TSG-6对HCMV感染引起的TNF-α和IL-6表达水平的抑制作用

TSG-6 inhibited the HCMV infection-induced expression of TNF-α and IL-6

目的研究肿瘤坏死因子α刺激基因-6（tumor necrosis factor α stimulated gene 6, TSG-6）在人巨细胞病毒（human cytomegalovirus, HCMV）感染所致的炎症过程中的作用及机制。 方法用HCMV感染人胚肺成纤维细胞（MRC-5）和视网膜上皮细胞（ARPE-19），并设正常细胞对照组和灭活病毒感染组。采用qRT-PCR和Western blot检测各组TSG-6 mRNA和蛋白表达水平。将重组人TSG-6蛋白（recombinant human TSG-6 protein, rhTSG-6） 100 ng/ml作用于HCMV感染的MRC-5细胞作为rhTSG-6干预组，采用ELISA方法检测细胞培养物上清中TNF-α和IL-6表达水平，用IFA及Western blot检测NF-κB核定位及其蛋白表达水平，并设正常细胞对照和病毒对照。 结果HCMV感染MRC-5细胞72 h时，TSG-6 mRNA表达量为细胞对照组的19倍，在感染96 h时TSG-6 mRNA表达达高峰，为细胞对照组的376倍；HCMV感染ARPE-19细胞96 h时，TSG-6 mRNA表达量为细胞对照组的15倍，在120 h时为细胞对照组的354倍。Western blot结果证实，TSG-6蛋白表达水平在相应时间点也随之升高。rhTSG-6作用于HCMV感染的MRC-5细胞后与病毒对照组相比，TNF-α、IL-6水平明显下降（P〈0.05），且TSG-6能抑制核转录因子（nuclear factor-kappaB, NF-κB）的核移位及蛋白的表达。 结论TSG-6参与HCMV引起的炎症反应，抑制HCMV相关炎性因子水平的表达并影响NF-κB的核定位。

ObjectiveTo investigate the roles of tumor necrosis factor alpha stimulus gene-6 （TSG-6） in the inflammatory responses induced by human cytomegalovirus （HCMV） infection and the possible mechanism. MethodsHuman lung fibroblast cells （MRC-5） and human retinal pigment epithelial cells （ARPE-19） were infected with HCMV strains. A normal cell control group and an inactivated virus infection group were set up as well. qRT-PCR analysis and Western blot assay were performed to measure the expression of TSG-6 at mRNA and protein levels in each group. The recombinant human TSG-6 protein （rhTSG-6） was administrated to the HCMV-infected MRC-5 cell cultures at the concentration of 100 ng/ml. The supernatants were harvested to measure the levels of TNF-α and IL-6 and the transcriptional activity of NF-κB by using ELISA, indirect immunofluorescence assay （IFA） and Western blot assay. Meanwhile, a normal cell control group and a virus control group were set up for comparison. ResultsThe expression of TSG-6 at mRNA level in HCMV-infected MRC-5 cells were 19 and 376 times of that in normal cells at the time points of 72 h and 96 h after infection. The expression of TSG-6 at mRNA level in HCMV-infected ARPE-19 cells were 15 and 354 times of that in normal cells at the time points of 96 h and 120 h after infection. Results of the Western blot assay further confirmed the increased expression of TSG-6 at protein level at the corresponding time points. Compared with the virus control group, the levels of TNF-α and IL-6 in the supernatants of HCMV-treated MRC-5 cell culture were significantly decreased upon the intervention of rhTSG-6 protein （P〈0.05）. Moreover, TSG-6 protein could suppress the expression and nuclear translocation of NF-κB. ConclusionTSG-6 was involved in the HCMV infection-induced inflammation responses and played a role in inhibiting the expression of HCMV infection-related inflammatory factors and affecting the nuclear localization of NF-κB.