人巨细胞病毒感染对THP-1源性巨噬细胞自噬的影响

Influence of HCMV infection on autophagy in THP-1 derived macrophage

目的了解人巨细胞病毒(human cytomegalovirus,HCMV)感染THP-1源性巨噬细胞后对细胞自噬的影响。方法构建HCMV感染THP-1源性巨噬细胞模型,分别设HCMV感染组(HCMV组)、热灭活HCMV感染组(Heat-HCMV组)、模拟感染对照组(M组)和雷帕霉素(rapamycin,400nmol)自噬阳性对照组(RAPA组),病毒感染复数=1。采用westernblot检验测量各组自噬相关蛋白Beclin1表达量和LC3转化量(LC3-II/LC3-I)在感染后1h、3h、6h、12h、24h和48h的动态变化,并用透射电镜观察感染后6h、12h和24h各组细胞胞质中自噬泡形成的数量。结果在HCMV感染组,HCMV感染THP-1源性巨噬细胞后1h,自噬相关蛋白Beclin1表达量明显增高,高于模拟感染组。LC3转化量在感染后6h明显增高,高于模拟感染组。感染后24h和48h,Beclin1表达量和LC3转化量都降低,且均低于RAPA阳性对照组。用热灭活HCMV处理THP-1源性巨噬细胞,Beclin1在1h就开始明显高表达,高于模拟感染组。LC3转化量在6h开始明显增高,高于模拟感染组。用透射电镜观察到在HCMV感染THP-1源性巨噬细胞后6h,自噬泡数目明显增加,但感染后24h明显降低。热灭活HCMV处理THP-1源性巨噬细胞后6h,自噬泡明显增加,并持续到48h。结论HCMV感染THP-1源性巨噬细胞早期可激活细胞自噬,其激活方式可能并不依赖于HCMV复制,而感染后期细胞自噬受到抑制,这可能与HCMV复制等因素有关,但需进一步实验证实。

ObjectiveTo study the autophagy in THP-1 derived macrophage after HCMV infection.MethodsThe cell model of HCMV-infected THP-1 derived macrophage was established. The experiment groups were HCMV infection group (HCMV group), heat inactivated HCMV infection group (Heat-HCMV group), mock control group (M group) and positive control group (autophagy induced by 400 nmol rapamycin, RAPA group). The multiplicity of infection was 1. Western blot assay was used to detect the dynamic changes of the autophagy-related protein (beclin1) and LC3 transformation (LC3-II/LC3-I) at 1 h, 3 h, 6 h, 12 h, 24 h and 48 h after HCMV infection. The numbers of autophagic vacuoles formed in cytoplasm were observed by transmission electron microscope at 6 h, 12 h and 24 h after infection.ResultsFor HCMV group, beclin1 increased significantly at 1 h after infection and was higher than that in M group. LC3 transformation increased dramatically at 6 h after infection and was higher than that in M group. However, the expression of beclin1 and LC3 transformation decreased obviously compared to the RAPA group at 24 h and 48 h after infection. For Heat-HCMV group, the expression of beclin1 increased significantly at 1 h after infection and was higher than that in the mock control group. LC3 transformation increased dramatically at 6 h after infection and was higher than that in M group. The number of autophagic vacuoles increased at 6 h after infection; however, it decreased significantly at 24 h after infection. For Heat-HCMV group, the numbers of autophagic vacuoles continued to increase at 48 h after infection.ConclusionsAt early stage of infection, HCMV can stimulate autophagy in THP-1 derived macrophages, which may not depend on the replication of HCMV. At the late phase of infection, the inhibition of autophagy in THP-1 derived macrophages may be related to the factors such as viral replication, but further experiments are needed to confirm it.