高致病性猪繁殖与呼吸综合征病毒及其非结构蛋白调控细胞凋亡的分子机制

发布时间：2018-01-06 03:38

  本文关键词：高致病性猪繁殖与呼吸综合征病毒及其非结构蛋白调控细胞凋亡的分子机制　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 猪繁殖与呼吸综合征病毒 细胞凋亡 机制 非结构蛋白 Nsp4 Nsp10

【摘要】：猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)的致病机理复杂,研究PRRSV感染对宿主细胞凋亡的调控可以为理解其致病机制提供科学依据。本研究分析了我国高致病性PRRSV调控细胞凋亡的现象及其分子机制,并进一步探讨了PRRSV的主要非结构蛋白(Nonstructural protein, Nsp)参与调控细胞凋亡的作用与机制以及不同致病性毒株诱导细胞凋亡的差异。采用western blot、流式细胞术、免疫荧光技术等对高致病性PRRSV感染MARC-145细胞的凋亡进行了定性检测和定量分析。结果显示,PRRSV感染后6h和8h由化学诱导剂—星孢菌素引起的PARP蛋白剪切受到明显抑制(P0.01),表明在感染早期PRRSV具有抑制细胞凋亡的作用；感染后24 h MARC-145细胞胞质内出现细胞色素c释放和明显的caspase-3活化与PARP蛋白剪切,表明在感染后期PRRSV可诱导细胞凋亡；感染后16 h PRRSV能够强烈激活细胞凋亡的外源性通路,且caspase-8前体蛋白明显降解,而在感染后24 h caspase-9和caspase-12开始活化,提示PRRSV感染可激活细胞的线粒体通路和内质网应激通路；Caspase-3/-8的活化集中于病毒感染的阳性细胞中,而旁细胞未发生明显的caspase活化。高致病性PRRSV感染可导致原代猪肺泡巨噬细胞(PAMs)的caspase-8活化和PARP蛋白的剪切,表明可诱导PAMs发生凋亡。应用慢病毒包装技术在MARC-145细胞中过表达PRRSV主要非结构蛋白,以western blot检测细胞中PARP蛋白剪切变化。同时经瞬时转染表达PRRSV非结构蛋白的质粒,利用激光共聚焦免疫荧光镜观察转染阳性细胞内caspase-3的活化,以分析PRRSV非结构蛋白在诱导细胞凋亡中的作用及其机制。结果表明,Nsp4和Nsp10能够明显引起MARC-145细胞内caspase-3的活化和PARP蛋白的降解,诱导细胞发生凋亡(P＜0.001);Nsp4可激活caspase-8/-9/-12,同时激活促凋亡蛋白Bim并降解抗凋亡蛋白Bcl-xL;Nsp10可激活细胞内caspase-8与caspase-9,促进胞质内BH3-only蛋白Bid表达并发生剪切形成tBid,引起线粒体外膜透化作用及下游caspase-9的活化,最终导致细胞内PARP蛋白的剪切。干扰细胞内Bid蛋白表达可致Nsp10诱导PARP蛋白剪切作用下降,而抑制caspase-8活化可导致Nsp10诱导的细胞凋亡被阻断,提示Nsp10诱导细胞凋亡的作用依赖于细胞内caspase-8和Bid的活化,且caspase-8的活化是Nsp10诱导细胞凋亡不可缺少的。相反,Nsp3、Nsp5和Nsp8能够明显抑制由星孢菌素引起的PARP蛋白的剪切而抑制细胞凋亡；Nsp3能激活PI3K/Akt通路中的Akt Ser473的磷酸化和BH3-only促凋亡蛋白Bad蛋白的Ser136磷酸化,同时促进抗凋亡蛋白Bcl-xL表达,从而抑制细胞凋亡。以PRRSV不同致病性毒株HB-1/3.9和JXwn06感染MARC-145细胞,采用western blot、流式细胞术等分析不同致病性毒株诱导宿主细胞凋亡的差异。结果表明,与高致病性毒株JXwn06相比,HB.1/3.9在感染后36 h能够明显激活caspase-9活化,形成明显的剪切带；感染后24 h-48h,HB-1/3.9感染活化的caspase-3细胞数量明显高于JXwn06组(P0.001)；感染后24 h,HB-1/3.9可引起细胞内PARP蛋白的明显降解,但仅在24 hpi与JXwn06存在明显差异(P0.001)。低致病性毒株HB-1/3.9感染后期能够明显激活细胞凋亡,其Nsp10蛋白同样具有诱导细胞凋亡的作用,作用机制与JXwn06 Nsp 10相同,但HB-1/3.9 Nsp10能更明显激活procaspase-8的降解,最终激活细胞内PARP降解。感染后24 h开始以HB.1/3.9为骨架的嵌合病毒Rv-HJn10诱导caspase-3活化细胞数量显著低于亲本病毒HB-1/3.9(C＜0.001);Rv-JHn10在24 h pi开始诱导凋亡细胞数量显著高于亲本病毒JXwn06(P0.001);感染后36 h开始Rv-JHn10活化细胞数量明显高于Rv-HJn10(P＜001)。以上结果表明,低致病性毒株HB-1/3.9诱导细胞凋亡的作用明显高于高致病性毒株JXwn06; Nsp10是决定PRRSV不同致病性毒株诱导凋亡差异的主要蛋白。综上所述,我们的研究表明：(i)高致病性PRRSV对细胞凋亡具有双向调控作用,即感染早期能够抑制细胞凋亡,而在感染后期可诱导感染细胞凋亡；(ii)PRRSV多个非结构蛋白可参与病毒对细胞凋亡的调控,其中,Nsp4和Nsp10能够促进感染细胞凋亡,而Nsp3、Nsp5和Nsp8具有抑制细胞凋亡的作用：(iii)不同致病性PRRSV毒株诱导细胞凋亡的差异与Nsp10密切相关。上述研究结果为阐明我国高致病性PRRSV的致病机制提供了科学依据,也丰富了PRRSV非结构蛋白在其致病机制方面的作用。
[Abstract]:Porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) the pathogenic mechanism is complex, the infection of PRRSV on apoptosis of host cells can provide a scientific basis for understanding its pathogenesis. This study analyzes China's highly pathogenic PRRSV cell apoptosis and its molecular mechanism of the phenomenon, and further discusses the the main non structural protein PRRSV (Nonstructural protein Nsp) the difference effects and