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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 pathogenic degeneration of wild-type rubella virus in mainland China by in vitro culture and infection of ECV304 vascular endothelial cells by rubella virus. Methods The ECV304 vascular endothelial cells and virus were inoculated by routine methods. The expression of envelope glycoprotein E1 gene of rubella virus was detected by RT-PCR and indirect immunofluorescence. Morphological changes of cells were observed by phase contrast microscope and electron microscope. Apoptosis was detected by TUNEL. Results Rubella virus envelope glycoprotein E1 gene expression was detected 2 to 3 days after infection. On the 4th day after infection, obvious cytopathic effects were observed by phase contrast microscopy, that is, cells were detached by cell suspension, the cell body became round and contracted, Shrinkage and chromatin condensation. Under the electron microscope, obvious virus particles were observed, nuclear chromatin was concentrated, marginalized, and mitochondria gathered around the nucleus to be lysosomal, vacuolated and autophagic. Apoptosis index (AI) detected by TUNEL method was significantly different between virus infected group and control group (P <0.01). Conclusion The wild type strain of rubella virus has a significant cytopathic effect in ECV304 cells and induces the apoptosis of ECV304 vascular endothelial cells in vitro.