肠道病毒71型诱导细胞自噬、凋亡及相互调控关系的研究

发布时间：2018-01-21 09:17

本文关键词： 肠道病毒71型自噬凋亡病毒复制　出处：《北京协和医学院》2011年硕士论文　论文类型：学位论文

【摘要】：近年来由肠道病毒71型(Enterovirus 71, EV71)感染而引起的手足口病发病人数在我国呈大幅上升趋势,缺乏合适的疫苗及有效的抗病毒药物是控制EV71感染的主要障碍。而EV71致病机制的阐明无疑是解除这两个障碍的有力手段。但到目前为止,EV71致病机制尚不明确。自噬和凋亡在病毒感染中起着关键的作用,一方面可以通过降解病毒成分,递呈病毒抗原,激活机体免疫反应,从而达到清除病毒的目的；另一方面,病毒也可利用对自噬和凋亡的调控逃逸机体的抗病毒作用,维持自身的存活和复制。研究表明,EV71可诱导细胞发生自噬,也可诱导细胞发生凋亡,但自噬和凋亡的相互调控机制尚不明确。病毒感染细胞后,可引起细胞死亡,但同时病毒又必须借助活的细胞完成自身的复制过程。而目前EV71感染诱导的细胞自噬和凋亡对EV71病毒复制和颗粒释放的影响尚不明确。本实验中,我们首先证实了EV71 (Fuyang-0805)病毒株能够诱导RD-A细胞发生自噬和凋亡,然后研究了EV71诱导细胞自噬及凋亡对病毒产生和释放的影响,在此基础上进一步探讨了EV71诱导细胞自噬和凋亡的相互调控关系。实验结果显示,EV71活病毒可诱导RD-A发生自噬,随着感染时间的延长和感染剂量的增加,自噬水平逐渐增强。自噬不同阶段的抑制剂能够抑制由EV71病毒感染诱导细胞自噬的相应阶段。10μM的3-甲基腺嘌呤(3-methyladenine,3-MA)能够抑制EV71诱导的细胞自噬,同时还能够降低细胞培养上清中EV71病毒的滴度。EV71的活病毒能够诱导细胞发生凋亡,随着感染时间的延长和感染剂量的增加,凋亡水平逐渐增加。凋亡蛋白酶通用抑制剂Z-VAD-FMK能够抑制EV71诱导的凋亡,同时也可以抑制EV71病毒颗粒的产生和释放。另外,我们通过自噬和凋亡抑制剂的使用以及siRNA干扰自噬和凋亡相关分子的表达,研究EV71诱导的自噬和凋亡的相互调控关系。结果证明,抑制自噬体的形成可以抑制细胞发生凋亡,而抑制自噬溶酶体的活性却促进了凋亡的发生。同时,抑制凋亡可以促进EV71诱导的细胞自噬的发生,促进凋亡可以抑制EV71诱导的细胞自噬,凋亡底物PARP (poly ADP-ribose polymerase, PARP)减少可以抑制EV71感染诱导的细胞自噬。以上结果提示,通过使用合适剂量的抑制剂或诱导剂调控EV71诱导的自噬和凋亡来影响病毒颗粒的释放,从而降低病毒毒力,缓解EV71病毒引发的疾病可能会为寻找新的抗EV71药物提供新的思路。
[Abstract]:In recent years, the number of HFMD cases caused by enterovirus 71 (EV71) infection in China is on the rise. The lack of appropriate vaccine and effective antiviral drugs are the main obstacles to the control of EV71 infection. The elucidation of the pathogenesis of EV71 is undoubtedly a powerful means to remove these two obstacles. But so far. The pathogenesis of EV71 is unclear. Autophagy and apoptosis play a key role in viral infection. On the one hand, autophagy and apoptosis can activate immune response by degrading viral components and presenting virus antigen. In order to achieve the purpose of eliminating the virus; On the other hand, the virus can also use the regulation of autophagy and apoptosis to escape the body's anti-virus effect, maintain its own survival and replication. Apoptosis can also be induced, but the mechanism of autophagy and apoptosis is not clear. Virus infection can cause cell death. But at the same time, the virus must use living cells to complete its own replication process, but the effect of autophagy and apoptosis induced by EV71 infection on the replication and particle release of EV71 virus is not clear. In this study, we first confirmed that EV71 Fuyang-0805) could induce autophagy and apoptosis in RD-A cells. Then the effects of EV71 induced autophagy and apoptosis on the production and release of virus were studied, and the relationship between autophagy and apoptosis induced by EV71 was further discussed. The results showed that EV71 virus could induce autophagy of RD-A with the increase of infection time and dose. Autophagy level gradually increased. Inhibitors at different stages of autophagy could inhibit the corresponding stage of autophagy induced by EV71 virus infection. 3-methyladenine. 3-MA) can inhibit autophagy induced by EV71 and decrease the titer of EV71 virus in cell culture supernatant. EV71 live virus can induce apoptosis of cells. With the prolongation of infection time and the increase of infection dose, the level of apoptosis gradually increased. Z-VAD-FMK, a common inhibitor of apoptotic protease, could inhibit the apoptosis induced by EV71. We also inhibit the production and release of EV71 virus particles. In addition, we interfere with the expression of autophagy and apoptosis-related molecules through the use of autophagy and apoptosis inhibitor and siRNA. To study the relationship between autophagy and apoptosis induced by EV71. The results showed that inhibiting the formation of autophagy could inhibit apoptosis, while inhibiting the activity of autophagy could promote apoptosis. Inhibition of apoptosis can promote autophagy induced by EV71, and promote apoptosis can inhibit autophagy induced by EV71. The reduction of apoptosis substrate PARP ADP-ribose polymerase (parp) inhibited autophagy induced by EV71 infection. These results suggest that EV71 induced autophagy and apoptosis can be controlled by appropriate dose of inhibitors or inducers to affect the release of virus particles and thus reduce the virulence of the virus. Alleviating the disease caused by EV71 may provide new ideas for finding new anti-EV71 drugs.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

【共引文献】