mechanisms involved in the regulation of cell apoptosis and apoptosis induced by different pathogenic strains. Using Western blot, flow cytometry and immunofluorescence technique were analyzed and the qualitative and quantitative detection of highly pathogenic PRRSV infection on apoptosis of MARC-145 cells. The results showed that PRRSV, 6h and 8h after infection by chemical inducers of PARP protein induced by shear staurosporine inhibited (P0.01), show that the infection Early PRRSV can inhibit cell apoptosis; infection occurred after 24 h MARC-145 cell cytoplasmic cytochrome c release and obvious activation of Caspase-3 shear and PARP protein, showed that in the late stage of infection PRRSV can induce cell apoptosis; 16 h after infection of PRRSV can strongly activate the extrinsic pathway of apoptosis, and caspase-8 protein precursor obvious degradation and activation after infection 24 h caspase-9 and caspase-12, suggesting that PRRSV infection can activate the mitochondrial pathway and endoplasmic reticulum stress pathway; the activation of Caspase-3/-8 in virus positive cells, and the adjacent cells did not change significantly caspase activation. The highly pathogenic PRRSV infection can cause primary porcine alveolar macrophages (PAMs) shear caspase-8 activation and PARP protein, that can induce apoptosis of PAMs. The application of lentiviral packaging technology in MARC-145 cells overexpressing PRRSV The main non structural protein, PARP protein in Western cells were detected in blot shear changes. At the same time by transient transfection of PRRSV expression plasmid of non structural protein, using laser scanning confocal fluorescence microscope to observe the activation of immune cells transfected with positive Caspase-3, the analysis of non structural protein PRRSV in inducing effect and mechanism of apoptosis. Results show that Nsp4 and Nsp10 can obviously cause the degradation activation and PARP protein in MARC-145 cells Caspase-3, induce cell apoptosis (P < 0.001); Nsp4 can activate caspase-8/-9/-12, and activation of Pro apoptotic protein Bim and degradation of anti apoptotic protein Bcl-xL; Nsp10 can activate Caspase-8 and caspase-9 cells, promote the expression of Bid protein in the cytoplasm of BH3-only the shear and the formation of tBid, caused by activation of mitochondrial membrane permeabilization and downstream of caspase-9, resulting in shear PARP protein in cells. Intracellular Bid protein interference The expression of PARP protein induced by white shear can be decreased Nsp10, and inhibit the activation of caspase-8 can lead to cell apoptosis induced by Nsp10 was blocked, suggesting the role of activation of Nsp10 induced apoptosis is dependent on intracellular Caspase-8 and Bid activation, and caspase-8 Nsp10 induced apoptosis is indispensable. On the contrary, Nsp3, Nsp5 and Nsp8 