摘要目的 探讨HIV派生的MicroRNA99(miRNA99)对巨噬细胞焦亡的影响及机制.方法 用佛波酯(PMA)刺激人单核白血病细胞(THP-1)分化成贴壁的巨噬细胞,流式细胞术测定细胞表面CD11b;将鉴定好的细胞分组,分为PBS组(空白对照)、LPS+ATP组(阳性对照)、miRNA99组(实验组);将miRNA99 mimics转染至细胞,采用Western blot(WB)方法测定活化caspase-1蛋白水平;采用ELISA法测定细胞上清液IL-18、IL-1β的水平;将巨噬细胞细胞又分为2组,miRNA99+TLR8 RNAi质粒组实验组、miRNA99+空质粒组,转染细胞,在共聚焦下观察细胞内荧光;采用WB方法检测TLR8蛋白及活化caspase-1蛋白水平;用ELISA法测定TLR8沉默后细胞培养上清液中IL-18、IL-1β的水平.结果 经PMA刺激24 h后细胞贴壁生长,伸出伪足,表面CD1lb阳性率在99％以上.LPS+ATP组与miRNA99组活化caspase-1蛋白水平、上清液中IL-18、IL-1β的含量明显高于对照组(P＜0.05),差异有统计学意义.转染TLR8 RNAi质粒后巨噬细胞内呈现绿色荧光,WB显示实验组TLR8蛋白表达明显减少.在TLR8质粒+miRNA99组,活化caspase-1蛋白水平及上清液中IL-18、IL-1β的含量明显低于空质粒+miRNA99组(P＜0.05),差异有统计学意义.结论 HIV派生的miRNA99能诱导巨噬细胞发生焦亡,miRNA99诱导焦亡的机制有TLR8途径的参与.

abstractsObjective To determine the impact and mechanism of HIV derived microRNA99 (miRNA99) on macrophages pyroptosis.Methods THP-1 cells were stimulated by phorbol ester (PMA) and then were cultured and differentiated into sidewall attached macrophages;the morphology and phenotype of CD11b were measured by microscopy and flow cytometry.TLR8 RNAi plasmid was transfected to macrophages and were detected by confocal fluorescence microscopy.The levels of IL-18 and IL-1β released by macrophages were measured by ELISA.Western blot(WB) was employed to examine TLR8 and cleaved caspase-1 protein expression in macrophages.Results THP-1 cells that were challenged with PMA (100 ng/ml) for 24 h became smooth and adherent.In addition,the expression of CD11b in macrophages was up to 99％.TLR8 protein expression in macrophages transfected with TLR8 plasmids was significantly lower than that in macrophages transfected with control plasmids.Levels of IL-18 and [L-1β secreted by macrophages were elevated in LPS+ATP group,miRNA99 group and control plasmid group,but not in control group and TLR8 RNAi plasmid group.Cleaved caspase-1 protein from macrophages of miRNA99 experimental group/LPS+ATP group and control plasmid group was significantly higher than that of control group and TLR8 RNAi plasmid group.Conclusions The present study demonstrates that HIV-derived miRNA99 could induce pyroptosis of macrophages via TLR8-dependent pathway.