中华急诊医学杂志
中華急診醫學雜誌
중화급진의학잡지
CHINESE JOURNAL OF EMERGENCY MEDICINE
2012年
7期
709-712
,共4页
曾振国%龚洪翰%李勇%聂贞蕴%揭克敏%詹以安%聂成%刘芬%丁成志%邵强%卿城%朱白鹭%钱克俭
曾振國%龔洪翰%李勇%聶貞蘊%揭剋敏%詹以安%聶成%劉芬%丁成誌%邵彊%卿城%硃白鷺%錢剋儉
증진국%공홍한%리용%섭정온%게극민%첨이안%섭성%류분%정성지%소강%경성%주백로%전극검
microRNA-146a%肺泡巨噬细胞%肿瘤坏死因子-α%实时荧光定量PCR%炎症
microRNA-146a%肺泡巨噬細胞%腫瘤壞死因子-α%實時熒光定量PCR%炎癥
microRNA-146a%폐포거서세포%종류배사인자-α%실시형광정량PCR%염증
microRNA-146a%Alveolar macrophages%Tumor necrosis factor-α%Real time-quantitative polymerase chain reaction%Inflammation
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mieroRNA-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1μg/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3h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3h后,细胞中TNF-α mRNA的表达即达到顶峰[(67.48±24.52)倍,P＜0.01],6h后开始降低[(29.53±4.26)倍,P＜0.05]；(3)LPS刺激6h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[12h:(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.
目的 觀察腫瘤壞死因子-α(TNF-α)和microRNA-146a在脂多糖(LPS)誘導的NR8383肺泡巨噬細胞中的動態錶達,分析二者在時間上的變化關繫,探討mieroRNA-146a對肺泡巨噬細胞炎癥反應的調控作用及其機製.方法 體外培養的肺泡巨噬細胞接種于六孔闆,貼壁後加入1μg/mL的LPS,刺激0、3、6、12 h後離心收集培養上清液和細胞.採用實時熒光定量PCR檢測細胞中microRNA-146a和TNF-α mRNA的錶達,酶聯免疫吸附實驗(ELISA)檢測培養上清液中TNF-α蛋白水平的錶達.microRNA-146a和TNF-α mRNA的相關性採用雙變量Pearson相關性分析.結果 (1)培養上清液中的TNF-α蛋白在LPS刺激3h後顯著升高[(359.80±57.54) pg/mL,P＜0.01],于12 h達高峰[(729.22±50.40) pg/ml,P＜0.01]; (2) LPS刺激3h後,細胞中TNF-α mRNA的錶達即達到頂峰[(67.48±24.52)倍,P＜0.01],6h後開始降低[(29.53±4.26)倍,P＜0.05]；(3)LPS刺激6h後,microRNA-146a錶達水平顯著增高[(5.33±0.81)倍,P＜0.01],併且持續增高[12h:(8.21±1.19)倍,P＜0.01]；(4)細胞中microRNA-146a 的錶達水平與TNF-α mRNA的含量呈負相關(r=-0.895,P＜0.01).結論 在LPS誘導的肺泡巨噬細胞中,microRNA-146a的錶達水平與TNF-α mRNA的含量呈負相關,推測microRNA-146a可能參與瞭肺泡巨噬細胞的炎癥反應調控.
목적 관찰종류배사인자-α(TNF-α)화microRNA-146a재지다당(LPS)유도적NR8383폐포거서세포중적동태표체,분석이자재시간상적변화관계,탐토mieroRNA-146a대폐포거서세포염증반응적조공작용급기궤제.방법 체외배양적폐포거서세포접충우륙공판,첩벽후가입1μg/mL적LPS,자격0、3、6、12 h후리심수집배양상청액화세포.채용실시형광정량PCR검측세포중microRNA-146a화TNF-α mRNA적표체,매련면역흡부실험(ELISA)검측배양상청액중TNF-α단백수평적표체.microRNA-146a화TNF-α mRNA적상관성채용쌍변량Pearson상관성분석.결과 (1)배양상청액중적TNF-α단백재LPS자격3h후현저승고[(359.80±57.54) pg/mL,P＜0.01],우12 h체고봉[(729.22±50.40) pg/ml,P＜0.01]; (2) LPS자격3h후,세포중TNF-α mRNA적표체즉체도정봉[(67.48±24.52)배,P＜0.01],6h후개시강저[(29.53±4.26)배,P＜0.05]；(3)LPS자격6h후,microRNA-146a표체수평현저증고[(5.33±0.81)배,P＜0.01],병차지속증고[12h:(8.21±1.19)배,P＜0.01]；(4)세포중microRNA-146a 적표체수평여TNF-α mRNA적함량정부상관(r=-0.895,P＜0.01).결론 재LPS유도적폐포거서세포중,microRNA-146a적표체수평여TNF-α mRNA적함량정부상관,추측microRNA-146a가능삼여료폐포거서세포적염증반응조공.
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 miR146a and TNF-α mRNA in cells were detected by real-time qPCR and the levels of TNF-α protein in the supematant 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 ＜0.01 ),and peaked 12 h later (729.22 ±50.40) pg/ml (P＜0.01 ) ; ②the expression of TNF-α mRNA peaked 3 h after LPS challenge (67.48 ±24.52) fold,P ＜0.01 ),and then decreased gradually; ③the expression of miR-146a mRNA increased continuously until 6 h or 12 h after LPS challenge 6 h:(5.33 ±0.81) fold,12 h:(8.21 ±1.19) fold,(P＜0.01),and it showed an upward tendency;④ the expression of miR-146a mRNA was negatively correlated with TNF-α mRNA ( r =-0.895,P ＜0.01).Conclusions The miR-146a mRNA showed a negative correlation with TNF-α mRNA present in lipopolysaccharide-stimulated alveolar macrophages,suggesting miR-146a mRNA involved in regulating the inflammatory response of alveolar macrophages.
