机构地区:[1]厦门大学附属中山医院皮肤科,361004 [2]厦门大学附属中山医院演武分院内科 [3]162-8640日本国立感染症研究所病毒一部
出 处:《中华微生物学和免疫学杂志》2012年第5期419-424,共6页Chinese Journal of Microbiology and Immunology
基 金:国家自然科学基金资助项目(30671884,30840070);福建省高校新世纪优秀人才支持计划资助项目(闽教科2007年20号);厦门市卫生局医学科研基金资助项目(WSK0618)
摘 要:目的应用构建的水痘-带状疱疹病毒(VZV)报告细胞系MV9G进一步研究白黎芦醇体外抑制VZV的作用机制。方法将无细胞VZV直接感染MV9G细胞(CFVs直接感染)或将带细胞VZV与MV9G细胞共培养(CAVs共培养)以激发MV9G细胞表达报告基因萤火虫荧光素酶。在CFVs直接感染前或CAVs共培养不同时间点加入白黎芦醇,通过比较药物对CFVs或CAVs激发荧光素酶的抑制强度分析白黎芦醇直接灭活病毒、抑制病毒黏附和穿透、抑制病毒在细胞内复制及其时间点和可逆性;通过比较药物作用前后VZV即刻早期蛋白62(IE62)mRNA拷贝数和IE62表达强度变化分析白黎芦醇对IE62转录和表达的抑制作用。结果白黎芦醇〉30.0μg/ml时MV9G细胞三磷酸腺苷(ATPs)含量随药物浓度升高而逐渐降低,ATPs降低50%时白黎芦醇浓度(CD50)约60.3μg/ml。CFVs与白黎芦醇(25.0μg/ml)预混37℃水浴孵育2h后直接感染MV9G细胞,CFVs激发荧光素酶下降50%。MV9G细胞在含白黎芦醇培养基中37%孵育2h后直接感染CFVs,CFVs激发荧光素酶随药物浓度升高而逐渐降低,但4℃孵育时无显著变化。在CAVs共培养中加入白黎芦醇后CAVs激发荧光素酶显著降低,药物抑制荧光素酶50%时浓度(IC50)约8.7μg/ml。分别在CAVs共培养3、6、9、12、24、30和36h时加入白黎芦醇,3~24h加药各组CAVs激发荧光素酶均显著高于对照组,但1h、30h和36h加药组与对照组问差异无统计学意义。CAVs共培养时撤除白黎芦醇后CAVs激发荧光素酶显著高于撤药前,尤以24h和72h撤药组明显。VZVIE62mRNA拷贝数和IE62抗体阳性细胞数随药物作用时间延长而逐渐降低。结论白黎芦醇细胞毒性较强,MV9G细胞可耐受最高浓度为30.0μg/ml。白黎芦醇部分灭活CFVs、抑制CFVs穿透MV9G细胞但对CFVs黏附MV9G细胞无影响,以浓度依赖方式可逆性抑制CAVs细胞内复制。白黎芦醇可能Objective To further investigate inhibitory mechanism (s) of resveratrol on varicellazoster virus (VZV) in vitro with our previously generated reporter cell line MV9G. Methods Cell-free VZVs were directly inoculated onto MV9G cells (CFVs direct-infection) or cell-associated VZVs were co-cultured with MV9G cells ( CAVs co-cuhure) to activate expression of reporter gene firefly luciferase in MV9G cells. Resveratrol was added before or after virus infection, roles of resveratrol on direct inactivation, on viral attachment to and penetration into MV9G cells, on intracellular viral replication and its IC50, inhibitory time points and reversibility were assayed by comparing the luciferase activities reduction by resveratrol. The reductions of VZV IE62 mRNA copies and IE62-antibody positive cells by resveratrol were further assayed. Results ATPs contents of MV9G cells in the presence of resveratrol over 30.0 μg/ml were concentration- dependently reduced, the CD50 of which was around 60.3 μg/ml. CFVs were premixed with 25.0 μg/ml resveratrol and incubated at 37℃waterbath for two hours and then directly inoculated onto MV9G cells, luciferases activated by resveratrol-treated CFVs were reduced to around half of the untreated controls. MV9G cells were pre-incubated with resveratrol at 37℃ for 2 h and then directly infected with CFVs at 37℃ for another 2 h, the CFVs-activated luciferase was concentration-dependently reduced, but no big change was observed in those pre-incubated at 4℃. MV9G cells were co-cultured with CAVs in the presence of resvertrol for 72 h, the CAVs-activated luciferases were markedly reduced in a concentration-dependent manner, the IC50 of which was around 8.7 μg/ml. Resveratrol was added in CAVs co-culture at 1, 3, 6, 9, 12, 24, 30, and 36 h post infection, the CAVs-activated luciferase in those resveratrol was added at 3, 6, 9, 12, and 24 h post infection were significantly higher than those of controls. Resveratrol was withdrawn from CAVs co- culture media, the CAVs-activa
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