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
9期
661-665
,共5页
刘奕君%曹殿青%莫桂熙%张良清
劉奕君%曹殿青%莫桂熙%張良清
류혁군%조전청%막계희%장량청
髓系细胞触发受体-1%微小RNA%转染%脓毒症
髓繫細胞觸髮受體-1%微小RNA%轉染%膿毒癥
수계세포촉발수체-1%미소RNA%전염%농독증
Triggering receptor expressed on myeloid cell-1%microRNA%Transfection%Sepsis
目的 探讨微小RNA-294(miR-294)靶向抑制髓系细胞触发受体-1(TREM-1)对脓毒症肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和高迁移率族蛋白B1(HMGB1)的影响.方法 利用生物信息学预测miR-294可能靶向调控TREM-1基因.体外培养小鼠巨噬细胞株RAW264.7,分成非炎症期和炎症期,每期各分为细胞对照组(NC组)、NC拟似物(mimic)和抑制剂(inhibitor)转染组(NCm组、NCi组)、miR-294 mimic和inhibitor转染组(miR-294m组、miR-294i组)5组.用双荧光素酶报告基因系统进行功能验证.以TurboFectTM小干扰RNA转染细胞48 h(非炎症期)后,用逆转录-聚合酶链反应(RT-PCR)检测细胞TREM-1 mRNA表达;以1 mg/L内毒素处理6h后(炎症期)收集上清液,用酶联免疫吸附试验(ELISA)检测TNF-α、IL-6、HMGB1浓度;用蛋白质免疫印迹试验(Western Blot)检测TREM-1蛋白表达.结果 ①双荧光素酶报告基因系统证实TREM-1是miR-294的靶点.②非炎症期:miR-294m组TREM-1 mRNA表达(2-△△Ct)较NC组、NCm组明显降低(0.673±0.049比1.000±0.003、0.915±0.039,f1=2.184、t2=5.421,均P＜0.001);miR-294m组TREM-1蛋白表达(灰度值)为NCm组的(50.00±1.19)％(t=41.586,P＜0.001).③炎症期:miR-294m组TNF-α(ng/L)较NC组明显降低(1 547.18±47.18比2 702.11±327.20,t＝4.212,P=0.010),IL-6(ng/L)较NC组、NCm组明显降低(505.28±33.33比837.66±69.43、918.72±119.39,t1=4.382、P1=0.015,t2=5.451、P2=0.021);TREM-1蛋白表达(灰度值)为NCm组的(51.33±0.88)％(t=63.368,P＜0.001);各组HMGB1比较均无差异.结论 miR-294能靶向抑制TREM-1表达,减少内毒素诱导的小鼠巨噬细胞株RAW264.7分泌TNF-α和IL-6.
目的 探討微小RNA-294(miR-294)靶嚮抑製髓繫細胞觸髮受體-1(TREM-1)對膿毒癥腫瘤壞死因子-α(TNF-α)、白細胞介素-6(IL-6)和高遷移率族蛋白B1(HMGB1)的影響.方法 利用生物信息學預測miR-294可能靶嚮調控TREM-1基因.體外培養小鼠巨噬細胞株RAW264.7,分成非炎癥期和炎癥期,每期各分為細胞對照組(NC組)、NC擬似物(mimic)和抑製劑(inhibitor)轉染組(NCm組、NCi組)、miR-294 mimic和inhibitor轉染組(miR-294m組、miR-294i組)5組.用雙熒光素酶報告基因繫統進行功能驗證.以TurboFectTM小榦擾RNA轉染細胞48 h(非炎癥期)後,用逆轉錄-聚閤酶鏈反應(RT-PCR)檢測細胞TREM-1 mRNA錶達;以1 mg/L內毒素處理6h後(炎癥期)收集上清液,用酶聯免疫吸附試驗(ELISA)檢測TNF-α、IL-6、HMGB1濃度;用蛋白質免疫印跡試驗(Western Blot)檢測TREM-1蛋白錶達.結果 ①雙熒光素酶報告基因繫統證實TREM-1是miR-294的靶點.②非炎癥期:miR-294m組TREM-1 mRNA錶達(2-△△Ct)較NC組、NCm組明顯降低(0.673±0.049比1.000±0.003、0.915±0.039,f1=2.184、t2=5.421,均P＜0.001);miR-294m組TREM-1蛋白錶達(灰度值)為NCm組的(50.00±1.19)％(t=41.586,P＜0.001).③炎癥期:miR-294m組TNF-α(ng/L)較NC組明顯降低(1 547.18±47.18比2 702.11±327.20,t＝4.212,P=0.010),IL-6(ng/L)較NC組、NCm組明顯降低(505.28±33.33比837.66±69.43、918.72±119.39,t1=4.382、P1=0.015,t2=5.451、P2=0.021);TREM-1蛋白錶達(灰度值)為NCm組的(51.33±0.88)％(t=63.368,P＜0.001);各組HMGB1比較均無差異.結論 miR-294能靶嚮抑製TREM-1錶達,減少內毒素誘導的小鼠巨噬細胞株RAW264.7分泌TNF-α和IL-6.
