CYP2C9基因高表达细胞株对三氯乙烯毒性影响

Construction of CYP2C9-overexpression Hepatocytes and Effects of CYP2C9-overexpression on Trichloroethylene Toxicity

目的建立细胞色素氧化酶P450 2C9(CYP2C9)基因高表达肝细胞株(L02),并观察其对三氯乙烯(TCE)毒性的影响。方法 PCR扩增CYP2C9基因并将其克隆到慢病毒表达载体中,然后转染293T细胞,收集病毒上清,感染正常L02细胞。采用荧光定量PCR和western blot筛选鉴定CYP2C9高表达细胞株。分别采用浓度为0.25、0.5、1.0、2.0、4.0、8.0、16.0mmol/L的TCE染毒剂量组对L02细胞和CYP2C9基因高表达细胞株染毒24h,同时设置二甲基亚砜(DMSO)溶剂对照组即0mmol/L染毒剂量组,观察L02细胞和CYP2C9基因高表达细胞株细胞增殖能力的变化以及凋亡基因Bcl-2和caspase-3表达变化。结果酶切和测序结果证明CYP2C9基因成功插入PCDH-MCS-EF1-GFP-T2A-PURO载体。CYP2C9高表达细胞株的CYP2C9 mRNA和蛋白相对表达水平分别较对照普通L02细胞升高107.3%和257.9%。TCE染毒后,2.0、4.0、8.0、16.0 mmol/L染毒剂量组CYP2C9基因高表达细胞抑制率与L02细胞相比,低于相同浓度TCE处理的L02细胞。TCE染毒后,通过荧光定量PCR和WB检测,发现TCE 2.0和8.0mmol/L染毒剂量组CYP2C9基因高表达细胞的Bcl-2表达水平低于相应染毒剂量组L02细胞,而TCE 0.0、0.5、2.0、8.0 mmol/L剂量组CYP2C9基因高表达细胞的caspase-3表达水平高于普通L02细胞。结论三氯乙烯对正常肝细胞和CYP2C9基因沉高表达细胞凋亡基因Bcl-2存在差异,提示CYP2C9与三氯乙烯在生物体内毒性存在一定关系。

Objective To construct the lentivirus vector with CYP2C9 gene overexpression,transduce it into normal human L02 hepatocytes and finally study trichlorethylene（TCE）toxicity in normal human L02 hepatocytes（L02 cells）and CYP2C9-overexpression hepatocytes.Methods Primers were designed according to cDNA sequence of CYP2C9 gene from GenBank.The gene was amplified using PCR and ligated into the lentiviral vector PCDH-MCS-EF1-GFP-T2A-PURO.293 Tcells were transfected with the recombinant vector,viral supernatant was collected,and L02 liver cells were then transfected.After puromycin screening,L02 cells with CYP2C9 gene transfection were constructed.Finally,gene sequencing,QPCR and western blot were performed for identification.Then the CYP2C9-overexpression hepatocytes and L02 cells were treated at various doses of TCE for 24 hours to observe cell viability and the expressions of apoptosis gene Bcl-2 and caspase-3.Results Sequencing and enzyme digestion results showed that the sequence contained in the recombinant vector was exactly the same as CYP2C9 gene from GenBank.CYP2C9 gene expression levels and CYP2C9 protein levels in the CYP2C9-overexpression hepatocyte increased by 107.3% and 257.9%,respectively,as compared with normal L02 cells.After TCE treatment,Bcl-2 expression levels in CYP2C9-overexpression hepatocytes were significantly lower than those in L02,while caspase-3 expression levels in CYP2C9-overexpression hepatocytes were higher than in L02.Conclusion The expression of apoptosis gene Bcl-2 is different in CYP2C9-overexpression hepatocytes and normal L02 cells after trichloroethylene treatment,suggesting that CYP2C9 may play a key role in trichloroethylene metabolism.