CYP3A4高表达肝细胞株建立及其对三氯乙烯毒性的影响  

Construction of CYP3A4 gene overexpressed hepatocytes and their effect on trichloroethylene toxicity

作　　者：毛侃琅[1,2] 徐新云[1] 何晓阳[2] 毛吉炎[1,2] 张然[1] 谢杏[1,2] 秦逍云[1,2] 

机构地区：[1]深圳市疾病预防控制中心卫生毒理学重点实验室/深圳市现代毒理学重点实验室,广东深圳518055 [2]深圳大学生命科学学院细胞生物学实验室/深圳市海洋生物资源与生态环境重点实验室,广东深圳518060

出　　处：《中国药理学与毒理学杂志》2013年第5期848-853,共6页Chinese Journal of Pharmacology and Toxicology

基　　金：深圳市科技计划重点项目(201101015)~~

摘　　要：目的建立CYP3A4高表达肝细胞株并观察其对三氯乙烯毒性的影响。方法 PCR扩增CYP3A4基因并将其克隆到慢病毒高表达载体中,将已经构建的慢病毒载体进行转染后,收集病毒上清,感染正常L02肝细胞。用嘌罗霉素进行筛选得到CYP3A4高表达肝细胞株,通过荧光定量PCR和Western蛋白质印迹法鉴定细胞株。用三氯乙烯0,0.25,0.5,1.0,2.0和4.0mmol·L-1对正常肝肝细胞和CYP3A4高表达肝细胞进行染毒12h,实时定量PCR检测凋亡基因bcl-2,胱天蛋白酶3,胱天蛋白酶8和胱天蛋白酶9以及癌基因c-fos,c-myc,K-ras和p53的表达。结果测序证明重组慢病毒高表达载体CYP3A4基因序列正确,荧光定量PCR检测CYP3A4高表达肝细胞株比正常肝细胞CYP3A4基因表达提高94倍。Western蛋白质印迹结果显示,CYP3A4高表达肝细胞株CYP3A4蛋白表达水平是正常肝细胞的2.36倍。CYP3A4高表达肝细胞bcl-2mRNA表达水平随三氯乙烯浓度增加呈下降趋势,与正常细胞相比,三氯乙烯0.25和0.5mmol·L-1使CYP3A4高表达肝细胞中的bcl-2mRNA明显升高(P<0.01);三氯乙烯0.5,1.0,2.0和4.0mmol·L-1处理使CYP3A4高表达肝细胞组的凋亡基因胱天蛋白酶3、胱天蛋白酶8和胱天蛋白酶9的mRNA表达水平明显升高(P<0.05);三氯乙烯0.5,1.0,2.0和4.0mmol·L-1处理的CYP3A4高表达肝细胞c-fos、c-myc和k-ras基因的表达显著升高(P<0.01),p53表达水平明显下降(P<0.01)。结论三氯乙烯对CYP3A4高表达肝细胞株中的凋亡基因和癌基因表达具有明显促进作用,说明CYP3A4是三氯乙烯在体内代谢的重要因素。OBJECTIVE To construct hepatocytes L02 cells with cytochrome P-450 （CYP）3A4 overexpression and to observe their effects on toxicity of trichloroethylene. METHODS CYP3A4 gene was amplified using PCR and ligated into the lentiviral vector pLVX-acGFP-CI. 293FT cells were transfected with the recombinant vector, viral supernatant was collected, and than L02 cells were transducted. After puromycin screening, CYP3A4-overexpressed hepatocytes strain was constructed, which was identified using quantitative PCR and Western blotting. Then, the constructed hepatocytes with CYP3A4 overexpression and normal L02 cells were treated with TCE 0, 0.25, 0.5, 1.0, 2.0 and 4.0 retool- L-1 for 12 h. The expression of apoptosis genes bc/-2, caspase3, caspase8, caspase9 and oncogenes c-fos, c-myc, K-ras and p53 was determined by real-time quantitative PCR. RESULTS Sequencing results showed that the recombinant vector contained the same sequence as CYP3A4 gene from Gen- Bank. Quantitative PCR indicated that CYP3A4 gene expression level in CYP3A4 overexpressed hepato- cytes was 94 times higher than in normal L02 cells. The Western blotting result revealed that CYP3A4 protein in CYP3A4-overexpressed hepatocytes was 2.36 times the level of normal L02 cells. After TCE 0.5, 1.0, 2.0 and 4.0 mmol. L-1 treatment, compared with normal L02 cells, p53 expression levels in CYP3A4 overexpressed hepatocytes were significantly derceased（ P 〈0.05）, while expression levels of other apoptosis genes caspase3, caspase8 and caspase9 expression levels and oncogenes （c-fos, c-myc and k-ras） significantly increased （ P 〈 0.05）. CONCLUSION Trichloroethylene significantly promotes the expression of apoptosis genes and oncogenes in CYP3A4 overexpressed hepatocytes, suggesting that CYP3A4 may play an important role in trichlorethylene metabolism in rive.

关 键 词：肝细胞 细胞色素P-450 CYP3A4 慢病毒感染 三氯乙烯 

分 类 号：R965[医药卫生—药理学]