CYP2C9基因沉默细胞株构建及其对三氯乙烯毒性的影响

Construction of CYP2C9-silent hepatocytes and effects of CYP2C9-silence on trichloroethylene toxicity

目的建立细胞色素氧化酶P450 2C9(CYP2C9)基因沉默正常肝细胞株(L02)并观察其对三氯乙烯(TCE)毒性的影响。方法设计合成短发卡RNA(shRNA)将其连接到PLVX-shRNA2P质粒中构建CYP2C9 shRNA慢病毒表达载体,感染L02细胞。采用荧光定量PCR和western blot筛选鉴定CYP2C9沉默细胞株。分别采用浓度为0.25、0.5、1.0、2.0、4.0、8.0、16.0 mmol/L的TCE染毒剂量组对L02细胞和CYP2C9基因沉默细胞株染毒24 h,同时设置二甲基亚砜溶剂对照组即0 mmol/L染毒剂量组,观察L02细胞和CYP2C9基因沉默细胞株细胞周期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插入PLVX-shRNA2P载体的CYP2C9基因干扰序列与设计的shRNA序列一致。CYP2C9沉默细胞株的CYP2C9 m RNA和蛋白相对表达水平分别较shRNAc沉默对照细胞下降76.0%和78.6%。TCE染毒后,0.5、2.0、8.0 mmol/L 3个染毒剂量组CYP2C9基因沉默细胞和L02细胞周期没有发生显著变化。TCE染毒后,2.0和8.0 mmol/L染毒剂量组CYP2C9基因沉默细胞的Bcl-2表达水平均高于相应染毒剂量组L02细胞,而0.5、2.0、8.0 mmol/L 3个染毒剂量组CYP2C9基因沉默细胞的caspase-3表达水平没有显著变化。结论三氯乙烯对正常肝细胞和CYP2C9基因沉默细胞凋亡基因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 Cytoehrome oxidase P450 2C9（CYP2C9）-silent hepatocyte（L02）, and to study the toxicity of trichlorethylene（TCE） on it. [Methodsl Short hairpin RNA（shRNA） was designed and ligated to PLVX-shRNA2P to construct lentiviral expression vector. L02 cells were transfected. CYP2C9-silenced cells were constructed and identified by quantitative PCR and western blot. Then the CYP2C9-silenced hepatocytes and L02 cells were treated at various does of TCE（0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mmol/L） for 24 hours, meanwhile the dimethyl sulfoxide solvent control group （0 retool/L） was set up to observe their cell cycle and the expressions of apoptosis gene Bcl-2 and caspase-3. [Results] Enzyme digestion and sequencing results showed that the sequence contained in the recombinant vector was exactly the shRNA which was designed. CYP2C9 gene expression levels and CYP2C9 protein levels in the CYP2C9-silenced hepatocyte were decreased by 76.0% and 78.6% respectively when compared with shRNAc-silenced cells. After TCE treatment, cell cycle in CYP2C9-silenced hepatocytes and L02 cells of 0.5, 2.0, 8.0 mmol/L dose groups had no significantly change. After TCE treatment, Bcl-2 expression levels in CYP2C9-silenced hepatocytes of 2.0 and 8.0 mmol/L dose groups were significantly lower than those of L02, while caspase-3 expression levels in CYP2C9-silenced hepatocytes of 0.5, 2.0, 8.0 mmol/L3 dose groups had no significantly change. [Conclusionl The expression of apoptosis gene Bcl-2 is different in CYP2C9-silenced hepatocytes and normal L02 cells after trichloroethylene treatment, these results indicates that CYP2C9 may play an important role in trichloroethylene metabolism in vivo.