脂质体RNAiMAX介导siRNA转染沉默胰岛素瘤NIT-1细胞血管紧张素Ⅱ1型受体基因的实验研究  

SILENCING ANGIOTENSINⅡ TYPE 1 RECEPTOR GENE IN NIT-1 INSULINOMA CELLS THROUGH TRANSFECTION OF SIRNAS BY LIPOFECTAMINE RNAIMAX

作　　者：莫丽雯[1] 卢德成[1] 李海滨[2] Mo Liwen Lu Decheng Li Haibin(Department of Endocrinology, the First Affiliated Hospital of Guan- gxi Medical University, Nanning 530021 , China)

机构地区：[1]广西医科大学第一附属医院内分泌科,南宁530021 [2]中国人民解放军第303医院,南宁530021

出　　处：《广西医科大学学报》2016年第5期770-773,共4页Journal of Guangxi Medical University

基　　金：广西自然科学基金资助项目(No.2013GXNSFAA019253)

摘　　要：目的:探讨脂质体RNAiMAX能否介导小干扰RNA(siRNA)转染入胰岛素瘤NIT-1细胞并实现血管紧张素Ⅱ1型受体(AT1R)的基因沉默,同时评价其细胞毒性。方法:针对AT1R,设计合成3对siRNA序列,转染NIT-1细胞,荧光显微镜下观察RNAiMAX能否介导荧光分子Cy3标记的siRNA转入细胞内,以及不同siRNA浓度对转染效率的影响;Real-time PCR检测AT1R mRNA的相对表达量,计算基因沉默效率并探讨不同干扰序列及干扰时间对其影响;流式细胞仪检测si-AT1R组、脂质体组和空白组的凋亡率。结果:荧光显微镜观察发现,在一定范围内,5个浓度siRNA转染时,细胞荧光强度均达90%以上,以40nmol/L的荧光强度最高;AT1R的3对序列(si-AT1R-1、si-AT1R-2、si-AT1R-3)转染24h沉默效率分别为86.07%、92.20%、74.67%,与阴性对照组比较差异有统计学意义(P<0.05),以si-AT1R-2的沉默效率最高,检测其24h、48h、72h的沉默效率,显示24h的沉默效率最高;各组细胞凋亡率比较,差异均无统计学意义(均P>0.05)。结论:脂质体RNAiMAX可以介导siRNA转入NIT-1细胞中并实现AT1R基因的沉默,40nmol/L si-AT1R-2转染24h可达到理想的沉默效果;此方法对细胞基本无毒性作用。Objective： To determine whether transfection of small interference RNAs （siRNAs） by lipo- fectamine RNAiMAX could silence angiotensin Ⅱ type 1 receptor （AT1R） gene in NIT-1 insulinoma cells. Methods： Three pairs of siRNA targeting to AT1R were designed and synthesized. It was transfected into NIT-1 cells by lipofectamine RNAiMAX. The transfection efficiency of Cy3-labeled siRNAs at different concentration was evaluated by fluorescence microscope. The mRNA level of AT1R gene was detected by Real-time PCR to calculate the gene silencing efficiency and determine the effect of different siRNA and treating times. The apoptosis rate of si-ATIR groups, control group and blank group flow cytometry. Results： The percentages of fluorescent cells were all over 90% in cells transfected with each siRNAs with five different concentrations, and the highest percentage was obtained at 40 nmol/L. The inhibitory effects of si-ARTR-1 to 3 were 86. 07%, 92. 20% and 74. 67 % at 24 h after transfection. The time course at 24 h, 48 h and 72 h were also analyzed. The maximum inhibitory effect was achieved by transfection with si-ATTR-2 at 24 h. There was no obvious significant difference in the apoptosis rate a-mong groups （ P 〈0. 05）. Conclusion： AT1R mRNA level was significantly decreased after transfection of targeted siRNAs indicates that using siRNA can be successfully transfected into NIT-1 cells and silencedAT1R gene by using lipofectamine RNAiMAX. This method is non-toxic for cells and is quent research.

关 键 词：NIT-1细胞 RNA干扰 血管紧张素Ⅱ1型受体 基因沉默 

分 类 号：R587.1[医药卫生—内分泌]