双功能3p-siRNA沉默HER2基因表达和激活RIG-I信号通路治疗胃癌的研究

发布时间：2018-02-15 00:08

  本文关键词： 胃癌 HER2 siRNA 3p-siRNA RIG-I 凋亡 增殖　出处：《第四军医大学》2017年博士论文　论文类型：学位论文

【摘要】：胃癌是全球范围内最常见的癌症之一,也是导致癌症相关死亡的常见原因。目前手术切除和放化疗仍是胃癌治疗的主要手段,因其治愈性差,复发率高,所以这就要求我们积极地寻找靶向药物。许多研究发现,免疫应答相关基因、代谢酶基因、DNA修复基因、肿瘤抑制基因和胃黏膜保护基因的变异或表达的改变与胃癌的发生具有很强的关联性。HER2为原癌基因,位于染色体17q21上,当原癌基因激活过度表达,使得细胞膜上的HER2受体表达增多,促进细胞癌变,最终导致癌症的发生。目前越来越多的证据表明HER2过表达与胃癌患者不良预后及复发率亦密切相关。荧光原位杂交技术和免疫组织化学方法发现胃癌患者中约有7%~35%的HER2阳性患者。临床前数据显示HER2特异单克隆抗体在体内体外实验中均具有显著的抗胃肿瘤效果,但在HER2阳性患者中应答率较低。目前RNAi已经被用于很多疾病的治疗研究中。新的研究发现,部分siRNA,特别是经过修饰的siRNA具有免疫激活的作用,可识别机体内固有的免疫受体。这一发现为癌症的治疗打开了新的视角。本研究通过设计合成HER2基因特异siRNA,利用生物学方法合成具有免疫激活能力的HER2 3p-siRNA,研究其对胃癌细胞HER2基因表达及免疫激活的双重影响,探究双功能HER2 3p-siRNA对胃癌细胞增殖和凋亡的影响及其分子机制。本课题包括以下三部分内容。第一部分:HER2 3p-siRNA的合成及鉴定实验主要目的是利用生物学方法设计、合成HER2 3p-siRNA,对合成产物进行鉴定。首先以NCBI数据库为基础,设计合成了3条HER2基因特异性siRNA,通过检测siRNA干扰后细胞内HER2的mRNA和蛋白表达水平,选择干扰效率最佳的siRNA 2,以siRNA2序列为模板合成HER2 3p-siRNA。生物学方法合成HER23p-siRNA,经琼脂糖凝胶电泳,验证合成的HER2 3p-siRNA与siRNA2具有相同的序列长度;荧光素酶活性检测结果显示HER2 3p-siRNA可显著激活IFN-β启动子的活性。第二部分:HER2 3p-siRNA特异性沉默HER2基因表达和激活RIG-I信号通路实验主要探究HER2 3p-siRNA在胃癌细胞中对HER2基因的干扰作用及免疫激活功能,同时探究双功能HER2 3p-siRNA对胃癌细胞凋亡及凋亡机制的研究。研究结果发现3p-siRNA可非特异性激活RIG-I,p IRF-3进而促进细胞内IP-10,和MHC-I的表达,同时,该3p-siRNA可以特性抑制细胞内HER2蛋白的水平,另外我们观察到该3p-siRNA可明显增加胃癌细胞凋亡并抑制细胞增殖。进一步的机制研究发现HER2 3p-siRNA能激活细胞内促凋亡蛋白Noxa和细胞色素C的表达,进而诱导Caspase-3和Caspase-9的活化,启动细胞凋亡。此外,Cisplatin与HER2 3psiRNA的联合使用能有效降低胃癌细胞增殖及增加细胞凋亡。第三部分:双功能HER2 3p-siRNA对荷瘤小鼠胃肿瘤增殖作用的体内研究通过小鼠体内实验探究HER2 3p-siRNA对肿瘤凋亡和生长或增殖的影响。首先我们在体外验证了HER2 3p-siRNA对小鼠胃癌细胞MFC的HER2干扰及免疫蛋白活化的影响。然后腹部皮下注射小鼠胃癌细胞MFC建立了小鼠体内肿瘤模型,通过尾静脉注射HER2 3p-siRNA,探究HER2 3p-siRNA对荷瘤小鼠肿瘤组织凋亡和生长的影响及其机制。细胞实验表明生物法合成的小鼠HER2 3p-siRNA不仅可抑制MFC细胞内HER2的表达,还同时促进RIG-I,pIRF-3,IFN-β,MHC-I和IP-10的表达,增加MFC细胞凋亡及降低增殖。动物体内观察到HER2 3p-siRNA能增加小鼠血清IFN-β及IFN-γ的浓度,显著抑制肿瘤的生长,且对小鼠肝脾肾肺的毒性较弱。肿瘤组织中可见HER2 3p-siRNA显著激活了IP-10,IFN-β,IFN-γ的表达,CD8+T细胞组织浸润能力增强。HER2 3p-siRNA和Cisplatin的联合使用可明显抑制荷瘤小鼠肿瘤组织的生长,为联合用药提供了新的思路。本实验合成了特异性靶向HER2基因的3p-si RNA,并通过体内体外实验探究HER2 3p-siRNA对胃癌细胞凋亡和增殖的影响,初步探究了其作用机制。这种兼具有靶基因沉默作用和免疫激活作用的双功能3p-siRNA具有改善胃肿瘤微环境、发挥免疫监视、解除免疫抑制的作用,该研究为HER2阳性胃癌患者寻找新的治疗方法奠定了实验基础,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.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2
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本文编号：1511950