目的:探讨巨细胞病毒(CMV)感染对人胚成纤维细胞(HF)微丝骨架影响及与病毒复制状态的可能关系。方法:显微镜观察细胞形态;W estern-b lot法分别检测细胞内β型肌动蛋白(β-actin)、球状肌动蛋白(G-actin)和纤维形肌动蛋白(F-actin)的含量;间接免疫荧光标记病毒即刻早期抗原(IE);微丝骨架探针观察HF细胞内感染状况及微丝骨架变化。结果:HF细胞CMV感染率大于95%,IE抗原主要位于细胞核。受染细胞变粗、变圆,甚至脱壁,呈时间依赖性。与未感染组相比,CMV感染后细胞内β-actin蛋白质含量有所下降,但无显著性差异(P>0.05);G-actin水平在感染24 h后增加(0.941±0.061 vs 0.714±0.119,P<0.05),但在感染后72 h、96 h却下降(0.218±0.035、0.230±0.055 vs 0.714±0.119,P<0.05);F-actin水平在感染后24 h、72 h、96 h呈渐进性下降(0.256±0.021、0.127±0.032、0.026±0.008 vs 0.373±0.050,P<0.05)。受染细胞内微丝排列紊乱,极性消失;细胞融合,荧光强度明显减弱。结论:CMV引起HF细胞内肌动蛋白质、微丝骨架形态及重组异常,可能有助于其侵袭宿主细胞并进一步活化及复制。
To investigate the infection of human embryonic fibroblast cell line(HF cells) with human cytomegalovirus (CMV) as well as the effects of CMV on cytoskeleton and apoptosis,RT-PCR assay was used to detect the mRNA expression of IE,β-actin and GAPDH of HF cells infected with CMV.CMV particles and microfilaments were detected with transmission electron microscope, and flow cytometry was used to detect the apoptotic cells.Shape of HF cells changed after infection of CMV.HF cells infected with CMV could express IE mRNA in a concentration-dependent manner.The expression of β-actin mRNA of HF cells infected with CMV decreased as compared with that of the uninfected,while the expression of GAPDH mRNA did not change much.CMV particles could be seen in the cells with transmission electron microscope.Microfilaments were ruptured and shortend after the infection of CMV.Necrotic and apoptotic cells increased after the infection of CMV.Our results showed that CMV can not only infect and replicate in human embryonic fibroblast cell line,but also affect the expression of β-actin mRNA and break microfilaments.Additionally,CMV can induce the apoptosis of HF cells.
