miR-146a在人牙髓细胞中的表达及其作用研究  

A study on the expression and function of miR-146a in human dental pulp cells

作　　者：舒珊[1] 洪丽莉[1] 陈丽虹[1] 韦曦[1] 

机构地区：[1]中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室,广州510055

出　　处：《中华口腔医学研究杂志（电子版）》2014年第3期1-5,共5页Chinese Journal of Stomatological Research(Electronic Edition)

基　　金：国家自然科学基金(81371133);广东省自然科学基金(S2012010008476);广东省科技计划(2011B050300007)

摘　　要：目的检测miR-146a在正常及脂多糖(LPS)刺激的人牙髓细胞(hDPC)中的表达,探讨miR-146a对hDPC分泌炎性因子的影响及其机制。方法 (1)实时荧光定量聚合酶链反应(PCR)方法测定正常及LPS刺激的hDPC miR-146a的表达水平。(2)Lipofectamine 2000脂质体分别转染化学合成的miR-146a类似物(miR-146a mimics)、miR-146a抑制剂(miR-146a inhibitor)及其相应无关序列小RNA对照,转染24 h后1μg/ml LPS刺激hDPC,4 h后实时荧光定量PCR检测白细胞介素6(IL-6)、IL-8 mRNA的表达,酶联免疫吸附试验(ELISA)检测细胞上清IL-6、IL-8的分泌;Western blot法检测miR-146a下游靶标白细胞介素1受体相关激酶1(IRAK1)及肿瘤坏死因子受体相关因子6(TRAF6)的蛋白表达水平。两组比较采用独立样本t检验,多组比较采用方差分析。结果 (1)经LPS刺激的hDPC miR-146a表达水平高于正常hDPC(12 h:t=8.488,P=0.014;24 h:t=39.661,P<0.001)。(2)转染miR-146a mimics的hDPC在1μg/ml LPS刺激下产生炎性因子IL-6、IL-8的水平低于阴性对照,差异有统计学意义(IL-6 PCR:P<0.001,IL-6 ELISA:P<0.001;IL-8 PCR:P<0.001,IL-8ELISA:P=0.011);过表达miR-146a后IRAK1及TRAF6蛋白水平明显下调(IRAK1:P=0.002;TRAF6:P<0.001)。结论 miR-146a在LPS刺激的hDPC中表达上调,转染miR-146a可下调其靶基因IRAK1及TRAF6表达并降低IL-6、IL-8分泌,推测miR-146a参与牙hDPC的炎症反应调控。Objective To investigate the expression of miR-146a in normal human dental pulp cells （hDPCs） and hDPCs stimulated by lipopolysaccharide （LPS）,and explore the possible role of miR-146a in pulpitis.Methods The expression levels of miR-146a in normal human dental pulp cells and hDPCs stimulated by LPS were detected by real-time PCR.Moreover,miR-146a mimics and miR-146a inhibitor,as well as their counterpart negative controls were transfected into cultured hDPCs by Lipofectamine 2000 for 24 h,followed by 1 μg/ml LPS stimulation for 4 h.MRNA of IL-6 and IL-8 were tested by real-time quantitative PCR,and IL-6 and IL-8 levels in the culture supernatant were detected by ELISA.Moreover,IRAK1 and TRAF6 which previously identified as actual target of miR-146a were examined by Western blot.Comparisons between groups were performed with t test or one-way ANOVA analysis.Results It was shown that miR-146a was significantly up-regulated in LPS treated hDPCs compared with normal hDPCs （12 h：t=8.488,P =0.014 ; 24 h：t=39.661,P ＜ 0.01）.Up-regulation of miR-146a could decrease secretion of IL-6 and IL-8 both in mRNA and supernatant levels （IL-6 PCR：P＜0.001,IL-6 ELISA：P＜0.001; IL-8 PCR：P＜0.001,IL-8 ELISA：P=0.011）.The expression of IRAK1 and TRAF6 of hDPCs transfected with miR-146a mimics was obviously down-regulated compared to those transfected with mimics control （IRAK1：P=0.002; TRAF6：P ＜ 0.001）.Conclusions MiR-146a is up-regulated in hDPCs induced by LPS.Up-regulation of miR-146a could not only down-regulate IRAK1 and TRAF6 expression,but also decrease IL-6 and IL-8 Ievels.This indicates miR-146a may be involved in the regulation of pulpitis.

关 键 词：MIR-146A 牙髓细胞 脂多糖 炎症因子 

分 类 号：R780.2[医药卫生—口腔医学]