摘要
目的通过体外培养和风疹病毒野生株感染ECV304血管内皮样细胞,初步探讨中国大陆流行的风疹病毒野生株的致病变性特征.方法常规方法培养ECV304血管内皮样细胞和病毒接种,RT-PCR和间接免疫荧光法检测风疹病毒包膜糖蛋白E1基因的表达,相差显微镜及电子显微镜下观察细胞受病毒感染后形态变化,脱氧核糖核酸末端转移酶法（TUNEL）检测细胞凋亡.结果感染后2～3 d即可检测到风疹病毒包膜糖蛋白E1基因表达;感染后4 d,相差显微镜观察呈现明显的细胞病变效应,即细胞悬浮脱壁、细胞体变圆缩小、细胞膜皱缩、染色质浓缩等;电镜下观察可见明显病毒颗粒,核染色质浓集、边缘化,线粒体聚集于细胞核周围呈溶酶体化、空泡化和自噬现象.TUNEL法检测细胞凋亡指数（AI）在病毒感染组与对照组间有显著性差异（P＜0.01）.结论风疹病毒野生株在ECV304细胞中有明显的致细胞病变效应,在体外细胞培养下可诱导ECV304血管内皮样细胞凋亡.
Objective To investigate the mechanisms for cytopathic effect （CPE） of wide-type rubella virus （RV）in ECV304 endothelial-like cells. Method ECV304 cells were cultured in MEM supplemented with 10% fetal calf serum, glutamine, sodium bicarbonate, penicillin and streptomycin at 37℃ with 5 % CO2 in a humidified incubator, and then grown in maintenance medium with 2% fetal calf serum to avoid overstimulating cell growth. Infected cells and control cells were harvested at day 2, 4, and 6 after infection for following experiments. RT-PCR and indirect immunofluorescence were used to detect RV infection. Phase-contrast and electron microscopy were performed to analyze morphology changes in infected and uninfected groups. TUNEL assay was carried out to study wide-type RV-induced apoptosis. Result In RV-infected ECV304 cells,cytopathic effects were first observed approximately at 2-3 days post-infection.The majority of cells with CPE changes were of detachment from the monolayer, rounding of cells, cell shrinkage and membrane blebbing were seen. At 4-6 days post-infection, the detached cells increased clearly ; the CPE regions on the monolayer were extended, and the infected cells in the CPE regions showed in spindly form with wide spacing. RT-PCR and indirect immunofluorescence results indicated RV infection in ECV304 endothelial-like cells. Using electron microscopy, RV particles and its cytopathic effects including the condensed chromatin, fragmented nuclei and clustering of mitochondria with dramatic changes around nuclear were further examined. TUNEL results showed that the difference of apoptosis index between the infected group and uninfected group was significant（ P 〈 0.01 ）. Conclusion The wide-type RV in China continent induced apparent cytopathic effects and apoptosis in ECV304 endothelial-like cells. The results provided more morphology evidences for the teratogenic mechanisms of RV.
出处
《解剖学报》
CAS
CSCD
北大核心
2006年第2期203-207,共5页
Acta Anatomica Sinica
基金
广州市重点科技攻关项目(2001-Z-005-01)
