A549细胞和Hep-2细胞表面糖链类型鉴定及其与H5N1型禽流感病毒结合的特性

【作者】 黄丽；

【导师】 尹洪超；

【作者基本信息】 北京协和医学院 ， 病理学， 2010， 硕士

【摘要】 背景：高致病性禽流感病毒H5N1可以感染人类并引起致命的呼吸道疾病。一旦禽流感病毒获得在人际传播的能力将很可能导致人类流感大流行。一般认为禽流感病毒对人类上呼吸道细胞亲和力低,可能是限制H5N1病毒人与人之间传播的因素。然而,禽流感H5N1病毒对人类呼吸道的亲嗜性和致病机理迄今仍不清楚。因此,研究H5N1病毒对人呼吸道上皮细胞的侵袭作用,对理解禽流感病毒的传播和致病具有重要的意义。目的：建立Hep-2细胞和A549细胞模型,研究H5N1型禽流感病毒对人呼吸道的亲嗜性和黏附作用。方法：用凝集素染色技术和流式细胞术检测A549细胞和Hep-2细胞表面流感病毒唾液酸受体SAa2,3Gal和SAa2,6Gal的表达。用FITC标记的H5N1病毒和间接免疫荧光法,检测H5N1病毒进入细胞情况。用western blotting法分析病毒进入A549细胞和Hep-2细胞的效率。用MTT法检测H5N1病毒处理后细胞的存活状况。结果：A549细胞和Hep-2细胞表面有大量SAa2,3Gal链,但SAa2,6Gal链含量却很少。Hep-2细胞表面SAa2,3Gal受体的表达水平高于A549细胞。H5N1病毒能够进入A549细胞和Hep-2细胞,而且H5N1病毒对A549细胞的亲嗜性更强。与A549细胞相比,Hep-2细胞对H5N1流感病毒诱导的细胞死亡更敏感。H5N1诱导的细胞死亡是非Caspases依赖性的,并且与PI3K活化无关。更多还原

【Abstract】 Background The H5N1 virus often leads to acute respiratory disease and death. The pandemic potential of H5N1 virus depends largely on its efficiency of human-to-human transmission. This inefficiency has been speculated to be determined in part by the tropism of influenza virus for the human upper respiratory tract. However, the tropism and pathogenicity of avian influenza A (H5N1) in huamn respiratory tract are poorly understood. Therefore, analysis of the interaction between influenza virus and human respiratory epithelial cells will be critically important to better understand the virus transmission and pathogenicity in humans.Objective To determine H5N1 virus attachment to the human respiratory tract, and to study the tropism of influenza virus, Hep-2 cells and A549 cells derived from human upper and lower respiratory tract were included.Methods Expression of SAa2,3Gal and SAa2,6Gal in A549 cells and Hep-2 cells was examined by using lectin fluorescence and flow cytometry. Binding experiments with FITC-labeled H5N1 virus and indirect immunofluorescence assay were perfomed to study the virus entry into A549 and Hep-2 cells. The entry efficiency was determined by Western blot analysis. Cell viability after H5N1 treatment was measured by MTT assay.Results SAa2,3Gal was prevalent in A549 cells and Hep-2 cells, while SAa2,6 Gal was little found. SAa2,3Gal expression was more regularly observed in Hep-2 cells, rather than A549 cells. The H5N1 virus tested could enter A549 cells and Hep-2 cells. However, viral entry efficiency differed between the two cell lines tested. A549 cells were found to be more susceptible to avian influenza than Hep-2 cells. H5N1-induced cell death was inefficient in A549 cells than Hep-2 cells. Furthermore, the cell death elicited by H5N1 virus was independent of Caspases or PI3K activition.Conclusion The expression of SAa2,3Gal on the cells tested corresponded with the attachment of the H5N1 virus. However, sialic acid only may not sufficient for entry into cells.更多还原