稳定干扰αv整合素表达的HT-29细胞系的建立  

Construction of colon cancer HT-29 cell line with αv integrin stably silenced

作　　者：何建明[1] 江恒[1] 庞学利[1] 肖红[1] 陈克力[1] 胡绍毅[1] 黄海辉[1] 梁后杰[1] 

机构地区：[1]第三军医大学西南医院肿瘤科,重庆400038

出　　处：《临床肿瘤学杂志》2011年第4期289-293,共5页Chinese Clinical Oncology

基　　金：国家自然科学基金资助项目(81000990);重庆市自然科学基金资助项目(CSTC2009;BB5339);第三军医大学青年创新人才基金资助项目(2009XQN32)

摘　　要：目的探讨逆转录病毒介导RNA干扰(RNAi)稳定抑制αv整合素表达的有效性,并建立稳定抑制αv整合素表达的结肠癌HT-29细胞系。方法设计并合成3对针对αv整合素基因的短发夹RNA(shRNA)序列,构建逆转录病毒质粒;脂质体转染逆转录病毒质粒至结肠癌细胞株HT-29;G418筛选,局部消化法挑选阳性克隆;Western blotting检测蛋白表达水平。结果琼脂糖凝胶电泳结果显示,构建成功的逆转录病毒载体被HindⅢ限制性内切酶切为两个分别长1950bp与5800bp片段。测序结果显示,针对αv整合素基因的3对shRNA序列皆成功插入逆转录病毒质粒;转染逆转录病毒载体的HT-29细胞持续表达绿色荧光蛋白,并对G418具抗性;Western blotting结果显示逆转录病毒介导的shRNA能有效地抑制αv整合素表达。结论逆转录病毒介导的shRNA能够高效、稳定地抑制αv整合素表达,进而可以建立稳定干扰αv整合素表达的结肠癌HT-29细胞系。Objective To explore efficiency and specificity of gene silence by retroviral delivery of short hairpin RNA（shRNA） targeting for αv integrin and to construct colon cancer HT-29 cell line with αv integrin stably silenced.Methods Three pairs of specific DNA sequences encoding shRNA targeting for αv integrin were synthesized.Retroviral vectors expressing αv integrin specific shRNA were constructed and were transfected colon cancer cell line HT-29 by liposome.Transfected cells were selected by G418.Clones were transferred by local trypsin digestion.Western blotting was employed to detect the expression level of target protein.Results Agarose gel electrophoresis of retroviral plasmid incised with restriction enzyme showed that constructed retroviral plasmids were incised into two segments,one was 1950bp and the other was 5800bp.Sequencing showed retroviral vectors which could express shRNA targeting αv integrin was constructed.HT-29 which was transfected with retroviral vector stably expressed enhanced green fluorescent protein gene and was resistant to G418.Result from Western blotting showed that expression level of αv integrin notably decreased by transfection constructed retrovirus vectors expressing shRNA.Conclusion Retroviral delivery of shRNA can effectively and stably silence αv integrin gene and colon cancer HT-29 cell line with αv integrin stably silenced was constructed.

关 键 词：结肠癌 整合素 逆转录病毒 RNAI 

分 类 号：R735.3[医药卫生—肿瘤]