中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2012年
9期
677-682
,共6页
陈毅雄%翁志宏%祁丹%张淑玲
陳毅雄%翁誌宏%祁丹%張淑玲
진의웅%옹지굉%기단%장숙령
肝硬化%肝星状细胞%Notch信号%小干扰RNA
肝硬化%肝星狀細胞%Notch信號%小榦擾RNA
간경화%간성상세포%Notch신호%소간우RNA
Liver cirrhosis%Hepatic stellate cell%Notch signaling%Small interfering RNA
目的 探讨肝星状细胞(HSC-T6)内是否存在功能激活的Notch信号分子以及调控Notch信号转导对HSC-T6活化的影响. 方法 用PCR方法检测Notch信号分子mRNA在HSC-T6细胞内的表达,TaqMan探针检测转化生长因子(TGF)β 1刺激HSC-T6后Notch信号分子的表达变化.细胞免疫荧光法检测Notch3和Jagged1在HSC-T6内的表达定位.用携带Notch3胞内域(ICD)的真核表达质粒pcDNA3.1-N3ICD和特异性干扰Notch3的siRNA(Notch3-siRNA)分别转染HSC-T6细胞,Western blot检测Notch3、α-平滑肌肌动蛋白(SMA)和Ⅰ型胶原的表达变化.对数据采用t检验和方差分析. 结果 HSC-T6细胞内存在功能激活的Notch信号转导.TGF β 1(2.0 ng/ml)刺激HSC-T6导致Jagged1、Notch3和HES-1的mRNA水平明显上调,分别提高了2.9、3.2、2.2倍,F值分别为2543.482,287.982和1719.851,P值均＜0.01.pcDNA3.1-N3ICD转染后促使HSC-T6合成α-SMA和Ⅰ型胶原蛋白增加,较对照组分别提高了5.8倍和4.5倍,t值分别为13.157和9.810,P值均＜0.01；相反,Notch3- siRNA明显抑制HSC-T6表达α-SMA和Ⅰ型胶原蛋白,较对照组分别下降了约90％和84％,t值分别为19.863和10.376,P值均＜0.01.而且,Notch3-siRNA能拮抗TGFβ1诱导HSC-T6表达α-SMA和Ⅰ型胶原的作用.结论 Notch信号通路可能通过调控肝星状细胞的活化参与肝纤维化的形成,选择性地干预Notch3信号转导可能成为抗肝纤维化的新途径.
目的 探討肝星狀細胞(HSC-T6)內是否存在功能激活的Notch信號分子以及調控Notch信號轉導對HSC-T6活化的影響. 方法 用PCR方法檢測Notch信號分子mRNA在HSC-T6細胞內的錶達,TaqMan探針檢測轉化生長因子(TGF)β 1刺激HSC-T6後Notch信號分子的錶達變化.細胞免疫熒光法檢測Notch3和Jagged1在HSC-T6內的錶達定位.用攜帶Notch3胞內域(ICD)的真覈錶達質粒pcDNA3.1-N3ICD和特異性榦擾Notch3的siRNA(Notch3-siRNA)分彆轉染HSC-T6細胞,Western blot檢測Notch3、α-平滑肌肌動蛋白(SMA)和Ⅰ型膠原的錶達變化.對數據採用t檢驗和方差分析. 結果 HSC-T6細胞內存在功能激活的Notch信號轉導.TGF β 1(2.0 ng/ml)刺激HSC-T6導緻Jagged1、Notch3和HES-1的mRNA水平明顯上調,分彆提高瞭2.9、3.2、2.2倍,F值分彆為2543.482,287.982和1719.851,P值均＜0.01.pcDNA3.1-N3ICD轉染後促使HSC-T6閤成α-SMA和Ⅰ型膠原蛋白增加,較對照組分彆提高瞭5.8倍和4.5倍,t值分彆為13.157和9.810,P值均＜0.01；相反,Notch3- siRNA明顯抑製HSC-T6錶達α-SMA和Ⅰ型膠原蛋白,較對照組分彆下降瞭約90％和84％,t值分彆為19.863和10.376,P值均＜0.01.而且,Notch3-siRNA能拮抗TGFβ1誘導HSC-T6錶達α-SMA和Ⅰ型膠原的作用.結論 Notch信號通路可能通過調控肝星狀細胞的活化參與肝纖維化的形成,選擇性地榦預Notch3信號轉導可能成為抗肝纖維化的新途徑.
