中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2015年
4期
296-302
,共7页
谢可炜%魏凯%顾明颖%向芃%顾乐怡
謝可煒%魏凱%顧明穎%嚮芃%顧樂怡
사가위%위개%고명영%향봉%고악이
足细胞%受体,Notch1%Sirtuin 1%γ-分泌酶
足細胞%受體,Notch1%Sirtuin 1%γ-分泌酶
족세포%수체,Notch1%Sirtuin 1%γ-분비매
Podocytes%Receptor,Notch1%Sirtuin 1%γ-secretase
目的 探索白藜芦醇是否影响足细胞中的Notch 1信号通路及其可能的机制.方法 分别使用RNA干扰(RNAi)技术和盐酸多西环素(doxycycline,Dox)诱导野生型(WT)和可诱导的Sirtuin 1(SIRT1)短发夹RNA(shRNA)转基因(CAGGS)足细胞SIRT1表达下降,重组小鼠Notch信号的Delta配体4(Delta-like ligand4,DLL4)诱导激活Notch1通路.实时荧光定量PCR法检测SIRT1、Notch1通路关键γ分泌酶mRNA及Notch1靶基因Hes1、Hey2的表达.Western印迹法检测足细胞内Notch1胞内段结构域(intracellular domain of Notch1,ICD1)、SIRT1、解聚素金属蛋白酶(ADAM) 10以及γ分泌酶复合物Presenilin 1和Nicastrin的表达.结果 白藜芦醇剂量依赖地诱导野生型小鼠足细胞Notch1信号胞内段ICD1表达以及下游产物Hes1和Hey2 mRNA表达增高.白藜芦醇可诱导Notch1蛋白水解酶γ分泌酶组分Nicastrin的mRNA和蛋白表达上升以及ADAM10蛋白的表达下降(P＜0.05),而这些变化在使用SIRT1RNAi后均被减弱(P＜ 0.05).重组小鼠DLL4可以增加Notch1通路靶基因Hes1和Hey2的mRNA水平,同时增加细胞核内ICD1的表达,盐酸多西环素孵育CAGGS足细胞显著降低SIRT1 mRNA水平(P＜0.05),48 h后SIRT1蛋白表达明显下降(P＜0.05),并且显著减弱DLL4的作用(P＜0.05).结论 白藜芦醇可以通过高表达分泌酶Nicastrin诱导足细胞中Notch1信号活化,SIRT1介导了白藜芦醇和DLL4诱导Notch1信号活化的作用.
目的 探索白藜蘆醇是否影響足細胞中的Notch 1信號通路及其可能的機製.方法 分彆使用RNA榦擾(RNAi)技術和鹽痠多西環素(doxycycline,Dox)誘導野生型(WT)和可誘導的Sirtuin 1(SIRT1)短髮夾RNA(shRNA)轉基因(CAGGS)足細胞SIRT1錶達下降,重組小鼠Notch信號的Delta配體4(Delta-like ligand4,DLL4)誘導激活Notch1通路.實時熒光定量PCR法檢測SIRT1、Notch1通路關鍵γ分泌酶mRNA及Notch1靶基因Hes1、Hey2的錶達.Western印跡法檢測足細胞內Notch1胞內段結構域(intracellular domain of Notch1,ICD1)、SIRT1、解聚素金屬蛋白酶(ADAM) 10以及γ分泌酶複閤物Presenilin 1和Nicastrin的錶達.結果 白藜蘆醇劑量依賴地誘導野生型小鼠足細胞Notch1信號胞內段ICD1錶達以及下遊產物Hes1和Hey2 mRNA錶達增高.白藜蘆醇可誘導Notch1蛋白水解酶γ分泌酶組分Nicastrin的mRNA和蛋白錶達上升以及ADAM10蛋白的錶達下降(P＜0.05),而這些變化在使用SIRT1RNAi後均被減弱(P＜ 0.05).重組小鼠DLL4可以增加Notch1通路靶基因Hes1和Hey2的mRNA水平,同時增加細胞覈內ICD1的錶達,鹽痠多西環素孵育CAGGS足細胞顯著降低SIRT1 mRNA水平(P＜0.05),48 h後SIRT1蛋白錶達明顯下降(P＜0.05),併且顯著減弱DLL4的作用(P＜0.05).結論 白藜蘆醇可以通過高錶達分泌酶Nicastrin誘導足細胞中Notch1信號活化,SIRT1介導瞭白藜蘆醇和DLL4誘導Notch1信號活化的作用.
