人巨细胞病毒感染对人胚肺成纤维细胞自噬的影响

Effect of human cytomegalovirus infection on autophagy of MRC-5 cells

目的:研究人巨细胞病毒(human cytomegalovirus,HCMV)感染对人胚肺成纤维(MRC-5)细胞自噬的影响。方法:用HCMV AD169株(MOI=1)感染MRC-5细胞,同时将未感染HCMV的MRC-5细胞设为空白对照组。分别在感染12、24、36、48和60 h后用蛋白质印迹法检测自噬相关蛋白Beclin1和LC3蛋白以及m TOR信号通路下游磷酸化关键分子4E-BP-1-P和p70S6K-P的表达;应用荧光定量PCR检测HCMV DNA拷贝数;流式细胞仪定量检测吖啶橙荧光颗粒阳性细胞率。结果:在MRC-5细胞病毒感染组中,LC3和Beclin1蛋白表达水平均先升高再下降,与流式细胞仪检测结果一致;同时,4E-BP-1-P、p70S6K-P蛋白的表达水平先降低后升高;HCMV DNA拷贝数也是先增加后降低。而对照组中无相应分子变化。结论:HCMV感染MRC-5细胞后,在病毒增殖复制的早期(12~24 h内)可促进细胞发生自噬,晚期(24~60 h)则自噬受到抑制。其机制可能与m TOR信号通路有关,且自噬在一定程度上有利于促进HCMV在细胞中的复制增殖。

Objective： To investigate the effect of human cytomegalovirus （HCMV） infection on auto-phagy of human embryonic lung fibroblasts （ MRC-5 ） cells. Methods： MRC-5 cells were infected with HCMV AD169 strain（MOI = 1） , and the normal MRC-5 cells without HCMV infection were set as control group. At the time points of 12 h,24 h, 36 h,48 h,60 h after infection, the expression of autophagy related proteins Beclinl, LC3 , 4E-BP-1-P and p70S6K-P were detected by Western-blotting, the copy number of HCMV DNA were detected by real time PCR. The positive rate of acridine orange fluorescent granules was detected by flow cytometry. Results ： The expression of Beclinl, LC3-II , LC3-I protein in HCMV infected MRC-5 cells increased first and then gradually decreased. At the same time, the expression of 4E-BP-1-P and p70S6K-P decreased first and then increased. Also, the copy number of HCMV DNA increased first and decreased last. The results of above experiments were confirmed by flow cytometry. Conclusion： HCMV infection in MRC-5 cells induced autophage in the early phase, and inhibited autophagy in the late phase, mTOR signaling pathway may be involved in this process. And autophagy, to some extent, may facilitate the replication of HCMV in MRC-5 cells.