中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2010年
6期
453-459
,共7页
白寿军%张亚敏%周巧丹%曾锐%李彩霞%裴广畅%许楚瓯%葛树旺%周欢%徐钢%刘晓城
白壽軍%張亞敏%週巧丹%曾銳%李綵霞%裴廣暢%許楚甌%葛樹旺%週歡%徐鋼%劉曉城
백수군%장아민%주교단%증예%리채하%배엄창%허초구%갈수왕%주환%서강%류효성
纤维化%肾小管%上皮细胞%β连环素%CIP4
纖維化%腎小管%上皮細胞%β連環素%CIP4
섬유화%신소관%상피세포%β련배소%CIP4
Fibrosis%Kidney tubules%Epithelial cells%Beta-catenin%Cdc42 interacting protein-4
目的 观察骨架调节蛋白CIP4(Cdc42 interacting protein-4)在肾纤维化过程中表达水平、细胞内定位及高表达的CIP4基因对人肾小管上皮细胞E钙黏蛋白(E-cadherin)、波形蛋白(vimentin)的表达和β连环素(β-catenin)酪氨酸磷酸化水平的影响.方法 体外实验以人近端肾小管上皮细胞(HK-2细胞)为研究对象,10 μg/L TGF-β1刺激72 h诱导HK-2细胞转分化;Western印迹法检测各组细胞内CIP4、E-cadherin、vimentin蛋白的表达;RT-PCR法检测细胞内CIP4 mRNA表达水平;激光共聚焦显微镜观察CIP4在细胞内定位.体内实验以SD大鼠为研究对象,5/6肾切除法制作慢性肾纤维化模型;常规检测BUN和Scr水平;Masson染色检测肾组织纤维化水平;免疫组化法检测肾组织内CIP4蛋白的表达和分布.脂质体法介导含野生型CIP4的重组真核表达质粒pcDNA3.1-CIP4或pcDNA3.1-Zeo(空载体)转染HK-2细胞,Wetern印迹法检查转染的效率.稳定转染成功后,Wetern印迹法检测正常组、pcDNA-CIP4转染组和空载体转染组细胞内E-cadherin、vimentin蛋白的表达和β-catenin酪氨酸磷酸化水平.结果 正常HK-2细胞表达E-cadherin和少量的CIP4,几乎不表达vimentin.TGF-β1干预组细胞vimentin蛋白表达增加(P＜0.05),E-cadherin蛋白表达减少(P＜0.05),CIP4 mRNA和蛋白表达均显著增多(P＜0.05).CIP4在正常细胞内大部分在细胞膜,少量在细胞质,在转分化的HK-2细胞表达显著增多,并向细胞质和细胞核聚集.假手术组大鼠肾功能正常,肾组织内未见明显纤维化组织,CIP4在肾小管表达较少,肾小球内几乎不表达;模型组大鼠BUN和Scr增高,肾组织内可见明显纤维化组织,CIP4在肾小管表达明显增加.pcDNA3.1-CIP4转染组较正常组和空载体转染组细胞内CIP4表达增多(P＜0.05),β-catenin酪氨酸磷酸化水平和vimentin蛋白表达增加(P＜0.05),而E-cadherin蛋白表达减少(P＜0.05).结论 CIP4高表达可能参与肾小管上皮细胞-间充质细胞转分化,促进肾间质纤维化.
目的 觀察骨架調節蛋白CIP4(Cdc42 interacting protein-4)在腎纖維化過程中錶達水平、細胞內定位及高錶達的CIP4基因對人腎小管上皮細胞E鈣黏蛋白(E-cadherin)、波形蛋白(vimentin)的錶達和β連環素(β-catenin)酪氨痠燐痠化水平的影響.方法 體外實驗以人近耑腎小管上皮細胞(HK-2細胞)為研究對象,10 μg/L TGF-β1刺激72 h誘導HK-2細胞轉分化;Western印跡法檢測各組細胞內CIP4、E-cadherin、vimentin蛋白的錶達;RT-PCR法檢測細胞內CIP4 mRNA錶達水平;激光共聚焦顯微鏡觀察CIP4在細胞內定位.體內實驗以SD大鼠為研究對象,5/6腎切除法製作慢性腎纖維化模型;常規檢測BUN和Scr水平;Masson染色檢測腎組織纖維化水平;免疫組化法檢測腎組織內CIP4蛋白的錶達和分佈.脂質體法介導含野生型CIP4的重組真覈錶達質粒pcDNA3.1-CIP4或pcDNA3.1-Zeo(空載體)轉染HK-2細胞,Wetern印跡法檢查轉染的效率.穩定轉染成功後,Wetern印跡法檢測正常組、pcDNA-CIP4轉染組和空載體轉染組細胞內E-cadherin、vimentin蛋白的錶達和β-catenin酪氨痠燐痠化水平.結果 正常HK-2細胞錶達E-cadherin和少量的CIP4,幾乎不錶達vimentin.TGF-β1榦預組細胞vimentin蛋白錶達增加(P＜0.05),E-cadherin蛋白錶達減少(P＜0.05),CIP4 mRNA和蛋白錶達均顯著增多(P＜0.05).CIP4在正常細胞內大部分在細胞膜,少量在細胞質,在轉分化的HK-2細胞錶達顯著增多,併嚮細胞質和細胞覈聚集.假手術組大鼠腎功能正常,腎組織內未見明顯纖維化組織,CIP4在腎小管錶達較少,腎小毬內幾乎不錶達;模型組大鼠BUN和Scr增高,腎組織內可見明顯纖維化組織,CIP4在腎小管錶達明顯增加.pcDNA3.1-CIP4轉染組較正常組和空載體轉染組細胞內CIP4錶達增多(P＜0.05),β-catenin酪氨痠燐痠化水平和vimentin蛋白錶達增加(P＜0.05),而E-cadherin蛋白錶達減少(P＜0.05).結論 CIP4高錶達可能參與腎小管上皮細胞-間充質細胞轉分化,促進腎間質纖維化.
