肾小管上皮细胞转分化中CIP4与β连环素的相互作用
신소관상피세포전분화중CIP4여β련배소적상호작용
The interaction between Cdc42-Interacting protein 4 and β-catenin in renal tubular epithelial-mesenchymal transition
  • 万方数据
    中华肾脏病杂志 중화신장병잡지

    2012年 6期 469-475 ,共7页

    许楚瓯%张亚敏%刘萍%周巧丹%曾锐%白寿军%裴广畅%韩敏%刘丽丽%徐钢

    허초구%장아민%류평%주교단%증예%백수군%배엄창%한민%류려려%서강

    纤维化%转化生长因子β1%β连环素%CIP4%上皮细胞-间质细胞转分化 纖維化%轉化生長因子β1%β連環素%CIP4%上皮細胞-間質細胞轉分化
    섬유화%전화생장인자β1%β련배소%CIP4%상피세포-간질세포전분화
    Fibrosis%Transforming growth factor betal%Beta catenin%Cdc42-interacting protein 4%Epithelial-mesenchymal transition
    目的 探讨在大鼠肾脏纤维化组织中CIP4(Cdc42-interacting protein 4)的表达和分布,以及在转化生长因子β1(TGF-β1)诱导的人肾小管上皮细胞(HK-2)-间质细胞转分化(EMT)模型中,CIP4与β连环素(β-catenin)之间的相互作用关系.方法 体内实验用SD大鼠5/6肾切除法建立肾纤维化模型,根据Masson染色结果观察肾脏组织纤维化程度;用免疫组织化学染色观察CIP4在模型组和假手术组的表达和分布.体外实验用HK-2细胞株作为研究对象,使用TGF-β1(10 μg/L)刺激72 h建立EMT模型.Western印迹法检测E钙黏蛋白(E-cadherin)和α平滑肌肌动蛋白(α-SMA)蛋白表达变化,同时检测CIP4和β-catenin的表达水平;免疫荧光法观察CIP4和β-catenin在两组细胞中的共定位关系;免疫共沉淀方法检测CIP4和3-catenin之间的相互作用关系;用脂质体2000将质粒pcDNA4.0-CIP4瞬时转染至HK-2细胞,Western印迹法检测高表达CIP4对上述指标的影响,以及高表达CIP4与TGF-β1刺激对β-catenin入核的影响.结果 体内实验,CIP4在假手术组和5/6肾切除组大鼠肾组织中都有表达,在纤维化的肾脏组织中表达上调,主要分布在肾小管中,且在上皮细胞侧基底膜处聚集.体外实验,与对照组相比,EMT模型组HK-2细胞CIP4和α-SMA蛋白表达明显增高,分别是对照组的1.8倍和2.5倍,同时E-cadherin表达量减少(均P＜0.05),β-catenin表达量无明显变化.免疫荧光结果显示对照组CIP4与β-catenin主要分布于细胞膜,且在细胞膜上存在部分共定位;模型组中,CIP4与β-catenin在细胞膜上分布减少,细胞核内表达增多,细胞核内存在部分共定位现象.免疫共沉淀结果表明在以上两组细胞中,CIP4和β-catenin均存在相互作用.单独转染CIP4目的基因质粒的HK-2细胞中,CIP4与α-SMA表达上调,分别为对照组的3.5倍与1.7倍,并伴有E-cadherin表达下降(均P＜0.05),与单独TGF-β1刺激相似;高表达CIP4与TGF-βl刺激均可上调β-catenin在核蛋白中的表达,分别为对照组的2.0倍和2.5倍(P＜0.05).结论 CIP4在5/6肾切除组大鼠肾脏组织中表达上调,且主要分布于肾小管上皮细胞侧基底膜处,其与β-catenin有部分共定位,且存在相互作用,提示CIP4可能通过促进β-catenin入核,参与EMT的进程.
    목적 탐토재대서신장섬유화조직중CIP4(Cdc42-interacting protein 4)적표체화분포,이급재전화생장인자β1(TGF-β1)유도적인신소관상피세포(HK-2)-간질세포전분화(EMT)모형중,CIP4여β련배소(β-catenin)지간적상호작용관계.방법 체내실험용SD대서5/6신절제법건립신섬유화모형,근거Masson염색결과관찰신장조직섬유화정도;용면역조직화학염색관찰CIP4재모형조화가수술조적표체화분포.체외실험용HK-2세포주작위연구대상,사용TGF-β1(10 μg/L)자격72 h건립EMT모형.Western인적법검측E개점단백(E-cadherin)화α평활기기동단백(α-SMA)단백표체변화,동시검측CIP4화β-catenin적표체수평;면역형광법관찰CIP4화β-catenin재량조세포중적공정위관계;면역공침정방법검측CIP4화3-catenin지간적상호작용관계;용지질체2000장질립pcDNA4.0-CIP4순시전염지HK-2세포,Western인적법검측고표체CIP4대상술지표적영향,이급고표체CIP4여TGF-β1자격대β-catenin입핵적영향.결과 체내실험,CIP4재가수술조화5/6신절제조대서신조직중도유표체,재섬유화적신장조직중표체상조,주요분포재신소관중,차재상피세포측기저막처취집.체외실험,여대조조상비,EMT모형조HK-2세포CIP4화α-SMA단백표체명현증고,분별시대조조적1.8배화2.5배,동시E-cadherin표체량감소(균P＜0.05),β-catenin표체량무명현변화.