靶向血管内皮生长因子的siRNA表达框架对喉癌细胞株Hep-2细胞生长的抑制作用  

SiRNA expression cassettes targeting VEGF effectively inhibits the growth of laryngeal squamous cell carcinoma cell lines

作　　者：陈雄[1] 孔维佳[1] 董继华[2] 张丹[1] 张甦琳[1] 韩月臣[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院耳鼻咽喉科,武汉430022 [2]华中科技大学同济医学院附属协和医院病毒室,武汉430022

出　　处：《中华耳鼻咽喉头颈外科杂志》2005年第10期759-763,共5页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：国家杰出青年基金资助项目(39925035);教育部高等学校骨干教师资助计划基金资助项目(200065);教育部高等学校博士学科点专项基金资助项目(2002173)

摘　　要：目的研究培养Hep-2细胞中,靶向血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)的小干扰RNA表达框架(smallinterferenceRNAexpressioncassettes,SECs)对肿瘤细胞生长的影响,检测VEGF基因表达的情况,筛选最佳干扰序列,探讨RNAi技术在肿瘤基因治疗中的应用前景。方法根据siRNA靶序列设计原则,设计出VEGF4个位点的干扰序列,然后进行合成,即为siRNA表达框架的下游引物。由于干扰试剂盒中采用RNA介导的U6聚合酶Ⅲ启动子和改良的终止序列,从而在靶细胞内可以高水平及精确地表达siRNA。我们将4个位点的PCR产物分别转染指数生长期的Hep-2细胞,48h开始观察和检测所产生的RNA干扰的效果。结果形态学检测:转染后的1366位点的细胞可见皱缩、变圆并开始脱落,而其他三个位点的细胞形态学上未见明显的改变;逆转录-聚合酶链反应产物进行琼脂糖凝胶电泳,与对照组相比,1366位点细胞VEGFmRNA明显被抑制,吸光度为0·05,而其他三个位点的抑制效果不显著,吸光度分别是0·25、0·30和0·18。Westernblot分析表明:与其他三个位点相比,1366位点的VEGF蛋白表达明显减弱;流式细胞仪检测表明:1366位点转染细胞的凋亡率为43%,而其他三个位点转染细胞的凋亡率改变不明显,分别为8·9%、6·5%和11·2%;本实验重复性强,同样的实验重复三次结果一致。结论研究表明靶向VEGF的1366位点的siRNA表达框架能高效抑制喉癌细胞株Hep-2细胞的生长。Objective To study the effects of siRNA expression cassettes （SECs） targeting VEGF in vitro on cultured Hep-2 cells, the observation of the expression of VEGF, the screening of the best interference sequences, and the exploration of the application of RNA interference on tumor genetberapy in the future. Methods On the basis of the principle of target sequence of siRNA, four interference sequences of VEGF were designed, and the downstream gene-specific primers of the SECs were synthesized. The RNAi transcription kit used U6 RNA-based polymerase Ⅲ promoter and modified terminator for high level, precise siRNA expression inside target cells. The Hep-2 cells was transfected in growth period of index with the PCR products of the four sites separately, and began to observe and measure the results of RNA interference in 48 hours. Results The transfected 1366-site cells creased, turned into the round shape and began to shed off, whereas morphology of the cells of other groups had not obviously changed. Performing the agarose gels electrophoresis with RT-PCR products, compared with the contrast groups, some cells VEGF mRNA of 1366-site were suppressed obviously, the ratio of OD was 0. 05 while the expression of VEGF of the cell of other groups had not obviously changed. Western blot revealed that VEGF expression was decreased obviously pest transfection using 1366-site SECs. Flow cytometry showed that apeptosis rate of 1366-site transfected cells is 43% ,and apoptosis rate of the rest three site transfected cells scarcely changed. Similar results were obtained in three independent experiments. Conclusions The study suggested that siRNA expression cassettes （SECs） targeting VEGF 1366-site can effectively inhibit the growth laryngeal squamous cell carcinoma cell lines （ Hep-2 ）.

关 键 词：喉肿瘤 癌 鳞状细胞 内皮生长因子 基因 抑制 肿瘤 肿瘤细胞 培养的 SIRNA表达 HEP-2细胞 血管内皮生长因子 肿瘤细胞生长 喉癌细胞株 

分 类 号：R739.65[医药卫生—肿瘤]