反转录病毒载体介导RNA干扰抑制EphA2在Hela细胞中的蛋白表达  

Retrovirus vector-mediated RNA interference inhibits EphA2 protein expression in Hela cells

作　　者：吴丹[1] 李珊珊[2] 索振河[3] 

机构地区：[1]上海交通大学医学院附属国际和平妇幼保健院,上海市200030 [2]郑州大学第一附属医院病理科,河南省郑州市450052 [3]威奥斯陆大学附属肿瘤医院癌症研究中心

出　　处：《中国组织工程研究与临床康复》2007年第37期7401-7404,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：教育部"十五";"211工程"重点学科建设项目[教重办(2002)第2号];河南省科技攻关计划项目(0424410033);挪威国家肿瘤医院访问学者基金~~

摘　　要：目的:课题前期发现EphA2和配体EphrinAl在宫颈鳞癌细胞和肿瘤相关血管内皮细胞均呈高表达,为基因治疗宫颈癌提供了理想的靶向。以此为基础,利用反转录病毒载体介导的RNA干扰抑制EphA2在Hela宫颈癌细胞中的蛋白表达。方法:实验于2006-01/06在河南省肿瘤病理重点实验室完成。①实验材料:PA317细胞、Hela229细胞购自上海生化细胞所,小鼠NIH3T3细胞为本室保存。EphA2兔抗人多克隆抗体,Actin兔抗人多克隆抗体(美国SantaCruz公司)。②实验方法:从EphA2的mRNA全序列中筛选出1个19nt的靶序列,设计双链发夹结构。以siRNA互补双链寡核苷酸,构建反转录病毒表达载体pSIREN-EphA2,提取质粒,用BglⅡ和EcoRⅠ双酶切进行鉴定。选择经酶切鉴定的正确克隆测序。挑选pSIREN-EphA2抗性克隆细胞扩增,检测转染后Hela细胞中EphA2蛋白的表达水平。结果:①pSIREN-EphA2重组质粒的酶切鉴定:双酶切后形成328bp和6182bp两条电泳条带。②pSIREN-EphA2重组质粒测序:结果与已知序列完全一致。③EphA2蛋白的表达:经Westernblot分析,重组pSIREN-EphA2转染的Hela细胞中EphA2蛋白表达水平明显降低。结论:通过反转录病毒载体介导的RNA干扰,能够抑制EphA2在Hela细胞中的蛋白表达,为以EphA2为靶点的宫颈癌基因治疗提供新的思路和手段。AIM： It has been found in previous study that EphA2 and EphrinAI protein expressions often high-expressed in tumor cells and vascular endothelial cells in squamous cervical carcinoma, suggesting that EphA2 and EphrinAI could be attractive new targets for therapy of cervical carcinoma. On this basis, by retrovirus vector-mediated RNA interference technique to suppress the endogenous EphA2 oncogene expression in cervical carcinoma Hela cells. METHODS： The experiment was performed at the Henan Tumor Pathology Key Laboratory from January to June 2006.①PA317 cell and Hela229 cell were bought from Shanghai Biochemical Cell Institute. Mouse NIH3T3 cell was collected from this laboratory. EphA2 Antibody and Actin Antibody were purchased from American Santa Cruz Company. ②One 19nt target sequence was selected from EphA2 mRNA to design double strands hairpin structure. Two complementary oligonucleotides with haipin loop for EphA2 siRNA were designed to construct pSIREN-EphA2 vector, and plasmid was extracted and identified by Bgl Ⅱ and EcoR Ⅰdouble enzyme digestion, and correct clone sequence was obtained. The pSIREN-EphA2 cells were selected. EphA2 protein expression was detected in transfected Hela cells. PESULTS：①The pSIREN-EphA2 vector was confirmed by restriction endonuclease digestion. By double-nuclease digestion, 328 bp and 6 182 bp two strip were formed. ②The confirmed pSIREN-EphA2 recombinant plasmid sequence was correct compared with known-sequence. ③The level of EphA2 protein was greatly decreased in Hela cells transfected by the recombinant pSIREN-EphA2. CONCLUSION： EphA2 protein expression in Hela cell line is successfully suppressed by the recombinant retroviru-mediated RNA interference technique, indicating that this may provide a new solution for EphA2-targeting therapeutic intervention of cervical carcinoma.

关 键 词：EPHA2 反转录病毒载体 RNA干扰 

分 类 号：R730[医药卫生—肿瘤]