三氯乙烯对CYP1A2高表达肝细胞株凋亡基因和癌基因的影响  

The effects of trichloroethylene on expression of apoptosis genes and oncogenes in hepatocytes with CYP1A2 overexpression

作　　者：毛侃琅[1,2] 徐新云[1] 毛吉炎[1,2] 袁建辉[1] 刘国红[1] 吴德生[1] 杨细飞[1] 周丽[1] 黄新凤[1] 

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,广东深圳518060

出　　处：《癌变．畸变．突变》2013年第1期7-12,17,共7页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：深圳市科技计划重点项目(201101015);深圳市重点实验室提升发展项目(CXB201005260068A)

摘　　要：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 the lentivirus vector with CYP1A2 gene overexpression, lentivirus was packaged, transduced into hepatocytes （L02 ceils）, then the hepatocytes with CYP1A2 overexpression were constructed. Subsequently hepatocytes with CYP1A2 overexpression （CYP1A20E cells） were treated with various doses of trichloroethylene（TCE）, and TCE toxicity was tested. METHODS： Primers were designed according to cDNA sequence of CYP1A2 gene from GenBank. The gene was amplified using PCR and ligated into the lentiviral vector pLVX-acGFP- C1. 293FT ceils were transfected with the recombinant vector, viral supernatant was collected, L02 liver ceils was then transduced. After puromycin screening, CYP1A2-overexpressing L02 cells were constructed, and identified using fluorescent quantitative PCR and Western blot. Then the constructed cells and normal L02 ceils were treated with various doses of TCE for 12 h before assessing the mRNA expression of apoptosis genes （Bcl-2, Caspase-3, 8, Caspase-9） and oncogenes （c-fos, c-myc, K-ras, p53）. RESULTS： The sequence contained in the recombinant vector was exactly the same as CYPIA2 gene from GenBank. Quantitative PCR indicated CYP1A2 mRNA expression level in the constructed cells was 317 times higher than that of normal L02 cells. CYP1A2 protein levels in the constructed cells was 3.41 folds higher than that of normal L02 cells. After down-regulated, whereas the other apoptosis genes TCE treatment, Bcl-2, c-myc and p53 expression levels were （Caspase-3, Caspase-8, Caspase-9） expression levels and oncogenes（c-fos, K-ras） expression levels were up-regulated when compared with the normal L02 cells. CONCLUSION： CYP1A2-overexpressing L02 cells were successfully constructed using lentiviral vector. TCE treatment upregulated the mRNA expression of some apoptosis genes and oncogenes in CYP1A2-overexpressing cells. These findings indicated that CYP1A2 might play an important role in TCE metabolism in vivo.

关 键 词：肝细胞 CYP1A2 慢病毒载体 三氯乙烯 凋亡基因 癌基因 

分 类 号：Q786[生物学—分子生物学]