稳定沉默NF kappa B p65 HT 29细胞系建立及其裸鼠种植瘤药物敏感性的检测  

Construction of HT 29 Cell Line with Stably Silenced NF kappa B p65 and Detection of Its Drug Sensitivity

作　　者：何建明[1] 江恒[1] 陈克力[1] 庞学利[1] 黄海辉[1] 肖红[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：《肿瘤预防与治疗》2009年第4期363-366,I0001,共5页Journal of Cancer Control And Treatment

基　　金：国家自然科学基金资助(项目批准号30171067)

摘　　要：目的:利用逆转录病毒介导的ARNA稳定沉默NF kappa B p65基因,观察沉默NF kappa B p65对HT29细胞药物敏感性的影响。方法:设计并合成针对p65基因的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筛选阳性细胞,局部消化法挑选单克隆;Western blot检测蛋白表达水平;于裸鼠皮下接种细胞形成种植瘤。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 Hind Ⅲ限制性内切酶切为两个分别长1950bp与5800 hp片段。测序结果显示,针对NF kappa B p65基因的shRNA序列成功插人逆转录病毒质粒。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 blot结果显示逆转录病毒介导的shRNA有效沉默NF kappa B p65基因。经化疗后,沉默NF kappa B p65基因HT 29组的裸鼠种植瘤显著性低于HT 29组。结论:逆转录病毒介导的shRNA能高效、特异、稳定沉默NF kappa B p65基因,提高结肠癌裸鼠种植瘤对奥沙利铂的药物敏感性。Objective： To explore efficiency and specificity of gene silence by retroviral delivery of shRNA targeting for NF kappa B 1365 and to detect the effect of NF kappa B p65 silencing on drug sensitivity of HT 29. Methods ： Specific DNA sequences encoding shRNA were synthesized. Retroviral vector expressing NF kappa B p65 specific shRNA was constructed and was transfected colon carcinoma cell line, HT 29, by liposome. Transfected cells were selected by G418. Clones were transferred by local trypsin diges- tion. Western blot was employed to detect the expression level of target protein. Colon carcinoma xenografts were used to detect drug sensitivity. Results： Agarose gel electrophorcsis of retroviral plasmid incised with restriction enzyme showed that constructed retroviral plasmid was incised into two segments, one was 1950bp and the other was 5800bp. Sequencing showed retroviral vector which could express shRNA targeting NF kappa B p65 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 blot showed that expression level of NF kappa B p65 notably decreased by transfection constructed retrovirns vectors expressing shRNA. Both treated with oxaliplatin, HT 29 with NF kappa B p65 silenced were significantly lighter than HT 29. Conclusion： Retroviral delivery of shRNA can effectively, specifically and stably silence NF kappa B p65 gene and enhance drug sensitivity to oxaliplatin of colon carcinoma xenografts.

关 键 词：结肠癌 NF KAPPA B 逆转录病毒 RNAi 

分 类 号：R735.35[医药卫生—肿瘤]