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200txl病毒滴度为103细胞培养半数感染量（CCID50）／ml的病毒液于人JurkatT细胞，不同感染时间后，采用RT—PCR方法检测TLR1～TLR10mRNA表达情况；采用Real—timePCR分析TLR7mRNA变化水平；采用Westernblot分析TLR7相关接头分子髓样分化因子（MyDS8）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—timePCR检测结果显示，感染6、12、24和48h，感染组TLR7mRNA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）；Westernblot结果显示，感染24h和48h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24h和48h时，IL-6的分泌水平分别为（302．86±38．11）、（[79．70±14．50）pg／ml，高于对照组24h和48h的分泌水平[（[76．42±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）。48h感染组TNF．01分泌水平为（[00．81±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。
Objective To investigate the expression of Toll-like receptor （TLR） mRNA in enterovirus 71（EV-AT1） 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 p,1 EV-A71 at 103 cell culture infective dose 50%（CCIDs0）/ml. The expression of TLRI-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 c~ （TNF-~x） 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.
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2016年第3期266-269,共4页
Chinese Journal of Preventive Medicine
基金
深圳市科技计划项目(201302169)；深圳市宝安区科技计划项目(2013089)；深圳市战略性新兴产业发展专项资金项
作者简介
通信作者：茌静，Email：chiing0905@163．com
