發(fā)布時間：2018-01-19 00:08

  本文關鍵詞： S100A6基因 短發(fā)夾RNA 真核表達質粒 RNA干擾 實時熒光定量PCR 靶點篩選　出處：《大連醫(yī)科大學》2012年碩士論文　論文類型：學位論文

【摘要】：研究背景：胃癌是最常見的消化道惡性腫瘤之一，化療是進展期胃癌最重要的治療措施，然而，由于腫瘤多藥耐藥(multidrug resistance，MDR)的產生，常常導致化療失敗，因此，深入研究胃癌MDR形成的機制，從而采取RNA干擾、基因過表達等方法逆轉胃癌MDR，將有可能給胃癌基因治療帶來新的突破。 RNA干擾(RNAinterference，，RNAi)是由雙鏈RNA(double strand RNA,dsRNA)誘發(fā)的序列特異的轉錄后基因沉默（PTGS）過程。期間，雙鏈RNA被特異的核酸酶降解成小干擾RNA(small interfering RNA，siRNA)，小干擾RNA與靶蛋白結合成RNA誘導的沉默復合物(siRNA-induced silencing complex，RISC)，RISC識別并降解靶mRNA，產生有效的基因沉默。RNA干擾技術具有相當高的特異性和高效性，并且操作簡單、周期短、成本低。目前，該技術已成為研究基因功能、認識疾病本質的重要工具，將來更有望用于疾病的基因靶向治療，具有相當廣闊的臨床應用前景。 此前,本課題組以人胃癌細胞株SGC7901、阿霉素誘導的耐藥細胞株SGC7901/ADR和丹參逆轉后的耐藥細胞株為研究對象，應用蛋白質組學技術對三者進行蛋白質組學的差異比較，并且通過高效液相色譜電噴霧電離飛行串聯質譜對差異蛋白質進行鑒定，結果發(fā)現，S100A6在SGC7901中呈現高表達，在SGC7901/ADR中表達下調，在丹參逆轉后的耐藥細胞株中再次呈現高表達；并采用免疫細胞化學方法、Western blot方法和RT-PCR技術再次驗證了S100A6的差異表達。本研究以S100A6高表達的人胃癌細胞株SGC7901為研究對象，運用RNA干擾技術，將構建S100A6-shRNA質粒載體并轉染入SGC7901細胞，篩選有效的RNA干擾片段，為有效下調S100A6在SGC7901細胞中的表達，并進一步探討S100A6與胃癌MDR的關系奠定基礎。 目的：構建人S100A6基因shRNA真核表達質粒，利用實時熒光定量PCR技術檢測其對S100A6基因的沉默效果，篩選出最有效的RNA干擾靶點，為進一步研究S100A6與胃癌多藥耐藥奠定基礎。 方法：本課題依據Gene Bank中人S100A6基因序列，針對S100A6基因設計合成具有高特異性的四條shRNA干擾片段，同時設計一無關序列作為陰性對照(negative control，NC)。將各干擾片段及陰性對照與帶有綠色熒光蛋白GFP及氨芐青霉素抗性的真核表達載體pRNAT-U6.1/GFP/Neo連接，陽性克隆鑒定及DNA測序鑒定重組質粒。用脂質體轉染法將重組質粒轉染到SGC7901細胞中，在熒光顯微鏡下觀察綠色熒光蛋白表達情況及轉染效率。轉染48小時后提取細胞總RNA，運用RT-QPCR技術進行檢測，從而篩選出最有效的RNA干擾片段，確定最佳干擾靶點。 結果：通過陽性克隆鑒定鑒定和DNA測序鑒定，成功構建四種針對S100A6的shRNA質粒及陰性對照質粒。將這五組質粒分別轉染SGC7901細胞后，在熒光顯微鏡下均可觀察到亮綠色熒光，得到轉染效率為40%。轉染后48小時提取細胞總RNA，熒光實時定量PCR檢測四條shRNA真核表達質粒在mRNA水平對S100A6抑制率分別為31.3%、20.6%、27.9%、22%，說明四組質粒均可使S100A6基因的mRNA表達量下降，其中S100A6-1對S100A6基因在mRNA水平上的抑制作用最明顯，抑制率為31.3%。四組質粒RNA干擾效率分別為78.25%、51.5%、69.75%、55%，其中S100A6-shRNA-1的干擾效率最高，為78.25%，說明S100A6-1為最佳篩選靶點。 結論：1、人S100A6基因shRNA真核表達質粒能特異性抑制人胃癌細胞株SGC7901中S100A6基因mRNA水平的表達。 2、S100A6-shRNA-1真核表達質粒對S100A6基因mRNA水平的干擾效率最高且正確有效。 3、S100A6-1的靶點序列作為最佳篩選靶點，可以用于后續(xù)實驗研究。
[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

本文編號：1441761