本文關鍵詞： 胃癌 HER2 siRNA 3p-siRNA RIG-I 凋亡 增殖　出處：《第四軍醫(yī)大學》2017年博士論文　論文類型：學位論文

【摘要】：胃癌是全球范圍內最常見的癌癥之一,也是導致癌癥相關死亡的常見原因。目前手術切除和放化療仍是胃癌治療的主要手段,因其治愈性差,復發(fā)率高,所以這就要求我們積極地尋找靶向藥物。許多研究發(fā)現(xiàn),免疫應答相關基因、代謝酶基因、DNA修復基因、腫瘤抑制基因和胃黏膜保護基因的變異或表達的改變與胃癌的發(fā)生具有很強的關聯(lián)性。HER2為原癌基因,位于染色體17q21上,當原癌基因激活過度表達,使得細胞膜上的HER2受體表達增多,促進細胞癌變,最終導致癌癥的發(fā)生。目前越來越多的證據表明HER2過表達與胃癌患者不良預后及復發(fā)率亦密切相關。熒光原位雜交技術和免疫組織化學方法發(fā)現(xiàn)胃癌患者中約有7%~35%的HER2陽性患者。臨床前數據顯示HER2特異單克隆抗體在體內體外實驗中均具有顯著的抗胃腫瘤效果,但在HER2陽性患者中應答率較低。目前RNAi已經被用于很多疾病的治療研究中。新的研究發(fā)現(xiàn),部分siRNA,特別是經過修飾的siRNA具有免疫激活的作用,可識別機體內固有的免疫受體。這一發(fā)現(xiàn)為癌癥的治療打開了新的視角。本研究通過設計合成HER2基因特異siRNA,利用生物學方法合成具有免疫激活能力的HER2 3p-siRNA,研究其對胃癌細胞HER2基因表達及免疫激活的雙重影響,探究雙功能HER2 3p-siRNA對胃癌細胞增殖和凋亡的影響及其分子機制。本課題包括以下三部分內容。第一部分:HER2 3p-siRNA的合成及鑒定實驗主要目的是利用生物學方法設計、合成HER2 3p-siRNA,對合成產物進行鑒定。首先以NCBI數據庫為基礎,設計合成了3條HER2基因特異性siRNA,通過檢測siRNA干擾后細胞內HER2的mRNA和蛋白表達水平,選擇干擾效率最佳的siRNA 2,以siRNA2序列為模板合成HER2 3p-siRNA。生物學方法合成HER23p-siRNA,經瓊脂糖凝膠電泳,驗證合成的HER2 3p-siRNA與siRNA2具有相同的序列長度;熒光素酶活性檢測結果顯示HER2 3p-siRNA可顯著激活IFN-β啟動子的活性。第二部分:HER2 3p-siRNA特異性沉默HER2基因表達和激活RIG-I信號通路實驗主要探究HER2 3p-siRNA在胃癌細胞中對HER2基因的干擾作用及免疫激活功能,同時探究雙功能HER2 3p-siRNA對胃癌細胞凋亡及凋亡機制的研究。研究結果發(fā)現(xiàn)3p-siRNA可非特異性激活RIG-I,p IRF-3進而促進細胞內IP-10,和MHC-I的表達,同時,該3p-siRNA可以特性抑制細胞內HER2蛋白的水平,另外我們觀察到該3p-siRNA可明顯增加胃癌細胞凋亡并抑制細胞增殖。進一步的機制研究發(fā)現(xiàn)HER2 3p-siRNA能激活細胞內促凋亡蛋白Noxa和細胞色素C的表達,進而誘導Caspase-3和Caspase-9的活化,啟動細胞凋亡。此外,Cisplatin與HER2 3psiRNA的聯(lián)合使用能有效降低胃癌細胞增殖及增加細胞凋亡。第三部分:雙功能HER2 3p-siRNA對荷瘤小鼠胃腫瘤增殖作用的體內研究通過小鼠體內實驗探究HER2 3p-siRNA對腫瘤凋亡和生長或增殖的影響。首先我們在體外驗證了HER2 3p-siRNA對小鼠胃癌細胞MFC的HER2干擾及免疫蛋白活化的影響。然后腹部皮下注射小鼠胃癌細胞MFC建立了小鼠體內腫瘤模型,通過尾靜脈注射HER2 3p-siRNA,探究HER2 3p-siRNA對荷瘤小鼠腫瘤組織凋亡和生長的影響及其機制。細胞實驗表明生物法合成的小鼠HER2 3p-siRNA不僅可抑制MFC細胞內HER2的表達,還同時促進RIG-I,pIRF-3,IFN-β,MHC-I和IP-10的表達,增加MFC細胞凋亡及降低增殖。動物體內觀察到HER2 3p-siRNA能增加小鼠血清IFN-β及IFN-γ的濃度,顯著抑制腫瘤的生長,且對小鼠肝脾腎肺的毒性較弱。腫瘤組織中可見HER2 3p-siRNA顯著激活了IP-10,IFN-β,IFN-γ的表達,CD8+T細胞組織浸潤能力增強。HER2 3p-siRNA和Cisplatin的聯(lián)合使用可明顯抑制荷瘤小鼠腫瘤組織的生長,為聯(lián)合用藥提供了新的思路。本實驗合成了特異性靶向HER2基因的3p-si RNA,并通過體內體外實驗探究HER2 3p-siRNA對胃癌細胞凋亡和增殖的影響,初步探究了其作用機制。這種兼具有靶基因沉默作用和免疫激活作用的雙功能3p-siRNA具有改善胃腫瘤微環(huán)境、發(fā)揮免疫監(jiān)視、解除免疫抑制的作用,該研究為HER2陽性胃癌患者尋找新的治療方法奠定了實驗基礎,提供了新的思路。
[Abstract]:Gastric cancer is one of the most common cancer worldwide, but also a common cause of cancer-related death. The main method of surgical resection and chemotherapy is the treatment of gastric cancer, because of its poor cure, high recurrence rate, so this requires us to actively search for targeted drugs. Many studies found that the immune response related genes and metabolic enzyme genes, DNA repair genes, changes in gastric cancer and tumor suppressor gene mutation and gene expression of gastric mucosal protection or.HER2 has strong relevance to the original cancer gene, located on chromosome 17q21, when proto oncogene activation over expression of the HER2 receptor on the cell membrane expression increased, promote cancerous cells, eventually leading to the occurrence of cancer. At present, more and more evidence that the overexpression of HER2 is closely associated with gastric cancer patients with poor prognosis and recurrence rate. The fluorescence in situ hybridization and immunohistochemistry. Found HER2 positive patients with gastric cancer in about 7%~35%. The pre clinical data showed that HER2 specific monoclonal antibody against gastric cancer has a significant effect both in vivo and in vitro, but the response rate in HER2 positive patients is low. At present, RNAi has been used to treat many diseases research. New research has found. Part of the siRNA, especially after the modification of siRNA with immune activation, can identify the body innate immune receptor. The treatment for cancer has opened up a new perspective. This study through the design and synthesis of HER2 gene specific siRNA, using biological synthesis method has immune activation ability of HER2 3p-siRNA, on the double effect the expression of HER2 gene in gastric cancer cells and immune activation, on the double function of HER2 3p-siRNA