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SARS-CoV 3a蛋白以及炭疽致死毒素诱导细胞自噬的研究

The Research of Autophagy Was Induced by SARS-CoV 3a and Anthrax Lethal Toxin

【作者】 章晟

【导师】 陈薇; 于长明;

【作者基本信息】 中国人民解放军军事医学科学院 , 微生物学, 2009, 博士

【摘要】 本研究通过透射电子显微镜,Western印迹法,自噬荧光标志蛋白和单丹磺酰尸胺(MDC)荧光染色等实验方法证明严重急性呼吸综合征冠状病毒(SARS-CoV)3a蛋白以及炭疽致死毒素(LT)在细胞内能够引起细胞自噬,并初步研究了SARS-CoV 3a蛋白引起细胞自噬的信号通路,以及细胞自噬对炭疽致死毒素毒性的影响。细胞自噬是真核细胞中普遍存在的一种通过溶酶体吞噬降解自身成分的机制。细胞自噬具有很多重要的生理功能,其中包括清除病原体的感染。但是由于细胞自噬在进化过程中具有高度的保守性,某些病原体通过进化,具有逃避细胞自噬的能力,甚至利用细胞自噬为病原体复制和发育提供有利条件。细胞自噬的检测有多种方法,包括电子显微镜观察、MDC荧光染色、Western印迹法检测以及自噬荧光标志蛋白的检测等方法。哺乳动物细胞内Atg8的同源物微管相关蛋白1轻链3(Microtubule-associated protein light chain 3,LC3),是细胞自噬检测的标志蛋白,在细胞自噬过程中会发生特异性的剪切和聚集现象,本研究首先从HeLa细胞的cDNA中扩增出LC3的基因,并克隆至pEGFP-C1载体中,构建了pEGFP-LC3表达质粒,转染HeLa细胞后,通过荧光显微镜观察和Western印迹法证明GFP-LC3蛋白能在HeLa细胞内表达。通过诱导转染pEGFP-LC3表达质粒的HeLa细胞发生细胞自噬,在荧光显微镜下可以观察到GFP-LC3荧光蛋白在细胞内出现点状聚集现象,说明构建的pEGFP-LC3表达质粒可用于检测细胞自噬。SARS-CoV相对于其他冠状病毒具有较强的致死性,这可能与其特有的非结构蛋白有关,3a蛋白是SARS-CoV中最大的一个非结构蛋白。通过文献调研,推测SARS-CoV 3a蛋白能诱导产生细胞自噬。首先通过pCI-3a表达质粒转染Vero E6细胞,检测细胞内是否诱导细胞自噬的产生,但是由于pCI-3a表达质粒在Vero E6细胞转染效率低,SARS-CoV 3a蛋白在细胞内表达水平很低,无法说明在细胞内SARS-CoV 3a蛋白能否诱导产生细胞自噬。为了提高SARS-CoV 3a蛋白的表达效率,将pcDNA5/FRT-3a表达质粒转染Flp-InTM CHO细胞,筛选出稳定表达SARS-CoV 3a蛋白的CHO细胞(CHO-3a)。通过电子显微镜观察,在CHO-3a细胞中观察到自噬泡的出现。将pEGFP-LC3转染CHO-3a细胞,可观察到有荧光蛋白的点状聚集现象,而正常CHO细胞中此现象相对较少。