면역형광결과현시대조조CIP4여β-catenin주요분포우세포막,차재세포막상존재부분공정위;모형조중,CIP4여β-catenin재세포막상분포감소,세포핵내표체증다,세포핵내존재부분공정위현상.면역공침정결과표명재이상량조세포중,CIP4화β-catenin균존재상호작용.단독전염CIP4목적기인질립적HK-2세포중,CIP4여α-SMA표체상조,분별위대조조적3.5배여1.7배,병반유E-cadherin표체하강(균P＜0.05),여단독TGF-β1자격상사;고표체CIP4여TGF-βl자격균가상조β-catenin재핵단백중적표체,분별위대조조적2.0배화2.5배(P＜0.05).결론 CIP4재5/6신절제조대서신장조직중표체상조,차주요분포우신소관상피세포측기저막처,기여β-catenin유부분공정위,차존재상호작용,제시CIP4가능통과촉진β-catenin입핵,삼여EMT적진정.
    Objective To investigate the expression and distribution of Cdc42-interacting protein 4 (CIP4) in renal fibrotic tissue,and the interaction between CIP4 and β-catenin in transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) model of HK-2 cell line.Methods In vivo,the model of renal fibrosis was induced by 5/6 subtotal nephrectomy in rat.Masson staining was used to evaluate the level of renal tissue fibrosis.The expression and distribution of CIP4 was detected by immunohistochemistry.In vitro,the EMT model of HK-2 cell line was induced by TGF-β1 (10 μg/L) for 72 h.Western blotting was used to observe the expression of E-cadherin,β-catenin,CIP4 and α-SMA.Colocalization and interaction of CIP4 and β-catenin were detected by immunofluorescence and immunoprecipitation respectively.Results Compared to sham group,CIP4 expression was increased in group of 5/6 subtotal nephrectomy,CIP4 was mainly distributed in basolateral side of renal tubular epithelia.In vitro,expressions of α-SMA and CIP4 were increased in HK-2 cells stimulated by TGF-β1 for 72 h (2.5and 1.8 folds,respectively) (all P＜0.05),expression of E-cadherin was decreased(P＜0.05).Partial colocalization between CIP4 and β-catenin was detected by immunofluorescence.In control group,CIP4 and β-catenin partially colocalized at the cell membrane.Mter the stimulation of TGF-β1,translocation to nucleus of CIP4 and β-catenin were increased,and partially colocalized in nucleus.The interaction between CIP4 and β-catenin was observed by immunoprecipitation in both control and TGF-β1 stimulated groups.Conclusions Expression of CIP4 in renal fibrotic tissue is increased,which is mainly distributed in basolateral side of renal tubular epithelia.CIP4 and βcatenin partially colocalize and interact with each other.CIP4 may play a role in EMT process through the interaction with β-catenin.
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