人S100A6基因真核表达质粒的构建及RNA干扰靶点筛选

发布时间：2018-01-19 00:08

  本文关键词： S100A6基因 短发夹RNA 真核表达质粒 RNA干扰 实时荧光定量PCR 靶点筛选　出处：《大连医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究背景：胃癌是最常见的消化道恶性肿瘤之一，化疗是进展期胃癌最重要的治疗措施，然而，由于肿瘤多药耐药(multidrug resistance，MDR)的产生，常常导致化疗失败，因此，深入研究胃癌MDR形成的机制，从而采取RNA干扰、基因过表达等方法逆转胃癌MDR，将有可能给胃癌基因治疗带来新的突破。 RNA干扰(RNAinterference，，RNAi)是由双链RNA(double strand RNA,dsRNA)诱发的序列特异的转录后基因沉默（PTGS）过程。期间，双链RNA被特异的核酸酶降解成小干扰RNA(small interfering RNA，siRNA)，小干扰RNA与靶蛋白结合成RNA诱导的沉默复合物(siRNA-induced silencing complex，RISC)，RISC识别并降解靶mRNA，产生有效的基因沉默。RNA干扰技术具有相当高的特异性和高效性，并且操作简单、周期短、成本低。目前，该技术已成为研究基因功能、认识疾病本质的重要工具，将来更有望用于疾病的基因靶向治疗，具有相当广阔的临床应用前景。 此前,本课题组以人胃癌细胞株SGC7901、阿霉素诱导的耐药细胞株SGC7901/ADR和丹参逆转后的耐药细胞株为研究对象，应用蛋白质组学技术对三者进行蛋白质组学的差异比较，并且通过高效液相色谱电喷雾电离飞行串联质谱对差异蛋白质进行鉴定，结果发现，S100A6在SGC7901中呈现高表达，在SGC7901/ADR中表达下调，在丹参逆转后的耐药细胞株中再次呈现高表达；并采用免疫细胞化学方法、Western blot方法和RT-PCR技术再次验证了S100A6的差异表达。本研究以S100A6高表达的人胃癌细胞株SGC7901为研究对象，运用RNA干扰技术，将构建S100A6-shRNA质粒载体并转染入SGC7901细胞，筛选有效的RNA干扰片段，为有效下调S100A6在SGC7901细胞中的表达，并进一步探讨S100A6与胃癌MDR的关系奠定基础。 目的：构建人S100A6基因shRNA真核表达质粒，利用实时荧光定量PCR技术检测其对S100A6基因的沉默效果，筛选出最有效的RNA干扰靶点，为进一步研究S100A6与胃癌多药耐药奠定基础。 方法：本课题依据Gene Bank中人S100A6基因序列，针对S100A6基因设计合成具有高特异性的四条shRNA干扰片段，同时设计一无关序列作为阴性对照(negative control，NC)。将各干扰片段及阴性对照与带有绿色荧光蛋白GFP及氨苄青霉素抗性的真核表达载体pRNAT-U6.1/GFP/Neo连接，阳性克隆鉴定及DNA测序鉴定重组质粒。用脂质体转染法将重组质粒转染到SGC7901细胞中，在荧光显微镜下观察绿色荧光蛋白表达情况及转染效率。转染48小时后提取细胞总RNA，运用RT-QPCR技术进行检测，从而筛选出最有效的RNA干扰片段，确定最佳干扰靶点。 结果：通过阳性克隆鉴定鉴定和DNA测序鉴定，成功构建四种针对S100A6的shRNA质粒及阴性对照质粒。将这五组质粒分别转染SGC7901细胞后，在荧光显微镜下均可观察到亮绿色荧光，得到转染效率为40%。转染后48小时提取细胞总RNA，荧光实时定量PCR检测四条shRNA真核表达质粒在mRNA水平对S100A6抑制率分别为31.3%、20.6%、27.9%、22%，说明四组质粒均可使S100A6基因的mRNA表达量下降，其中S100A6-1对S100A6基因在mRNA水平上的抑制作用最明显，抑制率为31.3%。四组质粒RNA干扰效率分别为78.25%、51.5%、69.75%、55%，其中S100A6-shRNA-1的干扰效率最高，为78.25%，说明S100A6-1为最佳筛选靶点。 结论：1、人S100A6基因shRNA真核表达质粒能特异性抑制人胃癌细胞株SGC7901中S100A6基因mRNA水平的表达。 2、S100A6-shRNA-1真核表达质粒对S100A6基因mRNA水平的干扰效率最高且正确有效。 3、S100A6-1的靶点序列作为最佳筛选靶点，可以用于后续实验研究。
[Abstract]:Background : Gastric cancer is one of the most common malignant tumors of the digestive tract , chemotherapy is the most important therapeutic measure for advanced gastric cancer . However , because of multidrug resistance ( MDR ) of tumor , it often leads to chemotherapy failure . Therefore , it is possible to study the mechanism of MDR formation in gastric cancer , so as to reverse MDR of gastric cancer by means of RNA interference and overexpression of gene , which will lead to a new breakthrough in gene therapy for gastric cancer . RNA interference ( RNAi ) is a sequence - specific post - transcriptional gene silencing ( PTGS ) process induced by double strand RNA ( dsRNA ) . During the period , double - stranded RNA is degraded into small interfering RNA ( siRNA ) by specific nuclease , siRNA - induced silencing complex ( RISC ) , RISC recognition and degradation of target mRNA , resulting in effective gene silencing . In this study , the differentially expressed proteins were identified by high - performance liquid chromatography electrospray ionization flight tandem mass spectrometry . The results showed that the high expression level was detected by high performance liquid chromatography electrospray ionization flight tandem mass spectrometry . Objective : To construct the eukaryotic expression plasmid of the shRNA eukaryotic expression plasmid of human ' s protein A6 gene and to detect the silencing effect of the gene by real - time fluorescence quantitative PCR . The most effective target of RNA interference was selected to lay the foundation for further study on the multidrug resistance of tumor cells A6 and gastric cancer . Methods : Four shRNA interference fragments with high specificity were designed and synthesized according to the gene sequence in Gene Bank , and a unrelated sequence was designed as negative control ( NC ) . The recombinant plasmid pRNAT - U6.1 / GFP / Neo , positive clone identification and DNA sequencing were used to determine the expression of green fluorescent protein and the transfection efficiency . After 48 hours of transfection , the total RNA of the cells was extracted and the most effective RNA interference fragment was screened . The optimal interference target was determined . Results : Four shRNA plasmids and negative control plasmids were successfully constructed by positive clone identification and DNA sequencing . Conclusion : 1 . The eukaryotic expression plasmid of the shRNA eukaryotic expression plasmid can specifically inhibit the mRNA level expression in human gastric cancer cell line SGC - SGC . 2 銆

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