摘要
目的实验研究靶向转染survivin的小分子干扰RNA（small interfering RNA，siRNA）对膀胱癌生物学行为的影响。方法设计1对survivin编码基因序列特异的siRNA，用脂质体转染T24膀胱癌细胞株。采用流式细胞术检测转染效率和细胞凋亡；荧光实时定量PCR检测干扰前后survivin的mRNA表达；用survivin的siRNA片段插入空载体后建立重组载体pRNAT-U6、1／Neo-survivin。结果脂质体转染T24细胞后的转染效率为92．3％；survivin编码基因序列特异的siRNA能有效下调survivin基因的表达，呈时间和剂量依赖性，最大效应浓度为100nmol／L，此时survivin表达水平下调了75．91％，并显著地抑制了细胞生长，抑制率达55．29％，差异有统计学意义（P〈0．01）；细胞凋亡率亦增至45．70％，与对照相比差异有统计学意义（P〈0．01）。用限制性内切酶将空载体线性化，T4DNA连接酶将siRNA片段插入空载体中，对该重组表达载体进行鉴定，获得RNA干扰质粒载体pRNAT-U6．1／Neo-survivin。结论siRNA．survivin能显著下调膀胱癌细胞survivin基因表达水平，促进细胞凋亡，抑制肿瘤细胞增殖，可能成为膀胱癌基因治疗的新工具。成功构建的靶向survivin基因表达的RNA干扰质粒载体为进一步应用RNA干扰技术进行survivin基因功能研究奠定了基础。
Objective To study the influence of small interfering RNA （siRNA） targeting survivin on the biological behavior of bladder cancer. Methods One pair of survivin target sequence-specific siRNA was designed, then siRNA/liposome complex was used to transfect bladder cancer cell hne-T24. The efficiency of transfection and the apoptosis were detected by flow cytometry, The transcriptional level of survivin was analyzed using real time PCR, DNA sequence corresponding to siRNA targeting survivin was cloned into pRNAT-U6.1/Neo to produce plasmid targeting survivin. Results The ratio of 19,4 cells releasing fluorescence in total cells were 92.3 % ; siRNA-survivin efficiently downregulated survivin expression （mRNA） in a dose-and time-dependent manner. Its maximal effect was achieved at the concentration of 100 nmol/L, at which survivin expression level was down regulated by 75.91%. Similar results were found in the inhibition ratio of cell growth, which was 55.29% （ P 〈 0.01 ）. Simultaneously the apoptotic rate was markedly increased, which was 45.70%（ P 〈0.01）. After cutting the vector with BamH I and Hind Ⅲ and ligating the vector with the insert by using T4 ligase, the recombinant vector was confirmed by restriction digestion and DNA sequencing. The application of siRNA-survivin can markedly inhibit survivin expression in bladder cancer ceil hne, induce apoptosis and inhibit the growth of the tumor. It may be a new gene therapy tool for bladder cancer. The successful construction of the siRNA expressing plasmid will facilitate the apphcation of RNA interference technique, and lay a foundation for further studies on the function of survivin.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2007年第4期401-404,共4页
Chinese Journal of Medical Genetics
基金
江苏省重点人才基金（RC2003094）
江苏省卫生厅科技基金（H200517）
江苏省六大高峰人才项目（06-B-021）
江苏省科技厅社会发展基金（BS2005620）
