Objective To analyze the differential expression of microRNA-146a (miR-146a) in monocyte-macrophage cell line (THP-1 cells) after induction by Cryptococcus neoformans (C.neoformans,reference strain WM148) or Cryptococcus gattii (C.gattii,reference strain R265),and investigate the mechanism of miR-146a in regulating the inflammatory response of cryptococcal meningitis.Methods The cultured THP-1 cells were divided into two groups to be induced by C.neoformans or C.gattii,respectively.THP-1 cells were induced with inactivated WN148 (or R265) strains at multiplicity of infection (MOI) of 5 in all experiments.The supematant and the cell pellet were collected separately after incubation.The expression of miR-146a was measured by real-time quantitative PCR (qRT-PCR) technique.The levels of TNF-u and IL-6 release were assayed by ELISA.Results The expression of miR-146a increased significantly in the C.neoformans induction group compared to 0 h.It reached peak at 3 h (P＜0.01),and then declined gradually.The level of TNF-α increased in supematant and reached peak at 12 h.The expression of IL-6 did not change significantly at each time point.The expression of miR-146a and TNF-α increased gradually and reached peak at 12 h in the C.gattii induction group (P ＜0.01),but the change did not reach statistical significance at 3 h,6 h time points.The expression of IL-6 gradually increased,and reached peak at 12 h time point.Conclusions Following stimulation with C.neoformans or C.gattii,the expression ofmiR-146a in THP-1 cells showed different patterns over time.The expression levels of TNF-α and IL-6 showed different patterns.These findings suggest that there may be different regulatory mechanisms in the THP-1 cells-associated inflammatory response after stimulation by inactivated C.neoformans and C.gattii strains.%目的 观察并分析微小RNA-146a(miR-146a)在新生隐球菌(标准株WM148)、格特隐球菌(标准株R265)刺激人单核巨噬细胞系(THP-1细胞)中表达的差异,探讨miR-146a在隐球菌所致隐球菌脑膜炎中的调控作用及机制.方法 体外培养的人单核巨噬细胞系THP-1细胞分成新生隐球菌诱导组、格特隐球菌诱导组,按灭活菌数∶THP-1=5∶1的比例与THP-1细胞共孵育0、3、6、9和12h后离心收集上清液和细胞沉淀.采用实时荧光定量PCR(qRT-PCR)技术检测各组细胞miR-146a的表达情况,酶联免疫吸附试验(ELISA)法检测各组上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的释放量.结果 新生隐球菌诱导组细胞中miR-146a表达量与0h相比明显升高(P＜0.01),且在3h达到峰值,随后呈下降趋势;上清液中TNF-α的表达量在诱导后12h达到最高值,IL-6的表达各时间点没有明显变化.格特隐球菌诱导后,miR-146a的表达逐渐升高,12h达到最高值,但3h、6h点变化与0h比较差异并无统计学意义;上清液中YNF-α的表达12h到达峰值;IL-6的表达逐渐增加,12h达到最高值,变化无统计学意义.结论 新生隐球菌、格特隐球菌刺激THP-1细胞炎性反应后,miR-146a表达随时间延长呈现不同的规律,TNF-α、IL-6的动态变化表现不同的特点.研究表明新生隐球菌、格特隐球菌诱导THP-l炎性反应可能存在不同的调控机制.
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