H5N1型禽流感病毒NS1蛋白诱导宿主细胞凋亡及相关miRNA改变的分子机制

Avian H5N1 influenza virus causes a remarkably sever disease in humans, with an overall case fatality rate close to 60%. However, the mechanism(s) of H5N1-induced diseases and death are not well understood at the present time. It has been postulated that H5N1-induced apoptosis is an important factor in pathogenesis. The nonstructural protein NS1, have been implicated in influenza virus-induced apoptosis. A detail characterization of the NS1 protein and the mechanisms involved in the induction of apoptosis is essential for understanding the pathogenesis of influenza viruses, particularly H5N1. To verify the role of H5N1-encoded NS1 in inducing apoptosis, the NS1 gene was cloned and expressed in cells such as human airway epithelial cells (NCI-H292 cells). The apoptotic events posttransfection were examined by immunofluorescence assay, ?ow cytometric measurement of propidium iodide, annexin V staining, cytometric beads array(CBA), Western blot analyses with antibodies speci?c for proapoptotic and antiapoptotic proteins, co-immunoprecipitation, mitochondrial membrane potential detection, and microRNA array. Furthermore, to investigate the activation of mitochondrial-dependent pathway of the apoptosis triggered by NS1 and to determine the key microRNAs which may be involved to regulate the expression of Bcl-2 family proteins or any other apoptotic proteins by study on the profile of microRNA expression treated by NS1 or/and apoptotic stimulations.

近年来高致病性禽流感病毒（AIV）H5N1跨物种屏障直接感染人，致使人感染禽流感疫情不断出现，病死率近60％，造成的公共卫生危害形势严峻。然而，AIV致病机制仍不明确。本研究旨在阐明高致病性禽流感病毒H5N1非结构蛋白（NS1）诱导哺乳动物细胞的凋亡机制，采用真核表达质粒构建、细胞转染、蛋白表达、流式液相多重蛋白定量技术、线粒体膜电位检测、免疫荧光、免疫共沉淀以及microRNA生物芯片等技术手段，率先探讨NS1基因对线粒体依赖的内源性凋亡通道的激活机制，阐明Bcl-2家族成员在NS1诱导的内源性细胞凋亡通路激活过程中的表达变化及作用，明确在NS1这一凋亡信号刺激下参与凋亡调控的关键miRNA分子，为H5N1致病机制的最终揭示奠定基础。

近年来高致病性禽流感病毒（AIV）H5N1跨物种屏障直接感染人，致使人感染禽流感疫情不断出现，病死率近60％，其流行给人类社会带来恐慌，造成的公共卫生危害形势严峻，因此对H5N1的致病力研究亟待加强。然而，AIV致病机制仍不明确。.通过本项目的实施，主要探讨了高致病性禽流感病毒H5N1非结构蛋白（NS1）诱导哺乳动物细胞的凋亡机制，研究内容涉及高致病性禽流感病毒H5N1的NS1蛋白与内源性通路相关的细胞凋亡、不同亚型流感病毒NS1蛋白与病毒致病力的相关性、关键信号通路的miRNA等科学热点问题，采用真核表达质粒构建、细胞转染、蛋白表达、线粒体膜电位检测、免疫荧光、免疫共沉淀及miRNA生物芯片等多种技术手段，涉及实验技术新，率先阐述了H5N1的NS1基因对线粒体依赖的内源性凋亡通道的激活机制，在NS蛋白导致的Bcl和Bax平衡上立意新颖；明确可见在凋亡诱导剂作用下NS1对人呼吸道上皮细胞细胞色素C释放效应的促进和放大以及线粒体膜电位的显著降低等凋亡效应，且NS1对宿主细胞的凋亡诱导效应与病毒致病力密切相关；初步筛选出在NS1这一凋亡信号刺激下参与凋亡调控的10个关键miRNA分子；尤其是在NS1的诱导下，miRNA-21的表达丰度与抑凋亡蛋白Bcl-2的表达水平密切相关，miR-21的表达水平降低可调节Bcl-2蛋白表达水平下调，并进一步促进细胞色素C的释放，诱导细胞凋亡的发生。但miRNA-21是否通过直接影响或其他间接调节效应影响了Bcl-2的表达丰度，从而遏制其抑凋亡效应，促进了靶细胞的凋亡都是我们拟进一步探讨的问题。miRNA在凋亡信号中起着复杂的调节作用，对找到H5N1致宿主细胞凋亡的分子靶标有重要的参考意义。本研究将有助于我们阐明NS1在诱导细胞凋亡中的角色和功能，在分子水平深入探讨H5N1的致病机理，为禽流感病毒致病机制的最终揭示奠定基础。