목적 탐토미소RNA-294(miR-294)파향억제수계세포촉발수체-1(TREM-1)대농독증종류배사인자-α(TNF-α)、백세포개소-6(IL-6)화고천이솔족단백B1(HMGB1)적영향.방법 이용생물신식학예측miR-294가능파향조공TREM-1기인.체외배양소서거서세포주RAW264.7,분성비염증기화염증기,매기각분위세포대조조(NC조)、NC의사물(mimic)화억제제(inhibitor)전염조(NCm조、NCi조)、miR-294 mimic화inhibitor전염조(miR-294m조、miR-294i조)5조.용쌍형광소매보고기인계통진행공능험증.이TurboFectTM소간우RNA전염세포48 h(비염증기)후,용역전록-취합매련반응(RT-PCR)검측세포TREM-1 mRNA표체;이1 mg/L내독소처리6h후(염증기)수집상청액,용매련면역흡부시험(ELISA)검측TNF-α、IL-6、HMGB1농도;용단백질면역인적시험(Western Blot)검측TREM-1단백표체.결과 ①쌍형광소매보고기인계통증실TREM-1시miR-294적파점.②비염증기:miR-294m조TREM-1 mRNA표체(2-△△Ct)교NC조、NCm조명현강저(0.673±0.049비1.000±0.003、0.915±0.039,f1=2.184、t2=5.421,균P＜0.001);miR-294m조TREM-1단백표체(회도치)위NCm조적(50.00±1.19)％(t=41.586,P＜0.001).③염증기:miR-294m조TNF-α(ng/L)교NC조명현강저(1 547.18±47.18비2 702.11±327.20,t＝4.212,P=0.010),IL-6(ng/L)교NC조、NCm조명현강저(505.28±33.33비837.66±69.43、918.72±119.39,t1=4.382、P1=0.015,t2=5.451、P2=0.021);TREM-1단백표체(회도치)위NCm조적(51.33±0.88)％(t=63.368,P＜0.001);각조HMGB1비교균무차이.결론 miR-294능파향억제TREM-1표체,감소내독소유도적소서거서세포주RAW264.7분비TNF-α화IL-6.
Objective To investigate the effects of microRNA-294 (miR-294) on tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6) and high mobility group box 1 (HMGB 1) secretion in sepsis by targeting triggering receptor expressed on myeloid cell-1 (TREM-1).Methods miRNA-294 was predicted to regulate TREM-1 specially through bioinformatics analysis.Mice macrophage cell lines RAW264.7 were cultured in vitro,the cells were divided into non-inflammatory stage and inflammatory stage,and the cells in the two stages were subdivided into five groups as follows:normal control (NC),NC mimic transfection (NCm),NC inhibitor transfection (NCi),miR-294 mimic transfection (miR-294m) and miR-294 inhibitor transfection (miR-294i) groups.The ability of miR-294 was confirmed with dual-luciferase activity assay.At non-inflammatory stage,the cells were transfected with mimic or inhibitor of miR-294 or NC using TurboFectTM siRNA Transfection Reagent for 48 hours,mRNA expression of TREM-1 was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR).At inflammatory stage,6 hours after stimulation by lipopolysaccharide (LPS,1 mg/L),the concentrations of TNF-α,IL-6 and HMGB1 were determined by enzyme linked immunosorbent assay (ELISA),the protein expression of TREM-1 was determined by Western Blot.Results ① Dual-luciferase activity assay demonstrated that TREM-1 was the target of miR-294.② In non-inflammatory stage,the expression of TREM-1 mRNA (2-ΔΔ~) in miR-294m group was significantly lower than that of the NC and NCm groups (0.673 ± 0.049 vs.1.000 ± 0.003,0.915 ± 0.039,t1=2.184,t2=5.421,both P＜0.001),the expression of TREM-1 protein (gray scale) was (50.00 ± 1.19)％ of NCm group (t=41.586,P＜0.001).③ In inflammatory stage,the concentrations of TNF-α (ng/L) in miR-294m group was significantly lower than that of the NC group (1 547.18 ±47.18 vs.2 702.11 ± 327.20,t=4.212,P=0.010),the concentrations of IL-6 (ng/L) was significantly lower than that of the NC and NCm groups (505.28 ± 33.33 vs.837.66 ± 69.43,918.72 ± 119.39,t1 =4.382,P1=0.015; t2=5.451,P2=0.021),the level of TREM-1 protein (gray scale) was (51.33 ±0.88)％ of NCm group (t=63.368,P＜0.001).Conclusion miR-294 reduce TNF-α and IL-6 secretion in LPS-induced RAW264.7 through inhibiting the expression of TREM-1 specifically.
