羊口疮病毒119蛋白诱导细胞凋亡分子机制的研究

发布时间：2018-04-21 21:12

本文选题：羊口疮病毒 + 细胞凋亡　；参考：《南方医科大学》2017年硕士论文

【摘要】：羊口疮病毒(Orfvirus,ORFV),属痘病毒科副痘病毒属,是羊传染性脓疱病(俗称羊口疮)的病原体。ORFV是一种双链DNA病毒,全长138kb,GC含量64%,有132个预测基因,其中病毒的复制、形态和结构主要由中间保守区域调节;而两端则为相对不保守的基因,这些基因常常与宿主选择、免疫逃避,免疫调节及病毒毒力等相关。病毒在靶细胞复制过程中,会刺激宿主产生多种细胞因子和趋化因子,参与免疫调节、抗病毒等作用。目前已鉴定了一些ORFV的毒性基因,包括IL-10,CBP,VEGF和干扰素(IFN)抗性基因等,其靶标主要为宿主的细胞因子、趋化因子、NF-κB信号途径和凋亡途径等。这些毒性因子的协同作用使ORFV具有很强的免疫调节作用。ORFV125蛋白是文献报道的第一个ORFV编码的凋亡相关蛋白,该蛋白定位于线粒体,能在体外抑制UV诱导的细胞凋亡。也可与Bik,Bim等一系列BH3-only蛋白结合,中和这些蛋白的促凋亡活性。本课题组前期初步研究发现ORFV119蛋白定位于线粒体,可抑制细胞增殖和诱导细胞凋亡,但其具体的分子调控机制尚不清楚。因此,本研究拟对ORFV119蛋白的功能进行深入的研究,明确其调控宿主细胞凋亡的作用,并重点研究其发挥凋亡作用的具体途径和分子机制。本课题主要来源:国家自然科学基金(NO.31170147)下面分别对本课题的研究方法、研究内容和研究结论进行总结。研究方法:第一章中,主要利用CCK8细胞增殖实验、DAPI染色、TUNEL实验和流式细胞术检测等多种技术、从不同的角度验证ORFV119蛋白诱导细胞凋亡这一作用。第二章中主要采用Caspase激活/抑制实验和凋亡蛋白芯片技术初步探讨ORFV119诱导细胞凋亡的途径,之后通过Western blot和ELISA技术分析验证ORFV119蛋白诱导细胞凋亡过程中相关蛋白和细胞因子变化情况,进而阐明ORFV119蛋白诱导细胞凋亡的分子机制。研究内容:第一部分:ORFV119诱导细胞凋亡作用的验证在293T细胞中高表达ORFV119蛋白,不同时间点利用CCK8法检测细胞增殖情况;采用两种不同真核表达载体瞬时转染ORFV119蛋白,不同时间DAPI荧光染色后,激光共聚焦显微镜观察细胞核形态变化。转染119GFP和pEGFP-N1后24 h采用TUNEL法检测凋亡细胞DNA断裂情况。同时高表达ORFV119蛋白后24 h,AnnexinV-APC和7-AAD双染,流式细胞术检测凋亡细胞数。第二部分:ORFV119诱导细胞凋亡机制的研究首先高表达ORFV119蛋白后24 h和48 h,利用Caspase激活试剂盒检测ORFV119蛋白对不同Caspase成员的活化程度,初步探讨凋亡的分子机制。为了获得更多凋亡过程中蛋白或细胞因子的变化情况,进行了人凋亡蛋白芯片检测。之后在高表达ORFV119蛋白的24 h和48 h,应用Western blot和ELISA技术验证相应蛋白及细胞因子变化水平,进一步明确ORFV119诱导细胞凋亡的分子机制。研究结论:本研究通过DAPI荧光染色,流式细胞术,蛋白芯片,Western blot,ELISA等方法研究了羊口疮病毒ORFV119的分子功能及其对细胞凋亡的影响和相关分子机制。研究发现,ORFV119蛋白定位于线粒体;能抑制细胞增殖和诱导细胞凋亡;其诱导细胞凋亡作用主要为:ORFV119蛋白可使线粒体表面的促凋亡蛋白Bax和Bak上升,线粒体膜通透性增强,释放细胞色素C和促凋亡蛋白Smac/Diablo;同时下调cIAP-2和Bcl-2两个抑制凋亡蛋白,激活Caspase9;释放的细胞色素C和活化的Caspase9、Apaf-1因子形成凋亡小体,导致下游Caspase3和PARP的活化使细胞发生凋亡。同时ORFV119蛋白也可激活Caspase8,再直接活化Caspase3使细胞凋亡,也可使BID活化为tBID通过线粒体途径发挥凋亡调控作用。本研究明确了 ORFV119蛋白诱导细胞凋亡的作用并阐明其分子机制,使羊口疮病毒未知基因功能的探索更进一步。这些结果有助于研究羊口疮病毒的致病机制和免疫机理,为疾病的预防和控制提供理论基础,同时为新疫苗和药物的开发提供参考资料。
[Abstract]:Orfvirus (ORFV), the genus of the genus pox virus, the pathogen of infectious pustulosis (commonly known as sore),.ORFV is a double stranded DNA virus, with a total length of 138kb, GC content of 64%, and 132 predictive genes, in which the replication, morphology and structure of the virus are regulated by the middle conservative region, while both ends are relatively non conservative bases. These genes are often associated with host selection, immune escape, immunomodulation, and viral virulence. During the replication of the target cells, the virus stimulates the host to produce a variety of cytokines and chemokines, participate in immunomodulatory and antiviral functions. Some of the toxic genes of ORFV, including IL-10, CBP, VEGF and interferon (IFN), have been identified. The target is the cytokine, chemokine, NF- kappa B signaling pathway and apoptosis pathway. The synergistic effects of these toxic factors make ORFV with a strong immunoregulation effect,.ORFV125 protein is the first ORFV encoded apoptosis related protein reported in the literature, which is located in mitochondria and can inhibit UV in vitro. Induced apoptosis. It can also be combined with a series of BH3-only proteins, such as Bik, Bim, and other BH3-only proteins, and neutralize the apoptotic activity of these proteins. Preliminary studies in our group have found that ORFV119 protein is located in mitochondria, which inhibits cell proliferation and induces cell apoptosis, but its specific molecular regulation mechanism is not clear. Therefore, this study is intended to be on ORFV119 eggs. The function of white is studied in depth to clarify the role of its regulation of host cell apoptosis, and to focus on the specific ways and molecular mechanisms of its apoptosis. The main