过表达人RI抑制人膀胱癌T24细胞的侵袭,转移及EMT发生.docVIP

过表达人RI抑制人膀胱癌T24细胞的侵袭，转移及EMT发生 姚 雪，熊冬梅，舒 静，李 林，陈俊霞（400016 重庆，重庆医科大学基础医学院细胞生物学与遗传学教研室，分子医学与肿瘤研究中心） [摘要] 目的 检测过表达人核糖核酸酶抑制因子( ribonuclease inhibitor，RI) 基因的质粒pIRES2-EGFP-RI对人膀胱癌T24细胞侵袭转移及EMT现象的影响。方法 将连有RI的质粒pIRES2-EGFP-RI稳定转染T24细胞后利用G418筛选出阳性克隆。根据转染的质粒不同将各组分别命名为T24细胞组（未转染组），空质粒对照组（T24-0组）和实验组（T24-RI组）。RT-PCR检测T24细胞中RI的mRNA水平表达变化，细胞免疫荧光及Western blot检测RI蛋白水平的表达，利用HE染色观察其细胞形态的变化，Transwell方法检测细胞侵袭及转移能力的变化，Western blot检测侵袭转移及EMT相关蛋白Twist、MMP-9 和Snail及E-cadherin的表达，构建移植瘤模型，利用HE染色观测肺组织切片癌细胞转移情况，组织免疫化学方法检测移植瘤中RI、Twist、Snail 及E-cadherin的表达。结果 实验组人核糖核酸酶抑制因子的mRNA水平及蛋白水平表达显著升高(P＜0.05)，HE染色结果表明细胞铺展良好核质比明显减小，形态有间质形态向上皮形态转变，Transwell方法显示实验组细胞的的侵袭及转移能力较T24细胞组，空质粒对照组明显下降，Western blot结果表明Twist、MMP-9 和Snail较T24细胞组，空质粒对照组显著降低(P＜0.01),E-cadherin 较T24细胞组，空质粒对照组显著升高(P＜0.01)，HE染色肺组织切片显示实验组较两对照组间，癌细胞转移降低，组织免疫化学结果表明实验组RI、E-cadherin 蛋白明显升高，而Twist、Snail显著降低。结论 过表达人核糖核酸酶抑制因子( ribonuclease inhibitor，RI)载体提高了人核糖核酸酶抑制因子蛋白的表达水平，并抑制了人膀胱癌T24细胞EMT的发生，从而抑制了膀胱癌T24细胞的侵袭转移。 [关键词] 人核糖核酸酶抑制因子；T24细胞；细胞转移；细胞侵袭；EMT [中图法分类号] [文献标志码] A Over-expression of human ribonuclease inhibitor suppresses invasion, migration and EMT in human bladder cancer T24 cells Yao Xue, Xiong Dongmei, Shu Jing, Li Lin, Chen Junxia (Department of Cell Biology and Genetics, Molecular Medicine and Cancer Research Center, College of Basic Medical Sciences, Chongqing Medical University, Chongqing, 400016, China) [Abstract］Objective To observe the effect of over-expression of human ribonuclease inhibitor (RI) on invasion, migration and epithelial mesenchymal transition (EMT) in human bladder cancer T24 cells. Methods The vectors of pIRES2-EGFP (T24-0 group) and pIRES2-EGFP-RI (T24-RI group) were used to transfect human bladder cancer T24 cells, respectively, and the positive clones were screened by G418. The T24 cells without transfection were served as control (T24 group). The expression of RI gene at mRNA and protein levels was evaluated by RT-PCR, immunofluorescence and Western blot. HE staining was use