목적 탐색백려호순시부영향족세포중적Notch 1신호통로급기가능적궤제.방법 분별사용RNA간우(RNAi)기술화염산다서배소(doxycycline,Dox)유도야생형(WT)화가유도적Sirtuin 1(SIRT1)단발협RNA(shRNA)전기인(CAGGS)족세포SIRT1표체하강,중조소서Notch신호적Delta배체4(Delta-like ligand4,DLL4)유도격활Notch1통로.실시형광정량PCR법검측SIRT1、Notch1통로관건γ분비매mRNA급Notch1파기인Hes1、Hey2적표체.Western인적법검측족세포내Notch1포내단결구역(intracellular domain of Notch1,ICD1)、SIRT1、해취소금속단백매(ADAM) 10이급γ분비매복합물Presenilin 1화Nicastrin적표체.결과 백려호순제량의뢰지유도야생형소서족세포Notch1신호포내단ICD1표체이급하유산물Hes1화Hey2 mRNA표체증고.백려호순가유도Notch1단백수해매γ분비매조분Nicastrin적mRNA화단백표체상승이급ADAM10단백적표체하강(P＜0.05),이저사변화재사용SIRT1RNAi후균피감약(P＜ 0.05).중조소서DLL4가이증가Notch1통로파기인Hes1화Hey2적mRNA수평,동시증가세포핵내ICD1적표체,염산다서배소부육CAGGS족세포현저강저SIRT1 mRNA수평(P＜0.05),48 h후SIRT1단백표체명현하강(P＜0.05),병차현저감약DLL4적작용(P＜0.05).결론 백려호순가이통과고표체분비매Nicastrin유도족세포중Notch1신호활화,SIRT1개도료백려호순화DLL4유도Notch1신호활화적작용.
Objective To explore the relationship between resveratrol and Notch 1 signalling pathway in podocytes.Methods Interference RNA (RNAi) and doxycycline (Dox) were used to inhibit the Sirtuin (SIRT) 1 expression in the wild-type and inducible SIRT1 shRNA (CAGGS) podocytes respectively.Recombinant mouse delta-like ligand 4 (DLL4) was used to activate Notch1 signalling.The message RNA of SIRT1,Notch1 downstream gene Hes1 and Hey2,as well as the key enzymes of Notch1 signalling pathway were detected by using real-time PCR.Western blotting was used to detect intracellular domain of Notch 1 (ICD1),SIRT1,and metalloprotease (ADAM) 10 and components of γ-secretase complex protein expression.Results In WT murine podoytes,resveratrol up-regulated ICD1 protein production,as well as the mRNA of Hes1 and Hey2 in a dose-dependent manner.Treatment with resveratrol resulted Nicastrin mRNA and protein increase in podocytes (P ＜0.05),as well as inhibit ADAM10 expression (P ＜ 0.05),but all these changes were prevented after the use of SIRT1 RNAi(P ＜ 0.05).DLL4 up-regulated the expression of mRNA of Hes1 and Hey2,as well as ICD1 protein production in a dose-dependent manner.Treatment with doxycycline resulted decrease of SIRT1 gene and protein expression in CAGGS podocytes after 24 h and 48 h respectively(P ＜ 0.05),which weakend the role of DLL4 significantly(P ＜ 0.05).Conclusion Resveratrol induces Nicastrin expression,as well as activation of Notch1 signalling pathway in a SIRT1-dependent manner.