通过MDC荧光染色,CHO-3a细胞可见蓝色荧光,而正常CHO只观察到很微弱的蓝色荧光。Western印迹法结果显示CHO-3a细胞中LC3蛋白特异性的剪切的程度相对于CHO细胞有所增高,以上实验结果初步说明SARS-CoV 3a蛋白在CHO细胞中能够引起细胞自噬现象。通过酵母双杂交方法,发现SARS-CoV 3a蛋白与定位于内质网上的calcium- modulating cyclophilin ligand(CAML)蛋白具有相互作用,通过GST pull-down方法验证SARS-CoV 3a蛋白和CAML蛋白之间的相互作用。随后设计了针对CAML蛋白的siRNA,转染CHO-3a细胞,通过Western印迹检测和自噬荧光细胞质蛋白检测发现CHO-3a细胞中细胞自噬受到抑制。文献报道CAML蛋白能够引起内质网中的Ca2+释放到细胞质中,而细胞质中Ca2+浓度的增加会通过特定的信号通路诱导细胞自噬的产生,在CHO-3a细胞中加入Ca2+螯合剂BAPTA/AM降低细胞质中的Ca2+浓度,Western印迹法检测和自噬荧光标志蛋白观察结果显示细胞自噬受到抑制。有文献报道Ca2+浓度增加激活Ca2+-CaMKK-β-AMPK信号通路对mTOR起负调控,而引起细胞自噬,在CHO-3a细胞中加入AMPK抑制物compound C阻断Ca2+-CaMKK-β-AMPK信号通路的传递,Western印迹法检测和自噬荧光标志蛋白观察结果显示细胞自噬受到抑制。以上的结果初步说明SARS-CoV 3a蛋白与CAML蛋白相互作用,通过Ca2+信号通路引起细胞自噬。炭疽毒素由保护性抗原(PA)、致死因子(LF)和水肿因子(EF)组成。炭疽致死因子是炭疽杆菌致死的最主要毒力因子,但是相对于保护性抗原以及水肿因子,对致死因子的研究还不多,对致死因子的作用机制有待于进一步的深入。本研究采用电子显微镜观察,MDC荧光染色,Western印迹法检测炭疽致死毒素(LT)对于巨噬细胞J774.1细胞的作用,结果显示炭疽致死毒素在J774A.1细胞中能够引起细胞自噬现象,并且随着LT作用时间的增加,细胞自噬的程度也随之提高。炭疽致死毒素(LT)是由PA和LF构成,为了进一步了解LT的作用方式,分别用单独的PA、单独的LF、LF+PA以及LFn+PA分别作用于J774A.1细胞,通过Western印迹法和MDC荧光染色检测,结果显示只有LT能够引起细胞自噬,以上结果提示,LF被PA转运到细胞内才能引起细胞自噬。文献报道LF在巨噬细胞中能够引起氧自由基(ROS)的生成,本研究通过加入ROS清除剂-还原性谷胱甘肽(GSH)清除LT引起的ROS的产生,通过Western印迹法检测,结果显示细胞自噬受到抑制,提示ROS是细胞自噬的刺激因子。本研究通过在LT作用于细胞前诱导或抑制细胞自噬的产生,采用MTT法检测LT半数致死浓度(LC50)的变化情况,研究细胞自噬对于LT毒性的影响,结果显示诱导细胞自噬后LC50明显提高,提示细胞自噬可减弱LT对J774A.1细胞的毒性,说明细胞自噬是细胞抵御LT毒性作用的一种保护机制。

