摘要
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 CYPIA2-silenced hepatocyte through RNA interference technology and lentiviral vector, and then to study trichlorethylene （TCE） toxicity in normal human L02 hepatocyte （L02 cell） and CYP1A2-silenced hepatocyte. Methods Short hairpin RNA （shRNA） was designed and ligated to pLKO. 1-puro to construct lentiviral expression vector. L02 cells were transfected. After puromycin screening, CYP1A2-silenced cells were constructed and identified by quantitative poly- merase chain reaction and western blot. Then the CYP1A2-silenced hepatocytes and L02 cells were treated at various doses of TCE for 12 hours to observe the expressions of apoptosis genes （Bcl-2, Caspase-3, Caspase-8, Caspase-9） and oncogenes （c-fos, c-myc, K-ras, p53）. Results Sequencing results showed that the sequence contained in the recombinant vector was exactly the same as shRNA which was designed. CYPIA2 gene expression levels and CYP1 A2 protein levels in the CYP1A2-silenced hepato- cytes decreased by 75.9% and 82.4% respectively when compared with normal L02 cells （ P 〈 0. 01 ）. After TCE treatment, Bcl-2 expression levels in CYPIA2-silenced hepatocytes were significantly_ higher than those of L02 cells（ P 〈 0. 01 ） ; while the mRNA expression levels of Caspase-3 , Caspase-8 , Caspase-9 , c-fos, c-myc , k-ras and p53 decreased in CYPIA2-silcnced hepa- tocytes when compared with L02 cells （ P 〈 0.05 or P 〈 0. 01 ）. Conclusion CYPIA2-silenced reduces the activation of apoptosis genes and oncogenes of hepatocytes which were treated with TCE. CYP1A2 might play an important role in TCE metabolism in vivo.
出处
《中国职业医学》
CAS
北大核心
2013年第4期287-291,296,共6页
China Occupational Medicine
基金
深圳市科技计划重点项目(201101015)
