肿瘤特异性Surivivin启动子调控FAKshRNA靶向治疗肝癌实验研究.docVIP

肿瘤特异性Surivivin启动子调控FAKshRNA靶向治疗肝癌实验研究 　　【摘要】 目的：探?肿瘤特异性Surivivin启动子调控的FAK shRNA靶向治疗肝癌的疗效。方法：根据GenBank中Surivivin基因序列设计引物，在上下游引物5’端引入相应酶切位点，以HepG2基因组DNA为模板扩增Surivivin启动子基因片段。体外合成最小polyA转录中止信号，以pSUPER.retro.puro载体为媒介构建新的干涉载体pS-surP-shRNA-mpA（pS/surP），完成干涉靶点的选取并完成干涉载体的构建和鉴定；建立细胞培养和转染及稳定转染细胞系，完成FAK RNAi的干扰效应性检测，进行体外细胞增殖及凋亡实验。结果：重组质粒完成DNA提取后，PCR扩增，经琼脂糖凝胶电泳后获得1条预期大小的特异性皮带，质粒Surivivin启动子调控的FAK shRNA涉及符合要求。将目的片段与肿瘤特异性Surivivin启动子调控的FAK shRNA慢性载体表达载体连接的阳性克隆序列，获得DNA序列证实含有目的序列片段，提示质粒构建成功；PCR仪结果显示：Surivivin启动子调控的FAK shRNA在肝癌细胞中表达水平较低，抑制率能达到73.33%；Western blotting法结果显示：转染后，与未转染的HepG2细胞相比，转染后肝癌细胞Surivivin含量明显下降；流式细胞仪结果表明：细胞转染后，肿瘤特异性Surivivin启动子调控的FAK shRNA细胞凋亡率为24.39%，与未转染细胞的4.12%比较，差异有统计学意义（P0.05）。结论：肿瘤特异性Surivivin启动子调控的FAK shRNA靶向治疗肝癌可明显抑制肿瘤细胞的增殖，控制肿瘤的侵袭与转移。 　　【关键词】 肿瘤特异性； Surivivin启动； FAK shRNA； 靶向治疗 　　【Abstract】 Objective：To investigate the therapeutic effects of tumor specific Surivivin promoter targeting FAK and shRNA on hepatocellular carcinoma.Method：Primers were designed according to the Surivivin gene sequence in GenBank，and the corresponding restriction sites were introduced at the 5th and the end of primer DNA.The promoter fragment of Surivivin was amplified by HepG2 genomic DNA template.In vitro synthesis of polyA transcription termination signal to the minimum，pSUPER.retro.puro vector mediated interference vector pS-surP-shRNA-mpA （pS/surP），a new interference target selection and complete the construction and identification of interference vector.Establish the cell culture and transfection and stable transfection cell lines，to complete the interference effect of detecting FAK RNAi，cell proliferation and apoptosis experiments in vitro.Result：After DNA extraction，the recombinant plasmid was amplified by PCR，and 1 expected size specific bands were obtained by agarose gel electrophoresis.FAK shRNA regulated by plasmid Surivivin promoter met the requirement.FAK shRNA chronic carrier of the fragment and tumor specific Surivivin promoter and expression of positive clone