on proliferation and apoptosis of gastric cancer cells and its molecular mechanism. This paper includes the following three parts. Volume. The first part: the main purpose of HER2 3p-siRNA synthesis and identification experiment is designed by using biological method, synthesis of HER2 3p-siRNA, identification of synthetic products. Firstly, based on the NCBI database, 3 HER2 gene specific siRNA synthesized by mRNA and protein levels of intracellular HER2 expression after siRNA interference, selection the best jamming efficiency of siRNA 2, the siRNA2 sequence as a template to synthesize HER2 3p-siRNA. biological synthesis of HER23p-siRNA by agarose gel electrophoresis, HER2 3p-siRNA authentication and siRNA2 synthesis of sequences with the same length; luciferase activity assay showed that HER2 3p-siRNA could activate IFN- beta promoter activity. The second part: HER2 3p-siRNA silencing of HER2 gene expression and activation of RIG-I signaling pathway on HER2 3p-siRNA experiment in gastric cancer cells and free of interference of HER2 gene Immune activation function, and explore the research on gastric cancer cell apoptosis and the mechanism of the double function of HER2 3p-siRNA. The results showed that 3p-siRNA could be a nonspecific activation of RIG-I, P IRF-3 and IP-10 in cells, the expression of MHC-I, and at the same time, the characteristics of 3p-siRNA can inhibit HER2 protein level in the cells, we observed the 3p-siRNA can obviously increase the apoptosis of gastric cancer cells and inhibit cell proliferation. Further studies found that HER2 3p-siRNA can activate the intracellular expression of Pro apoptotic protein Noxa and cytochrome C, and activation induced by Caspase-3 and Caspase-9, to start apoptosis. In addition, the combined use of Cisplatin and HER2 3psiRNA can effectively reduce the gastric cancer cell proliferation and increased apoptosis the in vivo study. The third part: double function of HER2 3p-siRNA on the proliferation of gastric tumor bearing mice tumor by in vivo experiments on HER2 3p-siR Effect of NA on apoptosis and tumor growth and proliferation. We first examined the effect of HER2 3p-siRNA HER2 interference and immune protein on mouse gastric cancer cell MFC activation in vitro. Then subcutaneous injection of mouse gastric cancer cells MFC tumor mice model was established by intravenous injection of HER2, 3p-siRNA, HER2 and 3p-siRNA effect on the growth of the tumor tissue of mice with tumor cell apoptosis and its mechanism. Experiments show that the expression of mouse HER2 3p-siRNA synthesized by biological method can not only inhibit MFC cells in HER2, but also promote the RIG-I, pIRF-3, IFN- beta, the expression of MHC-I and IP-10, MFC increased cell apoptosis and decreased proliferation. The animal observed HER2 3p-siRNA can increase the concentration of mice serum IFN- and beta gamma IFN-, inhibit the tumor growth in mice, and the liver spleen and kidney lung toxicity observed in tumor tissue is weak. HER2 3p-siRNA was activated by IP-10, IF The expression of N- beta, IFN- gamma, CD8+T cell infiltration and enhance the ability of the combined use of.HER2 3p-siRNA and Cisplatin can significantly inhibit tumor growth in tumor bearing mice, and provides a new idea for the combination. This experiment synthesized targeting 3p-si RNA gene HER2, and through in vivo and in vitro experiments to explore the influence of the apoptosis and proliferation of gastric cancer cells HER2 3p-siRNA, a preliminary study of its mechanism. This has dual function 3p-siRNA activation of target gene silencing effect and immunity can improve gastric tumor microenvironment, immune surveillance, relieve immune inhibition, this paper provides an experimental basis for HER2 positive gastric cancer patients looking for new treatment methods that provides a new way of thinking.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.2
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本文編號：1511950