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Toll样受体7在肠道病毒71型感染人Jurkat T细胞诱导炎症因子中的作用
作者:茌静 何雅青 余光清 雷蕾

摘要:

目的  研究Toll样受体(TLRs)在肠道病毒71型(EV-A71)感染人T细胞中的表达情况,阐明其在EV-A71致炎症反应机制中的作用。方法  EV-A71毒株由深圳市CDC于2014年从手足口病患儿粪便中分离,接种200 μl病毒滴度为103细胞培养半数感染量(CCID50)/ml的病毒液于人Jurkat T细胞,不同感染时间后,采用RT-PCR方法检测TLR1~TLR10 mRNA表达情况;采用Real-time PCR分析TLR7 mRNA变化水平;采用Western blot分析TLR7相关接头分子髓样分化因子(MyD88)表达水平;采用ELISA检测培养上清细胞因子白细胞介素(IL)-6、IL-8和肿瘤坏死因子α(TNF-α)的分泌水平。结果  Real-time PCR检测结果显示,感染6、12、24和48 h,感染组TLR7 mRNA的相对表达水平分别为1.26±0.15、1.75±0.20、2.26±0.23和3.74±0.62,与对照组差异有统计学意义(t值分别为-2.96、-6.38、-9.57、-7.71,P值均<0.05);Western blot结果显示,感染24 h和48 h时,感染组MyD88蛋白表达水平与对照组相比分别增加1.34和2.17倍;细胞因子检测结果显示,感染24 h和48 h时,IL-6的分泌水平分别为(302.86±38.11)、(179.70±14.50)pg/ml,高于对照组24 h和48 h的分泌水平[(176.42±9.60)、(179.70±14.50)pg/ml],差异有统计学意义(t值分别为-5.57、-18.54,P值均<0.05)。48 h感染组TNF-α分泌水平为(100.81±9.81)pg/ml,高于对照组[(56.19±6.94)pg/ml],差异有统计学意义(t=-6.43,P=0.003)。结论  EV-A71主要通过TLR7被免疫活性细胞识别,进而激活TLR7相关信号通路,诱导产生大量促炎因子来参与感染致炎症反应的病理过程。

关键词:肠道病毒71型;Toll样受体;人Jurkat T细胞;细胞因子

Abstract:

Objective  To investigate the expression of Toll-like receptor (TLR) mRNA in enterovirus 71(EV-A71) infected human Jurkat T cells and clarify the role of TLRs in the pathogenesis of EV-A71 infection-induced inflammation.Methods  EV-A71 strains were isolated from feces of children patients with hand, foot and mouth disease in 2014 by Shenzhen Center for Disease Control and Prevention. Human Jurkat T cells were infected with 200 μl EV-A71 at 103 cell culture infective dose 50%(CCID50)/ml. The expression of TLR1-TLR10 mRNA in human Jurkat T cells was assessed at different exposure time by RT-PCR. Levels of TLR7 mRNA expression were detected by real-time PCR, and levels of myeloid differentiation factor 88 (MyD88) by western blot. The cytokine secretion of interleukin (IL)-6, IL-8 and Tumor Necrosis Factor α (TNF-α) was analyzed by ELISA assay.Results  The relative expression level of TLR7 mRNA in human Jurkat T cells were 1.26±0.15, 1.75±0.20, 2.26±0.23 and 3.74±0.62 in 6, 12, 24 and 48 h after EV-A71 infection, which the differences were significant with mock-infected group(t values were -2.96, -6.38, -9.57, -7.71; P<0.05). Western blot showed that the protein expression levels of MyD88 had increased 1.34 times and 2.17 times in 24 h and 48 h after EV-A71 infection compared with mock-infected group. After infected for 24 h and 48 h, the levels of IL-6 were (302.86±38.11), (179.70±14.50) pg/ml, which were significantly higher than mock-infected group (176.42±9.60), (179.70±14.50) pg/ml (t values were -5.57, -18.54, P<0.05). The levels of TNF-α in EV-A71 infected group (100.81±9.81) pg/ml was higher than that in mock-infected group (56.19±6.94) pg/ml, and the difference was significant (t=-6.43, P=0.003).Conclusion  TLR7 is the main pattern recognition receptor responsible for EV-A71 recognition in immune cells, which then leads to the activation of TLR7 downstream signaling and the production of proinflammatory cytokines.

Key words: Enterovirus 71;Toll-like receptors;Human Jurkat cells;Cytokines

发表日期:2016/3

引用本文:

图/表:

  • 10.3760/cma.j.issn.0253-9624.2016.03.015.F001:图1 肠道病毒71型感染人Jurkat T细胞后细胞病变效应(×400)

    10.3760/cma.j.issn.0253-9624.2016.03.015.F001:图1 肠道病毒71型感染人Jurkat T细胞后细胞病变效应(×400)

  • 10.3760/cma.j.issn.0253-9624.2016.03.015.T001:表1 两组人Jurkat T细胞上清液中IL-6和TNF-α分泌水平比较(,n=3)

    10.3760/cma.j.issn.0253-9624.2016.03.015.T001:表1 两组人Jurkat T细胞上清液中IL-6和TNF-α分泌水平比较(,n=3)

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