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目的研究CVB3损伤HeLa细胞高尔基体结构的机制,为进一步探讨CVB3的致病机制奠定基础。方法采用MOI为5的CVB3感染HeLa细胞,免疫荧光双标法检测病毒感染后不同时间点VP1和GM130在细胞中的定位情况,观察细胞高尔基带的变化;同时,选择CVB3感染后不同时间点收集细胞,Bradford法蛋白定量后,Western blot测定VP1、GM130和GRASP65蛋白的表达变化。结果正常HeLa细胞中GM130和GRASP65免疫荧光呈带状分布,高尔基体结构正常;而CVB3病毒感染HeLa细胞3h后,GM130的荧光呈现散点状分布,高尔基体结构损伤。与此同时,GM130和GRASP65蛋白表达量逐渐下降;而VP1蛋白表达量持续上升,且6h后一直维持在较高水平。结论 CVB3感染引起HeLa细胞蛋白GM130和GRASP65表达量下调,导致GM130和GRASP65荧光带弥散,高尔基带断裂,这可能是CVB3感染导致宿主细胞高尔基体结构损伤的机制之一。
Objective To investigate the mechanism of CVB3 damaging the Golgi structure of HeLa cells and lay a foundation for further study on the pathogenesis of CVB3. Methods HeLa cells were infected with CVB3 with an MOI of 5, and the localization of VP1 and GM130 in cells at different time points after infection with virus was detected by immunofluorescence double-labeled method. The change of Golgi band was observed. At the same time, The expression of VP1, GM130 and GRASP65 protein in the cells and Bradford method were determined by Western blot. Results The immunofluorescence of GM130 and GRASP65 in normal HeLa cells showed a ribbon-like distribution with normal Golgi structure. After 3 hours of infection with HepG2 virus, the fluorescence of GM130 showed a scattered distribution and the Golgi structure was damaged. At the same time, the expression of GM130 and GRASP65 protein gradually decreased; while the expression of VP1 protein continued to rise, and maintained at a high level after 6h. Conclusions The down-regulation of the expression of GM130 and GRASP65 in HeLa cells caused by CVB3 infection leads to the diffusion of GM130 and GRASP65 fluorescence bands and the rupture of the Golgi band, which may be one of the mechanisms of CVB3 infection leading to the structural damage of the Golgi apparatus.