安徽医科大学学报
安徽醫科大學學報
안휘의과대학학보
Acta Universitatis Medicinalis Anhui
2015年
9期
1243-1247
,共5页
江宇峰%伍超%吴佳俊%王极宇%马成骁%陈帆%吴德卫%杨明%郑凌云%杨雁
江宇峰%伍超%吳佳俊%王極宇%馬成驍%陳帆%吳德衛%楊明%鄭凌雲%楊雁
강우봉%오초%오가준%왕겁우%마성효%진범%오덕위%양명%정릉운%양안
大鼠肝星状细胞%丝裂原活化蛋白激酶抑制剂%Smad2%Smad3%Smad4
大鼠肝星狀細胞%絲裂原活化蛋白激酶抑製劑%Smad2%Smad3%Smad4
대서간성상세포%사렬원활화단백격매억제제%Smad2%Smad3%Smad4
HSCs%MAPK inhibitors%Smad2%Smad3%Smad4
目的 探究3种丝裂原活化蛋白激酶( MAPK)抑制因子对转化生长因子-β( TGF-β)活化的大鼠肝星状细胞( HSC)中Smad2/3磷酸化及Smad4 核转位的影响. 方法采用Ⅳ型胶原酶和链酶蛋白酶原位肝灌流法分离正常大鼠HSC并传代培养,分别用细胞外信号调节激酶( ERK)、c-Jun氨基末端激酶( JNK )、p38 抑制因子( PD98059、SP600125、SB203580 )与HSC共同培养,再加入TGF-β1活化细胞,采用免疫沉淀和 Western blot 法检测 pSmad2C、pSmad2L、pS-mad3L蛋白表达;采用细胞免疫荧光法检测Smad4 蛋白表达及细胞内定位情况. 结果 Western blot显示静息状态下HSC几乎不表达 pSmad2C 而低表达 pSmad2L、pSmad3L, TGF-β1刺激后显著上调上述磷酸化蛋白表达, p38 抑制因子(1、3 μmol/L)、ERK 抑制因子(1 μmol/L)对 Smad2C、Smad2L、Smad3L 磷酸化无显著影响;p38 抑制因子( 10μmol/L)降低了 pSmad3L 蛋白表达;ERK 抑制因子( 3、10μmol/L)降低了pSmad2C蛋白表达;JNK抑制因子(1、3、10μmol/L)减少了 pSmad2C、pSmad2L、pSmad3L 蛋白表达.TGF-β1刺激可明显诱导Smad4核转位,而3种MAPK抑制因子不同程度地抑制TGF-β1 诱导的Smad4 转位入核. 结论 在 HSC 细胞中 TGF-β1 可能通过活化 JNK 通路促进Smad2/3磷酸化,活化ERK、JNK、p38 通路促进Smad4 核转位.
目的 探究3種絲裂原活化蛋白激酶( MAPK)抑製因子對轉化生長因子-β( TGF-β)活化的大鼠肝星狀細胞( HSC)中Smad2/3燐痠化及Smad4 覈轉位的影響. 方法採用Ⅳ型膠原酶和鏈酶蛋白酶原位肝灌流法分離正常大鼠HSC併傳代培養,分彆用細胞外信號調節激酶( ERK)、c-Jun氨基末耑激酶( JNK )、p38 抑製因子( PD98059、SP600125、SB203580 )與HSC共同培養,再加入TGF-β1活化細胞,採用免疫沉澱和 Western blot 法檢測 pSmad2C、pSmad2L、pS-mad3L蛋白錶達;採用細胞免疫熒光法檢測Smad4 蛋白錶達及細胞內定位情況. 結果 Western blot顯示靜息狀態下HSC幾乎不錶達 pSmad2C 而低錶達 pSmad2L、pSmad3L, TGF-β1刺激後顯著上調上述燐痠化蛋白錶達, p38 抑製因子(1、3 μmol/L)、ERK 抑製因子(1 μmol/L)對 Smad2C、Smad2L、Smad3L 燐痠化無顯著影響;p38 抑製因子( 10μmol/L)降低瞭 pSmad3L 蛋白錶達;ERK 抑製因子( 3、10μmol/L)降低瞭pSmad2C蛋白錶達;JNK抑製因子(1、3、10μmol/L)減少瞭 pSmad2C、pSmad2L、pSmad3L 蛋白錶達.TGF-β1刺激可明顯誘導Smad4覈轉位,而3種MAPK抑製因子不同程度地抑製TGF-β1 誘導的Smad4 轉位入覈. 結論 在 HSC 細胞中 TGF-β1 可能通過活化 JNK 通路促進Smad2/3燐痠化,活化ERK、JNK、p38 通路促進Smad4 覈轉位.
