脂多糖诱导大鼠肺泡巨噬细胞中microRNA-146a与TNF-α的相关性研究

【摘要】目的 观察肿瘤坏死因子-α（TNF-α）和microRNA-146a在脂多糖(LPS) 诱导的NR8383肺泡巨噬细胞中的动态表达，分析二者在时间上的变化关系，探讨microRNA-146a对肺泡巨噬细胞炎症反应的调控作用及其机制。方法 体外培养的肺泡巨噬细胞接种于六孔板，贴壁后加入1 μg/mL的LPS，刺激0、3、6、12 h后离心收集培养上清液和细胞。采用实时荧光定量PCR检测细胞中microRNA-146a和TNF-α mRNA的表达，酶联免疫吸附实验（ELISA）检测培养上清液中TNF-α蛋白水平的表达。microRNA-146a和TNF-α mRNA的相关性采用双变量Pearson相关性分析。结果 （1）培养上清液中的TNF-α蛋白在LPS刺激3 h后显著升高［（359.80±57.54 )pg/mL，P＜0.01］，于12 h达高峰［(729.22±50.40 )pg/ml，P＜0.01］；（2）LPS刺激3 h后，细胞中TNF-α mRNA的表达即达到顶峰［（67.48±24.52)倍，P＜0.01］，6 h后开始降低［（29.53±4.26)倍，P＜0.05］；（3）LPS刺激6 h后，microRNA-146a表达水平显著增高［（5.33±0.81)倍，P＜0.01］，并且持续增高［12 h：(8.21±1.19)倍，P＜0.01］；（4）细胞中microRNA-146a的表达水平与TNF-α mRNA的含量呈负相关（r=-0.895，P＜0.01）。结论 在LPS诱导的肺泡巨噬细胞中， microRNA-146a的表达水平与TNF-α mRNA的含量呈负相关，推测microRNA-146a可能参与了肺泡巨噬细胞的炎症反应调控。

【关键词】microRNA-146a；肺泡巨噬细胞；肿瘤坏死因子-α；实时荧光定量PCR；炎症

The relationship between microRNA-146a and TNF-α in lipopolysaccharide-stimulated alveolar macrophages of rats ZENG Zhen-guo,GONG Hong-han, LI Yong, NIE Zhen-yun, JIE Ke-min, ZHAN Yi-an, NIE Cheng, LIU Fen, DING Cheng-zhi, SHAO Qiang, QING Cheng, ZHU Bai-lu, QIAN Ke-jian.省略

【Abstract】Objective To determine kinetics of TNF-α and miR-146a (microRNA-146a) expressions in lipopolysaccharide (LPS)-induced NR8383 alveolar macrophages (AM) at different intervals and their relationships in order to explore regulatory effect and mechanism of miR-146a on alveolar macrophages inflammatory responses.Methods NR8383 alveolar macrophages were seeded in a 6-well plate, and stimulated with 1 μg/ml of LPS for 0 h, 3 h, 6 h and 12 h separately after 90 min. Cells were harvested and supernatant were collected 0 h, 3 h, 6 h and 12 h after incubation. The expressions of miR-146a and TNF-α mRNA in cells were detected by real-time qPCR and the levels of TNF-α protein in the supernatant of cells were assayed by enzyme-linked immunosorbent assay (ELISA). Pearson correlation analysis was used to analyze the correlation between miR-146a and TNF-α mRNA. Results ①The level of TNF-α protein in the supernatant of cell was significantly increased 3 h after LPS challenge (359.80±57.54) pg/ml (P