流感病毒感染神经胶质细胞条件培养上清对星形胶质细胞的免疫生物学影响  

Immunobiological effects of influenza virus infected astrocytes derived conditional media on normal astrocytes in vitro

作　　者：王革非[1] 李蕊[1] 周健祥[1] 蒋治武[1] 代剑平[1] 李康生[1] 

机构地区：[1]汕头大学医学院微生物学与免疫学教研室,广东省感染病与分子免疫病理重点实验室,广东汕头515041

出　　处：《热带医学杂志》2015年第4期473-475,483,共4页Journal of Tropical Medicine

基　　金：国家自然科学基金(81001322;31170852;31300761);广东省高等学校优秀青年教师培养计划(Yq2013077)

摘　　要：目的利用人流感病毒H1N1和H3N2感染小鼠星形胶质细胞获得条件培养上清刺激正常星形胶质细胞,研究人流感病毒感染星形胶质细胞后释放的细胞因子,是否会诱导正常胶质细胞的免疫病理学变化。方法从新生小鼠大脑皮质分离纯化星形胶质细胞,经纯度鉴定后,用人流感病毒H1N1和H3N2体外感染,分别于感染早期(6 h)和感染中晚期(24 h)收获条件培养上清,刺激正常星形胶质细胞,CCK-8法检测细胞存活率,Western blotting法检测星形胶质细胞胶质纤维酸性蛋白(GFAP)表达状况,ELISA法检测细胞不同时间分泌的炎症因子TNF-α、IL-1β、IL-6水平变化。结果流感病毒感染星形胶质细胞的条件培养上清,可显著抑制正常胶质细胞的活性,诱导GFAP蛋白表达的上调,而炎症因子TNF-α、IL-1β、IL-6在不同时间点的蛋白表达水平发生不同程度改变,总体呈下降趋势。结论体外实验中,人流感病毒感染星形胶质细胞后释放的细胞因子会诱导正常胶质细胞发生较为明显的免疫病理学变化,但未见明显的细胞因子级联现象。Objective To investigate the immunopathological effects of influenza virus infected astrocytes derived proinflammatory cytokines on normal astrocytes.Methods Astrocytes were isolated from cerebral cortex of neonatal mouse and cultured in vitro.After identification of astrciyets purity,the cells were infected by influenza H1N1 and H3N2 viruses.At postinfection early（6 hour） and middle（24 hour） period,the infected astrocytes supernatant was harvested,then the viral particles in supernatant were removed by ultrafiltration.The primary mouse astrocytes were stimulated by viral particles free supernatant（conditioned supernatant）.The cell survive rate was detected using CCK-8;the expression of astrocyte GFAP was determined using Western blotting;the levels of inflammatory factor TNF-α,IL-1β and IL-6 at different post-infection time were tested using ELISA.Results After conditioned supernatant stimulation,the activity of normal astrocytes was suppressed remarkably.The up-regulation of GFAP protein expression was induced by conditioned supernatant.While,the secretion levels of inflammatory cytokines TNF-α,IL-1β and IL-6 from conditioned supernatant stimulated astrocytes showed a general tendency of decline.Conclusion In vitro,proinflammatory cytokines secreted by influenza virus infected astrocyte could induce immunopathological effects on normal astrocytes.

关 键 词：星形胶质细胞 流感病毒 炎症因子 

分 类 号：R373.13[医药卫生—病原生物学]