细胞色素氧化酶2E1基因沉默对三氯乙烯致L02肝细胞毒性的影响  

Effects of trichlorethylene on hepatotoxicity in cytochrome2El-silenced hepatocytes

作　　者：徐新云[1] 毛吉炎[1] 毛侃琅[1] 刘国红[1] 慈捷元[1] 杨细飞[1] 吴德生[1] 黄海燕[1] 张然[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,518055

出　　处：《中华劳动卫生职业病杂志》2013年第3期172-177,共6页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：深圳市科技计划重点项目（201101015）

摘　　要：目的通过RNA干扰技术，构建细胞色素氧化酶（CYP）2E1基因慢病毒表达载体，研究CYP2E1基因沉默对三氯乙烯（TCE）致肝细胞毒性的影响。方法设计合成shRNA片段连接到慢病毒载体，筛选单菌落，经聚合酶链式反应（PCR）和测序鉴定后提取质粒，转导L02肝细胞中，筛选CYP2E1缺陷型细胞，利用荧光定量PCR和免疫蛋白印迹法（Westernblot）鉴定干扰效果。以不同浓度（0、0．25、0．50、1．00、2．00、4．00mmol/L）TCE分别对L02肝细胞和CYP2E1基因沉默细胞染毒，测定TCE对2种细胞的存活率及IC50，应用流式细胞仪测定细胞凋亡率，采用实时荧光定量PCR检测细胞凋亡基因和癌基因mRNA表达水平。结果正常L02肝细胞对TCE的IC50为15．1mmol／L，CYP2E1沉默细胞对TCE的IC50为23．6mmol／L。随TCE剂量增加两组细胞凋亡率升高，2．0、4．0mmol／LTCE染毒剂量时，CYP2E1沉默细胞的凋亡率明显低于L02肝细胞组，差异有统计学意义（P〈O．05或P〈0．01）。TCE染毒后CYP2E1沉默细胞的抗凋亡基因Bcl-2表达水平比L02肝细胞升高15％-60％，凋亡基因caspase-3和caspase-9表达水平比L02肝细胞下降30％-60％，差异均有统计学意义（尸〈O．01）。抑癌基因p53表达水平明显高于L02肝细胞，升高81％-278％；癌基因c．fos和k-ras明显低于L02肝细胞组，下降20％～68％，差异均有统计学意义（P〈0．01）。结论利用慢病毒介导RNA干扰技术成功构建了CYP2E1基因沉默细胞。沉默CYP2E1基因可降低TCE对肝细胞毒性，抑制部分凋亡基因与癌基因表达，提示CYP2E1基因在三氯乙烯代谢过程中发挥重要作用，与三氯乙烯毒性存在一定关系。Objective To prepare cytochrome（CYP）2El-silenced hepatocytes by lentivirus-mediated RNA interference technology and to investigate the hepatotoxicity of trichlorethylene （TCE） in CYP2EI-si- lenced hepatocytes. Methods Short hairpin RNA fragments were designed and synthesized and were then lig- ated into the lentiviral vector; single colonies were screened; the plasmid was extracted after PCR and sequence identification and then transferred into L02 hepatocytes; the CYP2El-silenced hepatocytes were selected; real- time quantitative PCR and Western blot were used to evaluate the interference effects. The obtained CYP2E1- silenced hepatocytes, as well as normal L02 hepatocytes, were treated with TCE （0, 0.25, 0.50, 1.00, 2.00, and 4.00 mmol/L）. The cell viability and half maximal inhibitory concentration （ICs0） of TCE were measured; the apoptotic rate of cells was measured by flow cytometry; the mRNA expression levels of apoptosis genes and oncogenes were measured by real-time quantitative PCR. Results The IC50s of TCE for L02 hepatocytes and CYP2El-silenced hepatocytes were 15.1 mmol/L and 23.6 mmol/L, respectively. The apoptotic rate increased as the dose of TCE rose in the two types of cells; the CYP2El-silenced hepatocytes had a significantly lower apop- totic rate than L02 hepatocytes when they were exposed to 2.0 and 4.0 mmol/L TCE （P〈0.05 or P〈0.01 ）. The mRNA expression level of bcl-2 （anti-apoptosis gene） in CYP2El-silenced hepatocytes was 15%-60% higher than that in L02 hepatocytes （P〈0.01 ）, while the mRNA expression levels of caspase-3 and caspase-9 （apopto- sis genes） in CYP2El-silenced hepatocytes were 30%-60% lower than those in L02 hepatocytes （P〈0.01）. The mRNA expression level of p53 （cancer suppressor gene） in CYP2El-silenced hepatocytes was 81-278% higher than that in L02 hepatocytes （P〈0.01）, while the mRNA expression levels of c-fos and k-ras （oncogenes） in CYP2El-sileneed hepatocytes were 20-68% lower than those in L02 hepatocytes （P�

关 键 词：三氯乙烯 细胞色素氧化酶2E1 RNA干扰 慢病毒载体 基因沉默 

分 类 号：R9[医药卫生—药学]