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目的:构建CYP1A2基因高表达载体,转染到L02肝细胞,建立CYP1A2高表达细胞株并检测三氯乙烯对该细胞株凋亡基因和癌基因的影响。方法:根据GenBank提供的CYP1A2基因cDNA序列设计引物,PCR扩增该基因并将其克隆到慢病毒高表达载体pLVX-acGFP1-C1中,将已经构建的慢病毒载体对293FT细胞进行转染,收集病毒上清,感染正常L02肝细胞。用嘌呤霉素进行筛选得到CYP1A2高表达L02细胞株,通过荧光定量PCR和Westernblot对细胞株进行鉴定。用不同剂量三氯乙烯对正常L02肝细胞和CYP1A2高表达细胞进行染毒12h,采用PCR方法检测凋亡基因(Bcl-2、Caspase-3、Caspase-8、Caspase-9)和癌基因(c-fos、c-myc、K-ras、p53)mRNA表达的变化。结果:测序证明重组慢病毒高表达载体CYP1A2基因序列与GenBank提供的CYP1A2序列一致,荧光定量PCR检测显示CYP1A2高表达细胞株CYP1A2mRNA表达比正常L02肝细胞提高317倍,Westernblot结果显示CYP1A2高表达细胞株CYP1A2蛋白表达水平比正常L02肝细胞高3.41倍。三氯乙烯染毒后,CYP1A2高表达细胞中凋亡基因Bcl-2mRNA表达水平与正常肝细胞无显著性差异(P>0.05),而Caspase-3、Caspase-8和Caspase-9mRNA表达水平高于正常肝细胞(P<0.05或P<0.01)。CYP1A2高表达细胞中癌基因c-myc和p53mRNA表达水平低于正常肝细胞(P<0.05或P<0.01),而c-fos和K-rasmRNA则高于正常肝细胞(P<0.05或P<0.01)。结论:三氯乙烯对CYP1A2高表达细胞凋亡基因和癌基因表达有显著影响,提示CYP1A2是三氯乙烯在体内代谢的重要因素,与三氯乙烯毒性存在一定关系。
OBJECTIVE: To construct CYP1A2 high expression vector and transfect it into L02 hepatocytes to establish CYP1A2 high expression cell line and to detect the effect of trichlorethylene on the apoptosis gene and oncogene of this cell line. Methods: The primers were designed according to the cDNA sequence of CYP1A2 gene provided by GenBank. The gene was amplified by PCR and cloned into the lentiviral vector pLVX-acGFP1-C1. 293FT cells were transfected with the lentiviral vector and collected. Virus supernatant, infected with normal L02 hepatocytes. Puromycin was used to screen the CYP1A2-overexpressing L02 cell line, and the cell lines were identified by fluorescence quantitative PCR and Western blot. The normal L02 hepatocytes and CYP1A2 high expressing cells were exposed to different doses of trichlorethylene for 12 hours. The expressions of Bcl-2, Caspase-3, Caspase-8, Caspase-9 and c -fos, c-myc, K-ras, p53) mRNA expression. Results: Sequencing showed that the CYP1A2 gene sequence was highly consistent with the CYP1A2 sequence provided by GenBank. The CYP1A2 mRNA expression in CYP1A2-overexpressing cell line was 317-fold higher than that of normal L02 hepatocyte by fluorescence quantitative PCR. Western blot showed that the CYP1A2-overexpressing cell line CYP1A2 protein expression level of 3.41 times higher than normal L02 hepatocytes. After exposure to trichlorethylene, the expression level of Bcl-2mRNA in CYP1A2-overexpressing cells was not significantly different from that in normal hepatocytes (P> 0.05), while the expression levels of Caspase-3, Caspase-8 and Caspase- Normal hepatocytes (P <0.05 or P <0.01). The expression levels of c-myc and p53 mRNA in CYP1A2-overexpressing cells were significantly lower than those in normal hepatocytes (P <0.05 or P <0.01), while the expressions of c-fos and K-rasmRNA were higher in normal CYP1A2 cells than in normal hepatocytes 0.01). CONCLUSION: Trichlorethylene has a significant effect on the expression of CYP1A2 highly expressed apoptotic genes and oncogenes, suggesting that CYP1A2 is an important factor in the metabolism of trichlorethylene in vivo and has a certain relationship with the toxicity of trichlorethylene.