BrdU标记的人巨细胞病毒感染HEL细胞的方法学研究

目的 以标记在人巨细胞病毒(HCMV)DNA上的BrdU为示踪剂,研究病毒在受染HEL细胞中的移动过程;同时结合病毒蛋白pp65的表达探讨病毒复制、增殖的过程。方法 以BrdU标记的HCMV(MOI=4)感染HEL细胞,分别选取感染后2h、4h、6h、24h及48h 5个时间点的细胞,用抗BrdU单克隆抗体,研究病毒核酸的胞内定位;同时用抗HCMV蛋白pp65的单克隆抗体检测此蛋白的表达及分布。结果 免疫细胞荧光染色结果提示:在感染5个时间点,病毒DNA依次位于胞质、胞核及同时位于胞核和胞质;蛋白pp65的表达及分布规律为:胞内无表达、胞核分布、胞核与胞质同时分布及巨细胞和融合细胞内分布。结论 以BrdU为标记物标记双链DNA病毒核酸不仅为研究HCMV．的胞内移动提供了良好的模型,同时也为其他病毒的研究提供了良好的工具;本实验结合HCMV蛋白pp65的表达和分布直观地反应了HCMV感染HEL细胞并在其中复制、增殖的过程。

Research on the use of BrdU to track HCMV infection in HEL cells

Objective The genome of human cytomegalovirus(HCMV)was labeled with bromodeoxyuridine(BrdU)to follow the movement of HCMV in the infected HEL cells in combination with tracking of HCMV protein pp65 expression to study the process of viral replication and proliferation.Methods HEL cells were infected with HCMV(MOI=4)which was labeled with BrdU in advance.We inspected the distribution of the labeled viral nucleic acids with anti-BrdU monoclonal antibody in the infected cells at 2,4,6,24 and 48 hours post infection.At the same time point,we also inspected the expression and distribution of pp65 with anti-pp65 monoclonal antibody. Results At the five time points included in this study,immunofluorescence staining displayed the labeled viral genome first showed up in cytoplasm,then in nucleus and finally in both cytoplasm and nucleus.The expression of pp65 was absent in the beginning,then present sequentially in the nucleus and then both in the nucleus and cytoplasm.Finally giant cells and confluent cells were formed.Conclusion Labeling of double-stranded DNA with BrdU is a useful method to study the intracellular distribution of HCMV after initial infection and may serve as a tool for study of other viruses.By including the distribution of pp65 expression,this study directly observed the replication and proliferation of HCMV in HEL cells.