本文關(guān)鍵詞： S100A6基因 短發(fā)夾RNA 真核表達(dá)質(zhì)粒 RNA干擾 實(shí)時(shí)熒光定量PCR 靶點(diǎn)篩選　出處：《大連醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)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(jìn)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(chǎn)生，常常導(dǎo)致化療失敗，因此，深入研究胃癌MDR形成的機(jī)制，從而采取RNA干擾、基因過(guò)表達(dá)等方法逆轉(zhuǎn)胃癌MDR，將有可能給胃癌基因治療帶來(lái)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(zhuǎn)錄后基因沉默（PTGS）過(guò)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(jié)合成RNA誘導(dǎo)的沉默復(fù)合物(siRNA-induced silencing complex，RISC)，RISC識(shí)別并降解靶mRNA，產(chǎn)生有效的基因沉默。RNA干擾技術(shù)具有相當(dāng)高的特異性和高效性，并且操作簡(jiǎn)單、周期短、成本低。目前，該技術(shù)已成為研究基因功能、認(rèn)識(shí)疾病本質(zhì)的重要工具，將來(lái)更有望用于疾病的基因靶向治療，具有相當(dāng)廣闊的臨床應(yīng)用前景。 此前,本課題組以人胃癌細(xì)胞株SGC7901、阿霉素誘導(dǎo)的耐藥細(xì)胞株SGC7901/ADR和丹參逆轉(zhuǎn)后的耐藥細(xì)胞株為研究對(duì)象，應(yīng)用蛋白質(zhì)組學(xué)技術(shù)對(duì)三者進(jìn)行蛋白質(zhì)組學(xué)的差異比較，并且通過(guò)高效液相色譜電噴霧電離飛行串聯(lián)質(zhì)譜對(duì)差異蛋白質(zhì)進(jìn)行鑒定，結(jié)果發(fā)現(xiàn)，S100A6在SGC7901中呈現(xiàn)高表達(dá)，在SGC7901/ADR中表達(dá)下調(diào)，在丹參逆轉(zhuǎn)后的耐藥細(xì)胞株中再次呈現(xiàn)高表達(dá)；并采用免疫細(xì)胞化學(xué)方法、Western blot方法和RT-PCR技術(shù)再次驗(yàn)證了S100A6的差異表達(dá)。本研究以S100A6高表達(dá)的人胃癌細(xì)胞株SGC7901為研究對(duì)象，運(yùn)用RNA干擾技術(shù)，將構(gòu)建S100A6-shRNA質(zhì)粒載體并轉(zhuǎn)染入SGC7901細(xì)胞，篩選有效的RNA干擾片段，為有效下調(diào)S100A6在SGC7901細(xì)胞中的表達(dá)，并進(jìn)一步探討S100A6與胃癌MDR的關(guān)系奠定基礎(chǔ)。 目的：構(gòu)建人S100A6基因shRNA真核表達(dá)質(zhì)粒，利用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)其對(duì)S100A6基因的沉默效果，篩選出最有效的RNA干擾靶點(diǎn)，為進(jìn)一步研究S100A6與胃癌多藥耐藥奠定基礎(chǔ)。 方法：本課題依據(jù)Gene Bank中人S100A6基因序列，針對(duì)S100A6基因設(shè)計(jì)合成具有高特異性的四條shRNA干擾片段，同時(shí)設(shè)計(jì)一無(wú)關(guān)序列作為陰性對(duì)照(negative control，NC)。將各干擾片段及陰性對(duì)照與帶有綠色熒光蛋白GFP及氨芐青霉素抗性的真核表達(dá)載體pRNAT-U6.1/GFP/Neo連接，陽(yáng)性克隆鑒定及DNA測(cè)序鑒定重組質(zhì)粒。用脂質(zhì)體轉(zhuǎn)染法將重組質(zhì)粒轉(zhuǎn)染到SGC7901細(xì)胞中，在熒光顯微鏡下觀察綠色熒光蛋白表達(dá)情況及轉(zhuǎn)染效率。轉(zhuǎn)染48小時(shí)后提取細(xì)胞總RNA，運(yùn)用RT-QPCR技術(shù)進(jìn)行檢測(cè)，從而篩選出最有效的RNA干擾片段，確定最佳干擾靶點(diǎn)。 結(jié)果：通過(guò)陽(yáng)性克隆鑒定鑒定和DNA測(cè)序鑒定，成功構(gòu)建四種針對(duì)S100A6的shRNA質(zhì)粒及陰性對(duì)照質(zhì)粒。將這五組質(zhì)粒分別轉(zhuǎn)染SGC7901細(xì)胞后，在熒光顯微鏡下均可觀察到亮綠色熒光，得到轉(zhuǎn)染效率為40%。轉(zhuǎn)染后48小時(shí)提取細(xì)胞總RNA，熒光實(shí)時(shí)定量PCR檢測(cè)四條shRNA真核表達(dá)質(zhì)粒在mRNA水平對(duì)S100A6抑制率分別為31.3%、20.6%、27.9%、22%，說(shuō)明四組質(zhì)粒均可使S100A6基因的mRNA表達(dá)量下降，其中S100A6-1對(duì)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(zhì)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(shuō)明S100A6-1為最佳篩選靶點(diǎn)。 結(jié)論：1、人S100A6基因shRNA真核表達(dá)質(zhì)粒能特異性抑制人胃癌細(xì)胞株SGC7901中S100A6基因mRNA水平的表達(dá)。 2、S100A6-shRNA-1真核表達(dá)質(zhì)粒對(duì)S100A6基因mRNA水平的干擾效率最高且正確有效。 3、S100A6-1的靶點(diǎn)序列作為最佳篩選靶點(diǎn)，可以用于后續(xù)實(shí)驗(yàn)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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