EV71病毒对树鼩原代肾上皮细胞感染模型的建立

摘要

目的 建立EV71对树鼩原代肾细胞的感染模型.方法 胰蛋白酶消化法获得树鼩的原代肾细胞,用EV71感染树鼩肾细胞,测定1、2、4、6和8 d培养上清病毒滴度,分别用Western blot和间接免疫荧光法检测细胞中EV71病毒VP1蛋白的表达,以确定EV71病毒对树鼩原代肾细胞的感染性.结果 对分离得到的树鼩原代肾细胞进行传代纯化和形态鉴别,建立以树鼩原代肾细胞为主的细胞培养.用EV71病毒感染树鼩原代肾细胞,感染后48 ~ 96 h病毒滴度可达到1.3×106 TCID 50/mL,说明EV71病毒可有效感染树鼩原代肾细胞并有效增殖.Western blot检测发现,EV71病毒VP1蛋白可在感染后2 ~ 8 d的树鼩原代肾细胞中有效检出,间接免疫荧光法则在感染后2 ~ 6 d细胞的细胞质中检测到病毒VP1蛋白的分布.结论 在成功建立树鼩原代肾细胞培养的基础上,确定了EV71病毒对树鼩原代肾细胞的感染性和病毒增殖特性,初步建立了EV71树鼩原代肾细胞感染模型.%Objective To establish an enterovirus 71(EV71) infection model of tree shrew primary renal cells.Methods Tree shrew primary renal cells were obtained by trypsin digestion.After subculture and purification,EV71 virus was used to infect these primary cells.The culture supernatant of these EV71-infected cells was collected for virus titer detection at 1,2,4,6 and 8 days post-infection.The cells were collected for detection of EV71 VP1 protein by Western blot assay.Furthermore,the expression and location of VP1 protein in the infected cells were detected by indirect immunofluorescence assay.Vero cells were taken as positive control to evaluate the infectivity of EV71 virus to tree shrew primary renal cells.Results Morphologically,the cultured cells were proved to be majorly consisted of the primary renal cells after subculture and purification.The obtained primary cells were infected by EV71 virus.The virus titer was up to 1.3×106 TCID 50/mL during 48-96 h post-infection,proving that EV71 virus infected and proliferated in the tree shrew primary renal cells.Western blot showed that the viral VP1 protein was detected from infected primary cells at 2 to 8 d post infection.VP1 protein was also observed in the cytoplasm at 2 to 6 d post infection by indirect immunofluorescence.Compared with Vero cells,the infectivity of EV71 virus to tree shrew primary renal cells and its proliferation were confirmed.Conclusions Based on the successful establishment of cell culture of tree shrew primary renal cells,the infectivity to the obtained cells and proliferation of EV71 virus in the cells are confirmed.The model of EV71 virus-infected tree shrew primary renal cells is initially established.