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目的构建细胞色素氧化酶1A2(CYP1A2)基因沉默载体,转染L02人正常肝细胞(简称“L02细胞”),建立CYP1A2沉默细胞株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),连接到pLKO.1-puro质粒中构建CYP1A2 shRNA慢病毒表达载体,感染L02细胞。筛选CYP1A2沉默细胞株,采用荧光定量聚合酶链式反应和免疫印迹法鉴定。分别采用浓度为0.25、0.50、1.00、2.00和4.00 mmol/L的TCE(设为相应的染毒剂量组)对L02细胞和CYP1A2沉默细胞染毒12 h,同时设置二甲基亚砜溶剂对照组(即0.00 mmol/L染毒剂量组),观察凋亡基因[B淋巴细胞瘤-2基因(Bcl-2)、天冬氨酸特异性半胱氨酸蛋白酶(Caspase)-3、Caspase-8、Caspase-9]和癌基因(c-fos、c-myc、K-ras、p53)等基因表达的变化。结果 测序证明插入pLKO.1-puro载体的CYP1A2基因干扰序列与设计的shRNA序列一致。CYP1A2沉默细胞的CYP1A2 mRNA和CYP1A2蛋白相对表达水平分别较L02细胞下降75.9%和82.4%(P<0.01)。TCE染毒后,0.25～4.00 mmol/L 4个染毒剂量组CYP1A2沉默细胞Bcl-2表达水平均高于相应染毒剂量组L02细胞(P<0.01);部分0.25～4.00 mmol/L染毒剂量组Caspase-3、Caspase-8、Caspase-9和c-fos、c-myc、k-ras、p53相对表达水平均低于相应染毒剂量组的L02细胞(P<0.05或P<0.01)。结论 CYP1A2沉默降低TCE对L02细胞凋亡基因和癌基因的活化作用。
Objective To construct CYP1A2 gene silencing vector and transfect L02 human normal hepatocyte (“L02 cell”) to establish CYP1A2 silencing cell line and observe its effect on the toxicity of trichlorethylene (TCE). Methods Short hairpin RNA (shRNA) was synthesized and ligated into plasmid pLKO.1-puro to construct CYP1A2 shRNA lentivirus expression vector to infect L02 cells. The CYP1A2 silenced cell line was screened and identified by fluorescence quantitative polymerase chain reaction and Western blotting. L02 cells and CYP1A2 silencing cells were exposed to TCE (0.25%, 0.50, 1.00, 2.00 and 4.00 mmol / L, respectively) for 12 h and dimethyl sulfoxide solvent control group (0.00 mmol / L). The expression of Bcl-2, Caspase-3, Caspase-8 , Caspase-9] and oncogenes (c-fos, c-myc, K-ras, p53) Results Sequencing proves that the interference sequence of CYP1A2 inserted into pLKO.1-puro vector is consistent with the designed shRNA sequence. The relative expression level of CYP1A2 mRNA and CYP1A2 protein in CYP1A2-silencing cells decreased by 75.9% and 82.4% (P <0.01) compared with L02 cells respectively. After TCE exposure, the expression levels of Bcl-2 in CYP1A2-silenced cells were significantly higher than those in L02 cells exposed to 0.25 ~ 4.00 mmol / L 4 doses (P <0.01) The relative expression levels of Caspase-3, Caspase-8, Caspase-9 and c-fos, c-myc, k-ras and p53 in the dose group were lower than those in the corresponding dose group (P <0.05 or P <0.01) . Conclusion CYP1A2 silencing can reduce the activation of TCE on L02 apoptosis gene and oncogene.