CAJ | 학술논문

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(Western blot)鉴定干扰效果.以不同浓度(0、0.25、0.50、1.00、2.00、4.00 mmol/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15.1 mmol/L,CYP2E1沉默细胞对TCE的IC50为23.6 mmol/L.随TCE剂量增加两组细胞凋亡率升高,2.0、4.0 mmol/L TCE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＜0.05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(P＜0.01).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.
목적 통과RNA간우기술,구건세포색소양화매(CYP)2E1기인만병독표체재체,연구CYP2E1기인침묵대삼록을희(TCE)치간세포독성적영향.방법 설계합성shRNA편단련접도만병독재체,사선단균락,경취합매련식반응(PCR)화측서감정후제취질립,전도L02간세포중,사선CYP2E1결함형세포,이용형광정량PCR화면역단백인적법(Western blot)감정간우효과.이불동농도(0、0.25、0.50、1.00、2.00、4.00 mmol/L)TCE분별대L02간세포화CYP2E1기인침묵세포염독,측정TCE대2충세포적존활솔급IC50,응용류식세포의측정세포조망솔,채용실시형광정량PCR검측세포조망기인화암기인mRNA표체수평.결과 정상L02간세포대TCE적IC50위15.1 mmol/L,CYP2E1침묵세포대TCE적IC50위23.6 mmol/L.수TCE제량증가량조세포조망솔승고,2.0、4.0 mmol/L TCE염독제량시,CYP2E1침묵세포적조망솔명현저우L02간세포조,차이유통계학의의(P＜0.05혹P＜0.01).TCE염독후CYP2E1침묵세포적항조망기인Bcl-2표체수평비L02간세포승고15％～60％,조망기인caspase-3화caspase-9표체수평비L02간세포하강30％～60％,차이균유통계학의의(P＜0.01).억암기인p53표체수평명현고우L02간세포,승고81％～278％;암기인c-fos화k-ras명현저우L02간세포조,하강20％～68％,차이균유통계학의의(P＜0.01).결론 이용만병독개도RNA간우기술성공구건료CYP2E1기인침묵세포.침묵CYP2E1기인가강저TCE대간세포독성,억제부분조망기인여암기인표체,제시CYP2E1기인재삼록을희대사과정중발휘중요작용,여삼록을희독성존재일정관계.
Objective To prepare cytochrome(CYP)2E1-silenced hepatocytes by lentivirus-mediated RNA interference technology and to investigate the hepatotoxicity of trichlorethylene (TCE) in CYP2E1-si lenced hepatocytes.Methods Short hairpin RNA fragments were designed and synthesized and were then ligated into the lentiviral vector; single colonies were screened; the plasmid was extracted after PCR and sequence identification and then transferred into L02 hcpatocytes; the CYP2E 1-silenced hepatocytes were selected; realtime quantitative PCR and Western blot were used to evaluate the interference effects.The obtained CYP2E1silenced 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 CYP2E1-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 CYP2E 1-silenced hepatocytes had a significantly lower apoptotic 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 CYP2E1-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 (apoptosis genes) in CYP2E1-silenced hepatocytes were 30％～60％ lower than those in L02 hepatocytes (P＜0.01).The mRNA expression level of p53 (cancer suppressor gene) in CYP2E 1-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 CYP2E1-silenced hepatocytes were 20-68％ lower than those in L02 hepatocytes (P＜0.01).Conclusion CYP2El-silenced cells can be successfully prepared by lentivirus-mediated RNA interference technology.Silencing CYP2E1 gene can reduce the hepatotoxicity of TCE and inhibit the expression of some apoptosis genes and oncogenes,suggesting that CYP2E1 gene plays an important role in TCE metabolism and is related to the hepatotoxicity of TCE.