中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2014年
5期
300-303
,共4页
黄萍%刘芬%曾振国%黄彩雪%邵强%夏亮%揭克敏%钱克俭
黃萍%劉芬%曾振國%黃綵雪%邵彊%夏亮%揭剋敏%錢剋儉
황평%류분%증진국%황채설%소강%하량%게극민%전극검
微小RNA-146a%白细胞介素-1受体相关激酶1%肿瘤坏死因子受体相关因子6%肺泡巨噬细胞%炎症反应
微小RNA-146a%白細胞介素-1受體相關激酶1%腫瘤壞死因子受體相關因子6%肺泡巨噬細胞%炎癥反應
미소RNA-146a%백세포개소-1수체상관격매1%종류배사인자수체상관인자6%폐포거서세포%염증반응
MicroRNA-146a%Interleukin-1 receptor-associated kinase 1%Tumor necrosis factor receptor-associated factor 6%Alveolar macrophage%Inflammation
目的 观察转染微小RNR-146a(miR-146a)对肺泡巨噬细胞中白细胞介素-1受体相关激酶1(IRAK-1)和肿瘤坏死因子受体相关因子6(TRAF-6)表达的影响,为探讨miR-146a对肺泡巨噬细胞炎症反应的调控机制奠定基础.方法 将体外培养大鼠肺泡巨噬细胞NR8383分为miR-146a模拟物(mimic)组和阴性对照组,分别加入50 nmol/L Pre-miR miR-146a前体和Cy3标记的Pre-miR阴性对照进行转染,转染24 h后收集细胞,采用实时荧光定量逆转录-聚合酶链反应(RT-qPCR)检测细胞中miR-146a、IRAK-1 mRNA、TRAF-6mRNA的表达,采用蛋白质免疫印迹试验(Western Blot)检测细胞中IRAK-1和TRAF-6的蛋白表达.结果 miR-146amimic组miR-146a表达较阴性对照组上调了(24.55±6.14)倍(t=-6.643,P=0.003).细胞中IRAK-1和TRAF-6的mRNA表达分别为阴性对照组的(1.16±0.10)倍(t=2.701,P=0.054)和(1.19±0.16)倍(t=2.032,P=0.112).而IRAK-1和TRAF-6的蛋白表达分别为阴性对照组的73.0％(t=-9.353,P=0.001)和64.1％(t=-6.839,P=0.002).结论 miR-146a mimic能成功转染肺泡巨噬细胞NR8383; miR-146a过表达后,能有效下调肺泡巨噬细胞中IRAK-1和TRAF-6的表达,其机制可能是在蛋白翻译水平的抑制.
目的 觀察轉染微小RNR-146a(miR-146a)對肺泡巨噬細胞中白細胞介素-1受體相關激酶1(IRAK-1)和腫瘤壞死因子受體相關因子6(TRAF-6)錶達的影響,為探討miR-146a對肺泡巨噬細胞炎癥反應的調控機製奠定基礎.方法 將體外培養大鼠肺泡巨噬細胞NR8383分為miR-146a模擬物(mimic)組和陰性對照組,分彆加入50 nmol/L Pre-miR miR-146a前體和Cy3標記的Pre-miR陰性對照進行轉染,轉染24 h後收集細胞,採用實時熒光定量逆轉錄-聚閤酶鏈反應(RT-qPCR)檢測細胞中miR-146a、IRAK-1 mRNA、TRAF-6mRNA的錶達,採用蛋白質免疫印跡試驗(Western Blot)檢測細胞中IRAK-1和TRAF-6的蛋白錶達.結果 miR-146amimic組miR-146a錶達較陰性對照組上調瞭(24.55±6.14)倍(t=-6.643,P=0.003).細胞中IRAK-1和TRAF-6的mRNA錶達分彆為陰性對照組的(1.16±0.10)倍(t=2.701,P=0.054)和(1.19±0.16)倍(t=2.032,P=0.112).而IRAK-1和TRAF-6的蛋白錶達分彆為陰性對照組的73.0％(t=-9.353,P=0.001)和64.1％(t=-6.839,P=0.002).結論 miR-146a mimic能成功轉染肺泡巨噬細胞NR8383; miR-146a過錶達後,能有效下調肺泡巨噬細胞中IRAK-1和TRAF-6的錶達,其機製可能是在蛋白翻譯水平的抑製.
목적 관찰전염미소RNR-146a(miR-146a)대폐포거서세포중백세포개소-1수체상관격매1(IRAK-1)화종류배사인자수체상관인자6(TRAF-6)표체적영향,위탐토miR-146a대폐포거서세포염증반응적조공궤제전정기출.방법 장체외배양대서폐포거서세포NR8383분위miR-146a모의물(mimic)조화음성대조조,분별가입50 nmol/L Pre-miR miR-146a전체화Cy3표기적Pre-miR음성대조진행전염,전염24 h후수집세포,채용실시형광정량역전록-취합매련반응(RT-qPCR)검측세포중miR-146a、IRAK-1 mRNA、TRAF-6mRNA적표체,채용단백질면역인적시험(Western Blot)검측세포중IRAK-1화TRAF-6적단백표체.결과 miR-146amimic조miR-146a표체교음성대조조상조료(24.55±6.14)배(t=-6.643,P=0.003).세포중IRAK-1화TRAF-6적mRNA표체분별위음성대조조적(1.16±0.10)배(t=2.701,P=0.054)화(1.19±0.16)배(t=2.032,P=0.112).이IRAK-1화TRAF-6적단백표체분별위음성대조조적73.0％(t=-9.353,P=0.001)화64.1％(t=-6.839,P=0.002).결론 miR-146a mimic능성공전염폐포거서세포NR8383; miR-146a과표체후,능유효하조폐포거서세포중IRAK-1화TRAF-6적표체,기궤제가능시재단백번역수평적억제.
Objective To observe the effect of transfected microRNA-146a (miR-146a) on expression of interleukin-1 receptor-associated kinase 1 (IRAK-1) and tumor necrosis factor receptor-associated factor 6 (TRAF-6) in alveolar macrophages,and to explore the regulatory mechanism of miR-146a in the inflammatory response of alveolar macrophages.Methods Alveolar macrophages NR8383 were cultured and divided into two groups:transfected miR-146a mimic group was transfected 50 nmol/L Pre-miR miR-146a precursors and the negative control group was transfected Cy3-labeled Pre-miR negative control.Cells were collected at 24 hours after transfection.The miR-146a and the mRNA expression of IRAK-1 and TRAF-6 were detected by real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-qPCR),and the protein expression of IRAK-1 and TRAF-6 was assayed by Western Blot.Results Compared with negative control group,the expression of miR-146a was upregulated by (24.55 ± 6.14) fold compared with miR-146a mimic group (t=-9.353,P=0.001).The mRNA expressions of IRAK-1 and TRAF-6 in miR-146a mimic group were upregulated by (1.16 ± 0.10) fold (t=2.701,P=0.054) and (1.19 ± 0.16) fold (t=2.032,P=0.112),respectively,compared with that of negative control group,but the protein levels of IRAK-1 and TRAF-6 were decreased by 73.0％ (t =-9.353,P =0.001) and 64.1％ (t =-6.839,P =0.002),respectively.Conclusions miR-146a mimic was successfully transfected into the alveolar macrophage NR8383.The overexpression of miR-146a in alveolar macrophages can down-regulate the expression of IRAK-1 and TRAF-6 in protein translation levels,and its mechanism may be related with inhibition of protein translation.
