摘要
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。
We investigated the damage mechanism of Coxsackievirus B3（CVB3） in Golgi apparatus and discussed the pathogenic mechanism of its infection.After infecting HeLa cells with CVB3（MOI=5）,we observed the variation of Golgi ribbon by detecting the locations of viral protein VP1,cellular proteins Golgi matrix protein 130（GM130）,and Golgi reassembly stacking protein 65（GRASP65） in HeLa cells at different time points post-infection with double-label immunofluorescence.Total cellular proteins were collected for quantitative analysis by Bradford Protein Assay at different post-infection times.And then the expressions of viral protein VP1,cellular proteins GM130,and GRASP65 in HeLa cells were analyzed by Western blot.Results showed that there were banding-like immunofluorescence pattern for GM130 in normal HeLa cells,and the structure of Golgi apparatus was complete;while in the CVB3 infected HeLa cells,the immunofluorescence pattern of GM130 became scattered point-like and the structure of Golgi apparatus was damaged in the infected cells after 3 h post-infection.Meanwhile,the expression of cellular proteins GM130 and GRASP65 decreased gradually after the infection and the expression of viral protein VP1 maintained at a high level after 6 h post-infection.It＇s suggested that CVB3 infection down-regulates the expressions of GM130 and GRASP65 in HeLa cells and induces diffusion in immunofluorescence patterns of GM130 and GRASP65 as well as disruption in Golgi ribbon,which might be a possible pathogenic mechanism for Golgi apparatus structure damage in CVB3 infected host cells.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2012年第6期539-543,共5页
Chinese Journal of Zoonoses
基金
国家自然科学基金(30660010
81160196)
江西省自然科学基金(0540043)
江西省教育厅科技计划项目(2006323)
江西省科技厅2008年科技支撑计划项目"柯萨奇病毒VP1对高尔基体结构影响研究"~~
