摘要
目的观察转染微小RNA-146a（miR-146a）对大鼠肺泡巨噬细胞炎症因子肿瘤坏死因子-α（TNF-α）表达的影响，探讨转染miR-146a对肺泡巨噬细胞炎症反应的调控作用。方法体外培养大鼠肺泡巨噬细胞株NR8383，分别以25、50、100nmol／LCy^TM3 标记的Pre-miR^TM阴性对照转染细胞，选择转染效率最高的浓度作为实验浓度。将NR8383细胞分成两组，转染组转染50nmol／LPre-miRTMmiR-146a前体，对照组转染50nmol／LCyr^TM3标记的Pre-miRTM阴性对照，采用实时荧光定量逆转录-聚合酶链反应（RT-qPCR）检测转染后细胞内miR-146a的mRNA表达。用脂多糖（LPs）lmg／L刺激细胞6h后，采用酶联免疫吸附试验（EusA）测定细胞培养上清液内TNF-α蛋白表达，采用RT-qPCR检测细胞内TNF-αmRNA表达。结果以50nmol／LCy^TMm3标记的Pre-miRTM阴性对照转染细胞效率最高，约为80％。与对照组（作为1）相比，转染组细胞内miR-146a的mRNA表达上调了（24．55±6．14）倍（P〈0．01）。LPS刺激后，转染组细胞上清液TNF-α蛋白含量（ng／L）较对照组明显下降（211．5±30．4比616．6±423，P〈0．01），细胞内TNF-αmRNA表达下调了（47±6）％（P〈0．05）。结论转染miR-146a能下调肺泡巨噬细胞炎症因子TNF-α的表达，表明miR-146a能抑制LPS诱导肺泡巨噬细胞的炎症反应。
Objective To observe the effect of transfected microRNA-146a （miR-146a） on expression of tumor necrosis factor-α （TNF-α） in alveolar macrophages, and to analyze its regulatory mechanism in the inflammatory response of alveolar macrophages. Methods Cy^TM3 labeled with 25, 50, 100 nmol/L of Pre-miRTM, respectively, were transfected into rat alveolar macrophages NR8383 cultured in vitro. The highest transfection efficiency was selected to be the experimental concentration. NR8383 cells were divided into two groups： transfeeted group was tranfected with 50 nmol/L Pre-miR^TM miR-146a precursor, and control group with 50 nmol/L Cy^TM3 labeled Pre-miRTM as the negative control. The mRNA expression of miR-146a of cells was detected by real-time quantitative reverse transeription-polymerase chain reaction （RT-qPCR）. Then ceils were stimulated with lipopolysaecharide （LPS, 1mg/L） for 6 hours. The production of TNF-α protein in the supernatant of cells was assayed by enzyme-linked immunosorbent assay （ELISA）, and the expression of TNF-α mRNA of cells was detected by RT-qPCR. Results Transfeetion rate was highest in the 50 nmol/L CyTM3 labeled Pre-miRTM cells, and it reached 80%. Compared with control group （set at 1 ）, the expression of miR-146a increased by （24.55 ± 6.14） folds in transfected alveolar macrophages （P〈0.01）. After the cells were stimulated with LPS, the production of TNF-α protein （no/L） in the snpernatant of cell was decreased from 616.6 ± 42.3 to 211.5± 30.4 （P〈0.01）, and the expression of TNF-α mRNA was decreased by （47± 6） % （P〈0.05） in transfected alveolar maerophages when compared with that of the control. Conclusions Transfeetion alveolar macrophages with miR-146a precursors could down-regulate the expression of TNF- α. It is therefore suggests that up-regulation of miR-146a can inhibit inflammatory responses as induced by LPS, in alveolar maerophages.
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2013年第6期335-338,共4页
Chinese Critical Care Medicine
基金
国家自然科学基金（81060152）
国家自然科学基金（81160233）
国家自然科学基金青年基金项目（81101410）