【Abstract】 We used electron microscopy, Western blotting, GFP-LC3 monitor and monodansylcadaverin (MDC) staining to detect the autophagy in vivo, which was induced by the severe acute respiratory syndrome (SARS) coronavirus 3a protein and anthrax lethal toxin. We investigated the signaling pathway of autophagy which was induced by SARS-CoV 3a protein and the effect of autophagy on the intoxication of anthrax lethal toxin.Autophagy is one of the major pathways involved in the lysosomal degradation of cellular components in eukaryote. This process has an important role in various biological events such as eliminate intracellular pathogen. As autophagy is evolutionarily conserved in eukaryotes, some pathogen was able to escape autophagy, moreover some pathogen subvert the cellular autophagy pathway to form advantage on replication and release through evolution.Electron microscopy, Western blotting, MDC staining and GFP-LC3 monitor have been used to monitor autophagy in vivo. Microtuble?associated protein 1 light chain 3 (LC3), a mammalian homolog of yeast Atg8, has been used as a specific marker to monitor autophagy. Upon induction of autophagy, LC3 is conjugated to phosphatidylethanolamine and targeted to autophagic membranes. We constructed the expression plasmid encoding GFP-LC3, cDNA of HeLa cell was used as template and LC3 gene was amplified by PCR, then the gene was cloned into pEGFP-C1 vector, to generated the pEGFP-LC3 plasid. HeLa cells was transiently transfected with pEGFP-LC3 plasmid, the expression of GFP-LC3 protein in vivo was measured by fluorescence microscopy and Western blotting. We observed the GFP-LC3 puncta in HeLa cells when autophagy was induced. So we can use the pEGFP-LC3 plasmid to monitor the autophagy in cell.The non-structural proteins of SARS-CoV vary widely among different coronavirus species, which may be the reason of SARS-CoV was more lethal than other coronavirus species. 3a protein is the biggest non-structural protein of SARS-CoV. We speculated the SARS-CoV 3a protein could induce autophagy in vivo by references. Firstly, we transiently transfected pCI-3a plasmid into Vero E6 cells to monitor the autophagy in vivo, but we couldn’t measure the autophagy in Vero E6 cells because the expression of SARS-CoV 3 protein in Vero E6 cells was very low. To improve the expression of SARS-CoV 3a protein in vivo, we transfected pcDNA5/FRT-3a plasmid into Flp-InTM CHO cells and screened the stable expression of SARS-CoV 3a transformant named CHO-3a. We detected autophagic vacuoles in CHO-3a cells. After transfection with pEGFP-LC3 plasid, we observed the percentage of cells display fluorescent punctate GFP-LC3 in CHO-3a cells much more than in CHO cells. We observed the blue fluorescence in CHO-3a cells through by fluorescence microscopy that was stained by MDC, but the phenomenon in CHO cells was very low. Western blotting show the conversion of LC3-I to LC3-II was increased in CHO-3a cells relative to CHO cells. The SARS-CoV 3a protein induced autophagy in CHO cells by these experimentsSARS-CoV 3a protein could bind to calcium-modulating cyclophilin ligand (CAML), as revealed by yeast two-hybrid analysis. The interaction of SARS-CoV 3a protein and CAML was identified by GST pull-down assay. The autophagy could be inhibited by siRNA design for CAML measured by western blotting and GFP-LC3 monitor. CAML expression elevates the cytosolic Ca2+ concentration and the rise in the cytosolic Ca2+ concentration is a potent inducer of autophagy. The autophagy was inhibited by BAPTA/AM -a Ca2+ chelator, measured by western blotting and GFP-LC3 mintor. The Ca2+ induced Ca2+-CaMKK-β-AMPK signaling pathway and AMPK could induce autophagy by the negative regulation of mTOR. The autophagy was inhibited by compound C which was the AMPK inhibitor, measured by western blotting and GFP-LC3 monitor. According to above results, the interaction of SARS-CoV 3a and CAML could induce autophagy through a Ca2+ signaling pathway.Anthrax toxin contains three subunits: protective antigen (PA), lethal factor (LF) and edema factor (EF).Anthrax lethal factor was the most important toxin factor, but the research was less than EF or PA. In this research, we detected autophagic vacuoles in J774A.1 cells which was induced by anthrax lethal toxin through by Electron microscopy, Western blotting, MDC staining and a proportional relationship between with LT treating time and the degree of autophagy. Lethal toxin consists of PA and LF, we used PA, LF, PA plus LF, PA plus LFn to treated J774A..1 cells. The results showed only that the PA plus LF could induce autophagy, so the cytosolic LF was the functional factor to induce autophagy. Lethal toxin was reported to induce the production of reactive oxygen species (ROS) in macrophages, glutathione (GSH) is an antioxidant that is widespread in the cells and it can eliminate ROS. The result of Western blotting showed autophagy was inhibited by GSH and it’s suggested that the ROS was the sitmulating facter of the autophagy. The median lethal concentration (LC50) of LT was measured by MTT assay. The LC50 of anthrax lethal toxin was influenced by autophagy, Autophagy was induced in murine macrophage by anthrax lethal toxin and was used as a means of weaken anthrax lethal toxin in murine macrophage.

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