巨细胞病毒感染细胞内微丝骨架重组异常

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。

Impaired microfilament cytoskeleton rearrangement in cytomegalovirus infected cells

OBJECTIVE: To investigate the effect of cytomegalovirus (CMV) infection on actin and microfilament in human embryo fibroblast cells (HF) and its relationship with CMV replication. METHODS: Cell morphology was observed after the infection of CMV. Western-blot was used to measure the expression levels of beta-actin, G-actin and F-actin proteins. CMV immediately early antigen (CMV IE) in HF cells was analyzed by indirect immunofluorescence assay. Microfilament alteration was determined by cytoskeleton fluorescence probe. RESULT: CMV IE was demonstrated in more than 95% of HF cells after infection, which was primarily located in nucleus. The shape of HF cells changed from thin shuttle like to round and thick ball like, even escaping from wall after infection by CMV. Compared with control group, the expression of G-actin protein increased at 24 h of CMV infection (0.941 +/-0.061 compared with 0.714 +/-0.119, P <0.05), then decreased at 72 h, 96 h respectively(0.218 +/-.035, 0.230 +/-0.055 compared with 0.714 +/-0.119, P <0.05). The levels of F-actin in infected cells gradually decreased at 24 h, 72 h and 96 h compared with control HF cells (0.256 +/-0.021, 0.127 +/-0.032, 0.026 +/-0.008 compared with 0.373 +/-0.050, P<0.05). In infected HF cells, microfilaments were found ruptured, arranged turbulently. Cells fused and fluorescence density of microfilament markedly reduced. CONCLUSION: Cytomegalovirus can induce alteration of actins and microfilament, which may be associated with its infection, replication and reactivity in host cells.