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目的分析人巨细胞病毒(HCMV)感染对人星形胶质瘤细胞凋亡的影响及其细胞内差异蛋白的表达。方法以HCMV AD169株感染U251细胞,复制HCMV体外感染模型,通过RT-PCR和免疫细胞化学技术检测HCMV IE蛋白和结构蛋白pp65的表达。用AnnexinⅤ-FITC和PI染色检测细胞凋亡,通过SELDI-TOF质谱分析感染细胞内差异蛋白的表达。结果HCMV感染后6 h,U251细胞经RT-PCR可扩增出242 bp的IE基因条带,感染后3 d,细胞核内有大量IE蛋白表达,核周及细胞浆内有大量pp65蛋白表达。病毒感染后3 d,约47%的细胞产生凋亡。HCMV感染后细胞内Caspase-8和磷脂酶A2的表达明显增加。结论HCMV感染可能通过上调Caspase-8和磷脂酶A2的表达而诱导U251星形胶质瘤细胞产生凋亡损伤。
Objective To analyze the effect of human cytomegalovirus (HCMV) infection on the apoptosis of human astrocytoma cells and the expression of intracellular differential proteins. Methods U251 cells were infected with HCMV strain AD169 and infected with HCMV in vitro. The expression of IE protein and pp65 was detected by RT-PCR and immunocytochemistry. Apoptosis was detected by Annexin V-FITC and PI staining, and the expression of differentially expressed proteins in infected cells was analyzed by SELDI-TOF mass spectrometry. RESULTS: Six hours after HCMV infection, a 242 bp IE gene band was amplified by RT-PCR in U251 cells. After 3 days of infection, a large amount of IE protein was expressed in nuclei and a large amount of pp65 protein in nucleus and cytoplasm. About 47% of the cells developed apoptosis 3 days after virus infection. HCMV infected cells significantly increased the expression of Caspase-8 and phospholipase A2. Conclusion HCMV infection may induce apoptosis of U251 astrocytoma cells by up-regulating the expression of Caspase-8 and phospholipase A2.