氧化应激通过细胞自噬促进人巨细胞病毒在细胞中的复制

Acceleration of HCMV Replication in HFF Cells by Oxidative Stress-induced Autophagy

目的通过观察氧化应激诱导的细胞自噬对人巨细胞病毒在细胞内的增殖作用,为人巨细胞病毒防治提供新的思路。方法利用PCR扩增LC3B编码片段,构建细胞自噬表达载体,观察细胞自噬的形成;收集感染HCMV(AD169)72 h后的细胞,用RT-qPCR方法检测细胞中UL122 mRNA水平和病毒颗粒水平;采用蛋白免疫印迹实验检测病毒蛋白表达;并利用TCID_(50)法检测自噬对感染性病毒增殖的情况。结果成功构建细胞自噬载体,可用于指示细胞自噬的形成;与正常培养的细胞相比,氧化应激可诱导细胞自噬,并通过此途径促进病毒UL122基因表达水平、pp65蛋白水平和病毒载量升高;同时,病毒滴度实验也显示细胞自噬可以促进HCMV在细胞中的复制。结论氧化应激可诱导细胞自噬的发生,同时促进HCMV复制增殖,而自噬抑制剂3-MA可以抑制氧化应激促进的HCMV复制,结果证实细胞自噬是氧化应激促进HCMV复制增殖的机制之一。

Objective To provide new insights into the prevention and treatment of human cytomegalovirus(HCMV)by observing the effect of autophagy induced by oxidative stress on the proliferation of HCMV in cells.Methods Using PCR to amplify the LC3B encoding fragment,an autophagy expression vector was constructed to observe the formation of cellular autophagy.Then,HFF cells infected with HCMV(AD169)for 72 hours were collected.The levels of UL122 expression and viral particles were detected by real-time quantitative PCR.And the viral protein pp65 expression level and the proliferation of virion were detected by Western blot and TCID_(50),respectively.Results Autophagy detection vectors were successfully constructed,which could be used to indicate the formation of cellular autophagy.Compared with normal cultured cells,oxidative stress could induce autophagy formation,and upregulated UL122 gene expression,pp65 protein levels and viral load by this pathway.The viral titer test also showed that autophagy could promote the replication of HCMV in cells.Conclusion Oxidative stress can induce autophagy and promote the replication and proliferation of HCMV,while autophagy inhibitor 3-MA can inhibit the replication of HCMV promoted by oxidative stress.It is confirmed that autophagy is one of the mechanisms of oxidative stress promoting the replication and proliferation of HCMV.