plcεshrna质粒构建及功能签定.docVIP

PAGE PAGE 1 PLCεshRNA质粒构建及功能签定 郭永灿，罗春丽* ,颜令，欧俐苹，赵 懿，蔡晓钟（重庆医科大学医学检验系 临床检验诊断学教育部重点实验室、重庆市重点实验室 重庆　400016） 摘 要：目的 构建PLCε基因的shRNA表达载体及其功能鉴定。方法 设计并合成针对PLCε基因mRNA的寡核苷酸序列，插入绿色荧光蛋白质粒真核表达载体pGenesil中构建重组载体pGenesil-PLCε。重组载体经酶切、测序鉴定后转染膀胱癌细胞， RT-PCR及western blot检测对膀胱癌细胞株PLCε的抑制作用，流式细胞术(FCM)及MTT其对膀胱癌细胞株增殖活力影响。结果 成功构建了针对两个PLCε基因的shRNA表达载体。RT-PCR、Western blot检测显示：两个重组质粒载体转染T24细胞株后对PLCε mRNA及对PLCε蛋白表达均有明显抑制，流式细胞术结果表明细胞阻滞于G0/G1期，MTT检测表明明显抑制T24细胞增殖活力。结论 针对PLCε基因的shRNA能有效抑制膀胱癌T24细胞中PLCε基因的表达及膀胱癌细胞增殖。 关键词：载体构建；膀胱癌；shRNA；PLCε 中图法分类号：R737 文献标识码：A Construction of PLCε shRNA expression vector and functional identification GUO Yong-can，LUO Chun-li*，Yan Ling，OU Li-ping，ZHAO Yi，CAO Xiao-Zhong， (Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing 400016, China) Abstract：Objective The aim of this study was to construct a shRNA expression plasmid against gene PLCε and function identification in bladder cancer line, 基金项目：重庆市教委科学技术研究（KJ080306） 作者简介：郭永灿（1973-）男，四川省泸州市人，硕士研究生，主要从事膀胱癌的基础研究。电话（0830）3114009，E-mail:guoyongcan_2004@163.com. 通讯作者：罗春丽，女，硕士生导师，教授，主要从事于膀胱癌的诊断与防治。电话：（023E-mail:luochunli79@126. Methods Potential RNAi oligonucleotides of PLCε were selected on appropriate web site. Then these oligonucleotides were synthesized and pGenesil-PLCε plasmids were constructed using pGenesil vector with green fluorescent protein（GFP）. The plasmids were digested by SalI and sequenced to confirm the inserted sequence. RT-PCR, Western blot analysis was taken to know about inhibition of PLCε gene expression and it’s fuction in bladder cancer cell line was analysised by MTT and FCM after the constructed plasmids had been transfected bladder cancer cells. Results It was confirmed by digesting and sequencing that the two recombinant plasmids had been constructed successfully. The result of RT-PCR and Western blot showed that pGenesil-PLCε plasmids had inhibited PLCε expression of T24 cells obviously, and and FCM sho