목적 탐구3충사렬원활화단백격매( MAPK)억제인자대전화생장인자-β( TGF-β)활화적대서간성상세포( HSC)중Smad2/3린산화급Smad4 핵전위적영향. 방법채용Ⅳ형효원매화련매단백매원위간관류법분리정상대서HSC병전대배양,분별용세포외신호조절격매( ERK)、c-Jun안기말단격매( JNK )、p38 억제인자( PD98059、SP600125、SB203580 )여HSC공동배양,재가입TGF-β1활화세포,채용면역침정화 Western blot 법검측 pSmad2C、pSmad2L、pS-mad3L단백표체;채용세포면역형광법검측Smad4 단백표체급세포내정위정황. 결과 Western blot현시정식상태하HSC궤호불표체 pSmad2C 이저표체 pSmad2L、pSmad3L, TGF-β1자격후현저상조상술린산화단백표체, p38 억제인자(1、3 μmol/L)、ERK 억제인자(1 μmol/L)대 Smad2C、Smad2L、Smad3L 린산화무현저영향;p38 억제인자( 10μmol/L)강저료 pSmad3L 단백표체;ERK 억제인자( 3、10μmol/L)강저료pSmad2C단백표체;JNK억제인자(1、3、10μmol/L)감소료 pSmad2C、pSmad2L、pSmad3L 단백표체.TGF-β1자격가명현유도Smad4핵전위,이3충MAPK억제인자불동정도지억제TGF-β1 유도적Smad4 전위입핵. 결론 재 HSC 세포중 TGF-β1 가능통과활화 JNK 통로촉진Smad2/3린산화,활화ERK、JNK、p38 통로촉진Smad4 핵전위.
Objective To investigate the effects of three MAPK inhibitors on phosphorylation of Smad2/3 and nu-clear translocation of Smad4 in TGF-β1-activated hepatic stellate cell ( HSC) . Methods HSC was isolated from normal rat liver by using collagenase IV and pronase-E digestion in situ and continuously cultured in vitro. The cells in log phase were pretreated with ERK inhibitor ( PD98059 ) , JNK inhibitor ( SP600125 ) and p38 inhibitor (SB203580) in corresponding group respectively, then activated by TGF-β1. The protein expressions of phospho-rylated (p)Smad2C, pSmad2L and pSmad3L were measured by immunoprecipitation(IP) and western blot assay. The protein expression and intracellular localization of Smad4 were assessed by cell immunofluorescence assay. Results The protein expressions of phosphorylated Smad2C, Smad2L and Smad3L were feeble in quiescent HSC, while markedly increased by TGF-β1 stimulation. The two concentrations of p38-specific inhibitor (1, 3μmol/L) and low concentration ERK-specific inhibitor (1μmol/L) had no significant effect on the phosphorylation of Smad2/3; the high concentration of p38-specific inhibitor ( 10 μmol/L ) significantly down-regulated elevated pSmad2 C by TGF-β1; the two concentrations of ERK-specific inhibitor ( 3 , 10 μmol/L ) could remarkably de-crease elevated pSmad2C by TGF-β1;the three concentrations of JNK-specific inhibitor (1, 3, 10μmol/L) inhib-ited the phosphorylation of Smad2C, Smad2L and Smad3L stimulated by TGF-β1. All three MAPK-specific inhibi-tors suppressed increased nuclear translocation of Smad4 protein in TGF-β1-stimulated HSCs. Conclusion The phosphorylation of Smad2/3 and nuclear translocation of Smad4 might be induced by TGF-β1 via activating ERK, JNK, p38 pathways in HSCs.