件,根据去甲斑蝥素及其水解产物去甲斑蝥酸化学结构中原子指纹特征,基于数据挖掘和统计分析技术,通过计算原子作为代谢位点的发生率,预测去甲斑蝥素及其代谢物去甲斑蝥酸代谢位点及可能代谢产物;采用Sybyl软件分别进行CYP1A2, CYP2A6, CYP2B6, CYP3A4, CYP2D6, CYP2C9, CYP2E1和CYP2C19酶与去甲斑蝥酸对接;然后选择其中得分较高者,进行人重组CYP450酶体外代谢实验,结果显示,CYP2E1、 CYP2C19和CYP2C9酶均参与介导去甲斑蝥酸代谢过程。人重组CYP2E1酶介导去甲斑蝥素体外代谢呈米氏动力学过程,Km为23.64μM, CL为0.689ml/mmol CYP2E1/min;最后对体内外代谢产物进行分离鉴定,结果证实,去甲斑蝥素进入体内后,首先水解为去甲斑蝥酸,进而发生氧桥水解、六元环不同方式断裂开环或脱羧,进一步发生氧化代谢,转化为小分子化合物排出体外,或与葡萄糖醛酸、甘氨酸、谷胱甘肽等发生结合反应,增加水溶性,利用消除,软件预测与体内、体外代谢实验结果基本一致。 CYP2E1酶活性区域小,有利于小分子化合物进入,但分子内作用力和分子间范德华力能量较高,易导致酶-药物复合物不稳定;CYP2E1酶介导内外源性化合物代谢时发生变构,易导致氧化应激,诱导肝损伤发生。环孢素诱导肝损伤发病率约为30%,但其机制尚不清楚。本研究对322名长期服用环孢素进行抗排异或免疫抑制相关治疗的患者全血样本进行了CYP2E1基因型检测,首先对200名患者进行13种SNPs检测,经Hardy-Weinberg平衡检验和遗传连锁分析,从中选择6个标签SNPs,其余患者仅进行标签SNP测定,采用病例-对照研究方法,对全部患者6种标签SNPs与环孢素诱导肝损伤进行关联分析,结果显示,患者服用环孢素后,环孢素血清谷浓度与肝损伤发生未呈现明显相关性;rs3813866(-1563TA)为环孢素诱导肝损伤易感基因(OR:2.325,95%CI:1.491-3.626). 进一步从细胞分子水平,采用点突变技术,构建rs3813866突变体质粒载体,转染至HepG-2细胞,探索易感SNP如何调控CYP2E1蛋白表达或酶活性,结果显示,rs381366突变可使CYP2E1启动子活性增强约2.46倍,mRNA水平上调约1.64倍,导致携带rs381366突变基因的患者服用环孢素后,易发生诱导肝损伤;环孢素可使CYP2E1启动子活性增强,上调启动子活性能力与药物浓度成正比,但环孢素对CYP2E1启动子活性的上调作用远小于rs381366突变的影响:环孢素对细胞中CYP2E1mRNA表达水平的影响与启动子活性相反,环孢素干预后,野生型和突变型CYP2E1mRNA均呈现下调趋势,且随环孢素浓度增加而下调明显,20μM环孢素干预后CYP2E1野生型mRNA水平下调约2倍,突变型mRNA下降可达20倍;环孢素干预可使野生型和突变型CYP2E1蛋白表达和酶活性略微增强,环孢素浓度越高,上调蛋白表达和酶活性能力越强,20μM环孢素干预后CYP2E1蛋白表达水平提高约1.72倍,酶活性增强27.28%,环孢素上调CYP2E1酶转录表达过程是否存在其它反馈调节机制仍待进一步研究。
[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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