siRNA靶向Her-2逆转录病毒载体构建及其在SKOV3中的表达  

Construction of Recombinant Retroviral Vector of siRNA Specific for Her-2 and the Expression of Recombinant Vector in SKOV3

作　　者：周颖[1] 凌斌[1] 高婷[1] 陈敏[1] 冯定庆[1] 程志祥[1] 朱园园[1] 赵卫东[1] 石永云[1] 王梅梅[1] 祝怀平[1] 

机构地区：[1]安徽省分子医学重点实验室安徽医科大学附属省立医院分子医学临床研究中心,230001

出　　处：《肿瘤防治研究》2007年第4期277-280,318,共5页Cancer Research on Prevention and Treatment

基　　金：安徽省自然科学基金资助项目(20050430715);安徽省教育厅资助项目(2005KJ347ZC)

摘　　要：目的构建并鉴定Her-2特异性siRNA逆转录病毒载体,将其导入SKOV3卵巢癌细胞中,并初步筛选有效抑制Her-2表达的细胞株。方法体外合成含针对Her-2特异基因序列的寡核苷酸并定向克隆入逆转录病毒载体RNAi-ReadypSIREN-RetroQ,构建的载体通过测序、酶切鉴定后,脂质体介导下转染到包装病毒细胞株PT67中,嘌呤霉素筛选并挑选细胞克隆,收获病毒上清,将病毒上清转染SKOV3中,经过嘌呤霉素筛选得到稳定抑制Her-2表达的SKOV3细胞株,并分别通过RT-PCR和免疫组化方法鉴定抑制Her-2表达的效果。结果成功构建了重组逆转录病毒载体,转染包装病毒细胞株获得的病毒上清成功转染了SKOV3,并且抑制了Her-2的表达。结论抑制Her-2表达的SKOV3细胞株的建立,为观察转染siRNA的逆转录病毒表达载体的肿瘤细胞恶性生物学行为变化奠定了实验基础。Objective To establish the stable SKOV3 cell lines with a persistent knockdown of Her-2 by transfecting the recombinant retroviral vectors of siRNA specific for Her-2 into SKOV3 cell line. Methotis We synthesized oligonucleotides for Her-2 in vitro, and cloned them into retroviral vector RNAi- Ready pSIREN-Retro-Q. Subsequently the plasmids were sequenced and digested to identify the successful construction of the recombinant retroviral vectors. The vectors-based RNAi were transfected into packing cell line PT67, which was selected by puromycin later. SKOV3 was infected by the virus supernatant of stable PT67 cell lines, and the stable SKOV3 cell lines with a persistent knockdown of Her-2 were established by selecting with puromycin, many of which showed significantly reduced levels of Her-2 mRNA and p185 by RT-PCR and immumohistochemistry methods, compared to wild type SKOV3 cells. Results The recombinant retroviral vectors were constructed successfully, and the stable SKOV3 cell lines with a persistent knockdown of Her-2 were established by the infection of virus supernatant secreted by the new stable PT67 cell lines. Conclusion The establishment of stable SKOV3 cell lines with a persistent knockdown of Her-2 contributes to the study of the changes of malignant biological activity of tumor cell lines by transfection of siRNA retroviral vector specific for Her-2.

关 键 词：HER-2 RNA干扰 逆转录病毒载体 

分 类 号：R737.31[医药卫生—肿瘤] Q343[医药卫生—临床医学]