RNA干扰技术抑制鼻咽癌细胞内源性绿色荧光蛋白的表达  

The inhibition of intrinsic expression of GFP in nasopharyngeal carcinoma by RNA interferencing

作　　者：王祎琴[1] 彭惠[2] 洪苏玲[1] 

机构地区：[1]重庆医科大学附属第一医院耳鼻咽喉头颈外科,重庆400016 [2]重庆医科大学附属第一医院眼科,重庆400016

出　　处：《重庆医科大学学报》2007年第8期822-825,834,共5页Journal of Chongqing Medical University

摘　　要：目的:建立稳定表达绿色荧光蛋白(GFP)的鼻咽癌细胞株;观察不同转染浓度的短发卡状RNA对鼻咽癌细胞内源性GFP的抑制效率及细胞毒性。方法:转染pEGFP-N1至鼻咽癌细胞株HNE1,G418筛选获得稳定表达GFP的HNE1-GFP细胞株;利用针对GFP基因的短发卡状RNA表达载体shGFP,以不同浓度shGFP转染HNE1-GFP,荧光显微镜观察细胞荧光强度,软件分析荧光抑制效率,MTT检测细胞毒性,RT-PCR检测GFPmRNA水平改变,Western Blot检测GFP蛋白水平改变。结果:绿色荧光稳定表达于传代HNE1细胞中;shGFP能够显著抑制该细胞中GFP的表达,抑制效率分别为49.7%,84.8%,86.7%且无细胞毒性;GFP在mRNA和蛋白水平的表达均有明显下降。结论:稳定表达GFP的鼻咽癌细胞株成功建立;RNA干扰载体可抑制鼻咽癌细胞中GFP的表达,其抑制效率有明显的浓度依赖性且无明显细胞毒性。该系统能够用于鼻咽肿瘤的RNA干扰机制研究中。Objective：To construct a nasopharyngeal carcinoma cell strain expressing GFP stably and observe the inhibition effect and cytotoxicity of short hairpin RNA targeting the intrinsic GFP. Methods：HNE1 cells transfected with pEGFP-N1 were followed by the screening with G418 to obtain the stable nasopharyngeal carcinoma cell strain expressing GFP（HNE1-GFP）; shGFP,a vector expressing short hairpin RNA targeting GFP,was transfected into HNE1-GFP with various levels. The fluorescence was observed by fluorescent microscope. The inhibition ratios and cytotoxicity of shRNA were analyzed by software and MTr,respectively;The protein and mRNA level of GFP were detected by Western Blot and RT-PCR,respectively. Results： The GFP gene was expressed stably within HNE1-GFP cell line;shGFP suppressed the intrinsic expression of GFP with the ratios of 49.7%,84.8% and 86.7% ,respectively, without significant cytotoxicity. Conclusion：A nasopharyngeal carcinoma cell strain expressing GFP stably is established successfully; The inhibition effect of RNA interference is demonstrated in nasopharyngeal carcinoma cells and it is characterized by the significant concentration dependence and rare cytotoxicity.

关 键 词：鼻咽肿瘤 绿色荧光蛋白 短发卡状RNA RNA干扰 

分 类 号：R739.63[医药卫生—肿瘤]