發(fā)布時間：2018-04-21 21:12

  本文選題：羊口瘡病毒 + 細胞凋亡　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：羊口瘡病毒(Orfvirus,ORFV),屬痘病毒科副痘病毒屬,是羊傳染性膿皰病(俗稱羊口瘡)的病原體。ORFV是一種雙鏈DNA病毒,全長138kb,GC含量64%,有132個預(yù)測基因,其中病毒的復(fù)制、形態(tài)和結(jié)構(gòu)主要由中間保守區(qū)域調(diào)節(jié);而兩端則為相對不保守的基因,這些基因常常與宿主選擇、免疫逃避,免疫調(diào)節(jié)及病毒毒力等相關(guān)。病毒在靶細胞復(fù)制過程中,會刺激宿主產(chǎn)生多種細胞因子和趨化因子,參與免疫調(diào)節(jié)、抗病毒等作用。目前已鑒定了一些ORFV的毒性基因,包括IL-10,CBP,VEGF和干擾素(IFN)抗性基因等,其靶標主要為宿主的細胞因子、趨化因子、NF-κB信號途徑和凋亡途徑等。這些毒性因子的協(xié)同作用使ORFV具有很強的免疫調(diào)節(jié)作用。ORFV125蛋白是文獻報道的第一個ORFV編碼的凋亡相關(guān)蛋白,該蛋白定位于線粒體,能在體外抑制UV誘導(dǎo)的細胞凋亡。也可與Bik,Bim等一系列BH3-only蛋白結(jié)合,中和這些蛋白的促凋亡活性。本課題組前期初步研究發(fā)現(xiàn)ORFV119蛋白定位于線粒體,可抑制細胞增殖和誘導(dǎo)細胞凋亡,但其具體的分子調(diào)控機制尚不清楚。因此,本研究擬對ORFV119蛋白的功能進行深入的研究,明確其調(diào)控宿主細胞凋亡的作用,并重點研究其發(fā)揮凋亡作用的具體途徑和分子機制。本課題主要來源:國家自然科學(xué)基金(NO.31170147)下面分別對本課題的研究方法、研究內(nèi)容和研究結(jié)論進行總結(jié)。研究方法:第一章中,主要利用CCK8細胞增殖實驗、DAPI染色、TUNEL實驗和流式細胞術(shù)檢測等多種技術(shù)、從不同的角度驗證ORFV119蛋白誘導(dǎo)細胞凋亡這一作用。第二章中主要采用Caspase激活/抑制實驗和凋亡蛋白芯片技術(shù)初步探討ORFV119誘導(dǎo)細胞凋亡的途徑,之后通過Western blot和ELISA技術(shù)分析驗證ORFV119蛋白誘導(dǎo)細胞凋亡過程中相關(guān)蛋白和細胞因子變化情況,進而闡明ORFV119蛋白誘導(dǎo)細胞凋亡的分子機制。研究內(nèi)容:第一部分:ORFV119誘導(dǎo)細胞凋亡作用的驗證在293T細胞中高表達ORFV119蛋白,不同時間點利用CCK8法檢測細胞增殖情況;采用兩種不同真核表達載體瞬時轉(zhuǎn)染ORFV119蛋白,不同時間DAPI熒光染色后,激光共聚焦顯微鏡觀察細胞核形態(tài)變化。轉(zhuǎn)染119GFP和pEGFP-N1后24 h采用TUNEL法檢測凋亡細胞DNA斷裂情況。同時高表達ORFV119蛋白后24 h,AnnexinV-APC和7-AAD雙染,流式細胞術(shù)檢測凋亡細胞數(shù)。第二部分:ORFV119誘導(dǎo)細胞凋亡機制的研究首先高表達ORFV119蛋白后24 h和48 h,利用Caspase激活試劑盒檢測ORFV119蛋白對不同Caspase成員的活化程度,初步探討凋亡的分子機制。為了獲得更多凋亡過程中蛋白或細胞因子的變化情況,進行了人凋亡蛋白芯片檢測。之后在高表達ORFV119蛋白的24 h和48 h,應(yīng)用Western blot和ELISA技術(shù)驗證相應(yīng)蛋白及細胞因子變化水平,進一步明確ORFV119誘導(dǎo)細胞凋亡的分子機制。研究結(jié)論:本研究通過DAPI熒光染色,流式細胞術(shù),蛋白芯片,Western blot,ELISA等方法研究了羊口瘡病毒ORFV119的分子功能及其對細胞凋亡的影響和相關(guān)分子機制。研究發(fā)現(xiàn),ORFV119蛋白定位于線粒體;能抑制細胞增殖和誘導(dǎo)細胞凋亡;其誘導(dǎo)細胞凋亡作用主要為:ORFV119蛋白可使線粒體表面的促凋亡蛋白Bax和Bak上升,線粒體膜通透性增強,釋放細胞色素C和促凋亡蛋白Smac/Diablo;同時下調(diào)cIAP-2和Bcl-2兩個抑制凋亡蛋白,激活Caspase9;釋放的細胞色素C和活化的Caspase9、Apaf-1因子形成凋亡小體,導(dǎo)致下游Caspase3和PARP的活化使細胞發(fā)生凋亡。同時ORFV119蛋白也可激活Caspase8,再直接活化Caspase3使細胞凋亡,也可使BID活化為tBID通過線粒體途徑發(fā)揮凋亡調(diào)控作用。本研究明確了 ORFV119蛋白誘導(dǎo)細胞凋亡的作用并闡明其分子機制,使羊口瘡病毒未知基因功能的探索更進一步。這些結(jié)果有助于研究羊口瘡病毒的致病機制和免疫機理,為疾病的預(yù)防和控制提供理論基礎(chǔ),同時為新疫苗和藥物的開發(fā)提供參考資料。
[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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.654

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本文編號：1784114