목적 관찰골가조절단백CIP4(Cdc42 interacting protein-4)재신섬유화과정중표체수평、세포내정위급고표체적CIP4기인대인신소관상피세포E개점단백(E-cadherin)、파형단백(vimentin)적표체화β련배소(β-catenin)락안산린산화수평적영향.방법 체외실험이인근단신소관상피세포(HK-2세포)위연구대상,10 μg/L TGF-β1자격72 h유도HK-2세포전분화;Western인적법검측각조세포내CIP4、E-cadherin、vimentin단백적표체;RT-PCR법검측세포내CIP4 mRNA표체수평;격광공취초현미경관찰CIP4재세포내정위.체내실험이SD대서위연구대상,5/6신절제법제작만성신섬유화모형;상규검측BUN화Scr수평;Masson염색검측신조직섬유화수평;면역조화법검측신조직내CIP4단백적표체화분포.지질체법개도함야생형CIP4적중조진핵표체질립pcDNA3.1-CIP4혹pcDNA3.1-Zeo(공재체)전염HK-2세포,Wetern인적법검사전염적효솔.은정전염성공후,Wetern인적법검측정상조、pcDNA-CIP4전염조화공재체전염조세포내E-cadherin、vimentin단백적표체화β-catenin락안산린산화수평.결과 정상HK-2세포표체E-cadherin화소량적CIP4,궤호불표체vimentin.TGF-β1간예조세포vimentin단백표체증가(P＜0.05),E-cadherin단백표체감소(P＜0.05),CIP4 mRNA화단백표체균현저증다(P＜0.05).CIP4재정상세포내대부분재세포막,소량재세포질,재전분화적HK-2세포표체현저증다,병향세포질화세포핵취집.가수술조대서신공능정상,신조직내미견명현섬유화조직,CIP4재신소관표체교소,신소구내궤호불표체;모형조대서BUN화Scr증고,신조직내가견명현섬유화조직,CIP4재신소관표체명현증가.pcDNA3.1-CIP4전염조교정상조화공재체전염조세포내CIP4표체증다(P＜0.05),β-catenin락안산린산화수평화vimentin단백표체증가(P＜0.05),이E-cadherin단백표체감소(P＜0.05).결론 CIP4고표체가능삼여신소관상피세포-간충질세포전분화,촉진신간질섬유화.
Objective To observe the expression and localization of CIP4 (Cdc42 interacting protein-4) in the renal fibrosis and the effect of CIP4 on the expression of E-cadherin,vimentin and β-catenin tyrosine phosphorylation. Methods In vitro, the human tubular epithelial cells (HK-2 cell line) were cultured with 10 μg / L TGF-β1 for 72 h. The protein expressions of CIP4, E-cadherin, vimentin and β-catenin tyrosine phosphorylation were measured by Western blotting; the expression of CIP4 mRNA was detected by RT-PCR. The intracellular distribution of CIP4 was observe by confocal microscope. In vivo, Masson staining was used to evaluate the level of renal fibrosis; the expression and distribution of CIP4 in renal tissue were detected by immunohistochemistry. HK-2 cells were transfected with pcDNA3. 1-CIP via lipofectamine 2000. The expressions of E-cadherin, vimentin and β-catenin tyrosine phosphorylation level in the transfected cells were detected by Western blotting. Results The expressions of CIP4 mRNA and protein were up-regulated in renal tubular EMT cells. Most of CIP4 protein localized in cell membrane, and some was in cytoplasm. After stimulation by TGF-β1, the expression of CIP4 protein both in cytoplasm and nucleus was greatly increased (P ＜0.05),especially in cytoplasm. In vivo, CIP4 was expressed in renal tubular epithelia, but little expressed in glomeruli. In renal from 5/6 nephrectomized rats, CIP4 expression was significantly increased. In the CIP4 transfectants, the expression of CIP4, vimentin and β-catenin tyrosine phosphorylation level were up-regulated (P ＜0.05), but E-cadherin expression was suppressed (P ＜0.05).Conclusion The overexpression of CIP4 is likely to take part in the epithelial-to-mesenchymal transition process, thereby promoting the renal fibrosis.
