目的构建CYP3A4基因沉默载体,转染L02肝细胞,建立CYP3A4沉默细胞株并观察其对三氯乙烯毒性的影响。方法根据GenBank提供的CYP3A4基因mRNA序列设计合成shRNA,将shRNA连接到pLKO.1-puro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CYP3A4沉默细胞株,通过荧光定量PCR和Western blot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CYP3A4沉默细胞进行染毒12h,观察凋亡基因(Bcl-2、Caspase-3、Caspase-8、Caspase-9)和癌基因(c-fos、c-myc、K-ras、p53)表达变化。结果测序证明插入pLKO.1-puro载体的干扰序列与设计的序列一致,荧光定量PCR检测CYP3A4沉默细胞CYP3A4基因表达比正常L02肝细胞下降77.3%,Western blot实验显示CYP3A4沉默细胞CYP3A4蛋白表达水平比正常L02肝细胞下降84.6%。CYP3A4沉默细胞经三氯乙烯染毒后Bcl-2表达水平显著高于L02细胞的表达水平;Caspase-3表达水平无明显改变;Caspase-8仅在TCE高剂量组表达下降;Caspase-9在TCE剂量≥1.0 mmol/L表达水平下降;癌基因c-fos、c-myc、k-ras和p53表达水平都有一定程度下降,部分剂量组存在显著差异(P<0.05或P<0.01)。结论三氯乙烯对正常肝细胞和CYP3A4沉默细胞凋亡基因和癌基因表达存在明显差异,提示CYP3A4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 a CYP3A4 gene silencing vector and transfect L02 hepatocytes to establish CYP3A4 silencing cell lines and observe its effects on trichlorethylene toxicity. Methods According to the CYP3A4 mRNA sequence provided by GenBank, shRNA was designed and synthesized. The shRNA was ligated into pLKO.1-puro. 293FT cells were transfected with the lentiviral vector and the supernatant was collected to infect normal L02 hepatocytes. Screening with puromycin CYP3A4 silenced cell lines were obtained by fluorescence quantitative PCR and Western blot cell lines were identified. The normal L02 hepatocytes and CYP3A4 silencing cells were exposed to different doses of trichlorethylene for 12 hours. The expressions of Bcl-2, Caspase-3, Caspase-8 and Caspase-9 and oncogene c- -myc, K-ras, p53) expression changes. Results Sequencing showed that the interference sequence inserted into pLKO.1-puro vector was consistent with the designed sequence. The CYP3A4 silencing cells showed a 77.3% down-regulation in CYP3A4 silencing cells compared with normal L02 hepatocytes by fluorescence quantitative PCR. Western blot showed that CYP3A4 protein expression level Normal L02 hepatocytes decreased by 84.6%. The expression level of Bcl-2 in CYP3A4 silencing cells after trichlorethylene exposure was significantly higher than that in L02 cells; the expression of Caspase-3 had no significant change; the expression of Caspase-8 decreased only in TCE high dose group; The expression of oncogene c-fos, c-myc, k-ras and p53 all decreased to a certain extent with dose ≥1.0 mmol / L. There were significant differences in some dose groups (P <0.05 or P <0.01). CONCLUSION Trichlorethylene has obvious difference on apoptosis gene and oncogene expression of normal hepatocytes and CYP3A4 silencing cells, suggesting that CYP3A4 is an important factor in the metabolism of trichlorethylene in vivo and has certain relationship with trichlorethylene toxicity.