to shear inhibition caused by staurosporine PARP protein and inhibit cell apoptosis; Ser136 phosphorylation of Nsp3 can activate the PI3K/Akt pathway in Akt Ser473 phosphorylation and BH3-only Bad protein, while promoting the anti apoptotic protein Bcl-xL expression, thereby inhibiting cell apoptosis. PRRSV with different pathogenicity strains HB-1/3.9 and JXwn06 infection MARC-145 cells, using Western blot analysis, flow cytometry and other different pathogenic strain induced apoptosis of host cells. The results show that the high pathogenic strain Compared to JXwn06, HB.1/3.9 in 36 h after infection could significantly activate the activation of caspase-9, forming obvious shear band; 24 h-48h after infection, the infection of HB-1/3.9 activated caspase-3 cell number was significantly higher than that of JXwn06 group (P0.001); 24 h after infection, HB-1/3.9 can cause significant degradation of PARP protein in the cell, there are obvious differences but only in 24 HPI and JXwn06 (P0.001). Low pathogenic strain HB-1/3.9 infection stage could significantly activate apoptosis, Nsp10 protein also induce the apoptosis of tumor cells and the mechanism of JXwn06 Nsp 10, but HB-1/3.9 Nsp10 could significantly activate procaspase-8 degradation, eventually activate intracellular degradation of PARP. 24 h after infection begins with HB.1/3.9 for the chimeric virus Rv-HJn10 skeleton induced activation of Caspase-3 cells was significantly lower than that of the parental virus HB-1/3.9 (C < 0.001); Rv-JHn10 in 24 h pi to induce the number of apoptotic cells significantly JXwn06 higher than the parent virus (P0.001) infection; 36 h after activation of Rv-JHn10 cell number was significantly higher than Rv-HJn10 (P < 001). The above results suggest that low pathogenic strain HB-1/3.9 induces cell apoptosis was significantly higher than that of the highly pathogenic JXwn06 strain; Nsp10 is a main protein PRRSV with pathogenic strain induced apoptosis difference. In summary, our study shows that: (I) the highly pathogenic PRRSV has two-way regulating effect on cell apoptosis, namely early infection can inhibit apoptosis, and in the late stage of infection can induce apoptosis in infected cells; (II) a plurality of non structural protein PRRSV may be involved in the regulation of apoptosis of the virus, among them, Nsp4 and Nsp10 to promote apoptosis in infected cells, while Nsp3, Nsp5 and Nsp8 can inhibit cell apoptosis: (III) of different pathogenic PRRSV strains induced apoptosis and Nsp10 is closely related to the results of the study. It provides a scientific basis for elucidating the pathogenesis of highly pathogenic PRRSV in China, and also enriches the role of PRRSV unstructured protein in its pathogenesis.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65
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本文编号：1386143