목적 탐토간성상세포(HSC-T6)내시부존재공능격활적Notch신호분자이급조공Notch신호전도대HSC-T6활화적영향. 방법 용PCR방법검측Notch신호분자mRNA재HSC-T6세포내적표체,TaqMan탐침검측전화생장인자(TGF)β 1자격HSC-T6후Notch신호분자적표체변화.세포면역형광법검측Notch3화Jagged1재HSC-T6내적표체정위.용휴대Notch3포내역(ICD)적진핵표체질립pcDNA3.1-N3ICD화특이성간우Notch3적siRNA(Notch3-siRNA)분별전염HSC-T6세포,Western blot검측Notch3、α-평활기기동단백(SMA)화Ⅰ형효원적표체변화.대수거채용t검험화방차분석. 결과 HSC-T6세포내존재공능격활적Notch신호전도.TGF β 1(2.0 ng/ml)자격HSC-T6도치Jagged1、Notch3화HES-1적mRNA수평명현상조,분별제고료2.9、3.2、2.2배,F치분별위2543.482,287.982화1719.851,P치균＜0.01.pcDNA3.1-N3ICD전염후촉사HSC-T6합성α-SMA화Ⅰ형효원단백증가,교대조조분별제고료5.8배화4.5배,t치분별위13.157화9.810,P치균＜0.01；상반,Notch3- siRNA명현억제HSC-T6표체α-SMA화Ⅰ형효원단백,교대조조분별하강료약90％화84％,t치분별위19.863화10.376,P치균＜0.01.이차,Notch3-siRNA능길항TGFβ1유도HSC-T6표체α-SMA화Ⅰ형효원적작용.결론 Notch신호통로가능통과조공간성상세포적활화삼여간섬유화적형성,선택성지간예Notch3신호전도가능성위항간섬유화적신도경.
Objective To investigate whether Notch signaling is activated in hepatic stellate cells (HSCs),and to determine whether manipulation of the Notch signaling pathway can effect the activation of HSCs.Methods The expression of Notch signaling components in unactivated or TGF-β1-activated HSC-T6 cells was detected by Taqman Probe-based gene expression analysis.Differential expression of Notch3 and Jagged1 was detected by immunofluorescence analysis.Notch3-mediated expression of the myofibroblastic markers,α-SMA and collagen I,was detected in HSC-T6 cells transfected with pcDNA3.1-N3ICD or Notch3 siRNA by Western blotting.Results Notch signaling components were expressed in both unactivated and activated HSC-T6 cells,but the TGF-β1-treated cells showed significantly higher expression levels of Jagged1 (3.9-fold,F =2543.482),Notch3 (4.2-fold,F =287.982),and HES1 (3.2-fold,F =1719.851).Transfection-mediated over-expression of Notch3 led to significantly increased expression of α-SMA (6.8-fold,t =13.157) and collagen Ⅰ (5.5-fold,t =9.810) (both P ＜ 0.01).Transient knock-down of Notch3 expression by siRNA decreased expression of the myofibroblastic markers (α-SMA by approximately 90％,t =19.863 and collagen Ⅰ by 84％,t =10.376; both,P ＜ 0.01).Moreover,knock-down of Notch3 antagonized the TGF-β1-induced expression of αt-SMA and collagen Ⅰ.Conclusion Notch signaling may participate in liver fibrogenesis by regulating HSC activation.Selective interruption of Notch3 may represent a new anti-fibrotic strategy to treat liver fibrosis.