source of this topic is to summarize the research methods, research contents and conclusions of this subject under the National Natural Science Foundation (NO.31170147). In the one chapter, we mainly use CCK8 cell proliferation test, DAPI staining, TUNEL test and flow cytometry to verify the effect of ORFV119 protein induced apoptosis from different angles. The second chapter mainly uses Caspase activation / inhibition experiment and apoptotic protein chip technology to explore the pathway of ORFV119 induced apoptosis. Then the Western blot and ELISA techniques were used to verify the changes in the related proteins and cytokines induced by ORFV119 protein in the process of apoptosis, and then elucidate the molecular mechanism of ORFV119 protein induced apoptosis. The first part: the verification of ORFV119 induced apoptosis in 293T cells expressed the high expression of ORFV119 protein in 293T cells. The cell proliferation was detected by CCK8 method at the same time point; ORFV119 protein was transiently transfected with two different eukaryotic expression vectors. After DAPI fluorescence staining at different time, the morphological changes of nucleus were observed by laser confocal microscope. The 119GFP and pEGFP-N1 24 h were used to detect the DNA fracture in the dead cells by TUNEL method. Meanwhile, the ORFV119 eggs were highly expressed. After 24 h, AnnexinV-APC and 7-AAD double staining, the number of apoptotic cells was detected by flow cytometry. The second part: the mechanism of apoptosis induced by ORFV119 was first high expression of ORFV119 protein and 24 h and 48 h. The activation degree of ORFV119 protein to different Caspase members was detected by Caspase activation kit, and the molecular mechanism of apoptosis was preliminarily discussed. The changes of protein or cytokine during the process of apoptosis were detected, and the apoptosis protein chip was detected. Then 24 h and 48 h of high expression of ORFV119 protein were detected by Western blot and ELISA technology, and the molecular mechanism of ORFV119 induced apoptosis was further confirmed. Through DAPI fluorescence staining, flow cytometry, protein chip, Western blot, ELISA and other methods, the molecular function of ORFV119 and its effect on cell apoptosis and related molecular mechanism were studied. The study found that ORFV119 protein was located in the mitochondria; it could inhibit cell proliferation and induce apoptosis, and its induction of apoptosis was the main role. ORFV119 protein can increase the apoptosis protein Bax and Bak on the surface of mitochondria, enhance the permeability of mitochondrial membrane, release cytochrome C and apoptotic protein Smac/Diablo, and reduce the two inhibitory apoptotic proteins of cIAP-2 and Bcl-2 and activate Caspase9, and the released cytochrome C and activated Caspase9, Apaf-1 factor form apoptotic body, leading to the formation of apoptotic bodies. The activation of the downstream Caspase3 and PARP causes the cell apoptosis. At the same time, the ORFV119 protein also activates Caspase8, and then activates Caspase3 directly to make the cell apoptosis, and can also activate the BID into the mitochondrial pathway to regulate the apoptosis. This study clarifies the role of ORFV119 protein to induce apoptosis and clarifies its molecular mechanism to make the sheep oral ulcer. These results help to study the pathogenesis and immune mechanism of the virus and provide a theoretical basis for the prevention and control of the disease and provide reference for the development of new vaccines and drugs.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.654

【参考文献】