肠道病毒71型体外感染树鼩肾细胞特性观察

摘要

目的 探讨肠道病毒71型(EV71)对树鼩肾细胞的感染特性.方法 通过组织块贴壁法分离培养树鼩原代肾细胞,观察细胞形态并进行传代培养,利用细胞角蛋白18对树鼩肾细胞进行间接免疫荧光鉴定.用EV71感染树鼩肾细胞,通过倒置显微镜观察细胞病变情况,结合间接免疫荧光实验观察细胞中病毒蛋白的表达情况,采用实时荧光定量PCR技术检测病毒载量,以Vero细胞为对照,研究EV71对树鼩肾细胞的感染情况.结果 通过组织块贴壁法可以快速获得树鼩原代肾细胞,并可进行多次传代培养,细胞为梭形,贴壁呈铺路石状,免疫荧光鉴定为肾上皮细胞.EV71可以感染树鼩肾细胞,使之出现明显的细胞变圆、脱落或解体等病变,免疫荧光实验可以观察到病毒蛋白的分布,实时荧光定量PCR检测病毒载量最高可达105拷贝/mL,病变情况和增殖趋势与Vero细胞相似.结论 EV71可以体外感染树鼩肾细胞,该细胞模型可为EV71药物筛选和感染机制研究提供平台.%Objective To investigate enterovirus 71 (EV71) infection characters on the tree shrew kidney cells.Methods The primary kidney cells from tree shrew were isolated by tissue culture.The cell was passaged and morphology was observed regularly.The indirect immunofluorescence identification was performed by using keratin 18.Tree shrew kidney cells were infected with EV71,cell lesions were observed by inverted microscope,viral load was detected by real-time PCR.Vero cells were used as control.Results Tree shrew primary kidney cells can be rapidly prepared by tissue culture and can be passaged easily.The kidney cells are mainly spindle-shaped and show paving-stones structure after adherent.They were identified as kidney epithelial cell by immunofluorescence assay.Tree shrew kidney cells can be infect with EV71 and showed significant cytopathic effect (CPE) such as rounding,falling off and lysis.The viral protein was observed by immunofluorescence.Viral load of was up to 105 copies/mL.The CPE and proliferation trends were similar to that of Vero cell.Conclusion EV71 can infect tree shrew kidney cells under in vitro condition,and the cell assay can provide a platform for drug screening and infection mechanism research of EV71.