CAJ | 학술논문

目的通过体内外实验探讨PI3K/Akt信号对腹膜透析腹膜间皮细胞上皮-间充质转分化(EMT)的调节作用.方法 40只12周龄ICR小鼠腹腔置管行腹膜透析,制备腹膜间皮细胞的上皮-间充质EMT小鼠模型,成膜后分为PD模型组和对照组.采用实时定量PCR、Western印迹和组织免疫荧光等方法检测腹膜组织磷酸化丝氨酸/苏氨酸激酶(pAkt)的表达及EMT相关蛋白和mRNA的表达[包括紧密连接蛋白(ZO-1)、波形蛋白(Vimentin)、纤连蛋白(FN)].体外实验观察转化生长因子(TGF)β1对人腹膜间皮细胞(HPMCs) Akt磷酸化和核转位及ZO-1、Vimentin表达的影响.采用PI3K/Akt抑制剂LY294002预处理和负显性Akt(DN-Akt)质粒转染HPMCs,抑制P13K/Akt信号,观察其对TGF-β1诱导的HPMCs细胞ZO-1、Vimentin表达的影响.结果与对照组相比,模型组小鼠第28天壁层腹膜组织明显增厚,上皮细胞标志物ZO-1 mRNA和蛋白表达降低,间充质细胞标志物Vimentin和FN的mRNA和蛋白表达升高,Akt磷酸化水平(pAkt)也明显升高(均P＜0.01).体外实验发现:TGF-βl抑制HPMCs细胞ZO-1表达,增加Vimentin和pAkt表达(均P＜0.01),且呈浓度依赖性;LY294002、DN-Akt处理可抑制pAkt表达,对TGF-β1诱导的HPMCs ZO-1 mRNA和蛋白下调表达有明显回复作用,并抑制TGF-β1引起的Vimentin mRNA和蛋白的高表达(均P＜ 0.01).结论 PI3K/Akt信号参与调节PD腹膜间皮细胞EMT,以PI3K/Akt信号为靶点可望为腹膜纤维化的防治提供一种新途径.
목적 통과체내외실험탐토PI3K/Akt신호대복막투석복막간피세포상피-간충질전분화(EMT)적조절작용.방법 40지12주령ICR소서복강치관행복막투석,제비복막간피세포적상피-간충질EMT소서모형,성막후분위PD모형조화대조조.채용실시정량PCR、Western인적화조직면역형광등방법검측복막조직린산화사안산/소안산격매(pAkt)적표체급EMT상관단백화mRNA적표체[포괄긴밀련접단백(ZO-1)、파형단백(Vimentin)、섬련단백(FN)].체외실험관찰전화생장인자(TGF)β1대인복막간피세포(HPMCs) Akt린산화화핵전위급ZO-1、Vimentin표체적영향.채용PI3K/Akt억제제LY294002예처리화부현성Akt(DN-Akt)질립전염HPMCs,억제P13K/Akt신호,관찰기대TGF-β1유도적HPMCs세포ZO-1、Vimentin표체적영향.결과 여대조조상비,모형조소서제28천벽층복막조직명현증후,상피세포표지물ZO-1 mRNA화단백표체강저,간충질세포표지물Vimentin화FN적mRNA화단백표체승고,Akt린산화수평(pAkt)야명현승고(균P＜0.01).체외실험발현:TGF-βl억제HPMCs세포ZO-1표체,증가Vimentin화pAkt표체(균P＜0.01),차정농도의뢰성;LY294002、DN-Akt처리가억제pAkt표체,대TGF-β1유도적HPMCs ZO-1 mRNA화단백하조표체유명현회복작용,병억제TGF-β1인기적Vimentin mRNA화단백적고표체(균P＜ 0.01).결론 PI3K/Akt신호삼여조절PD복막간피세포EMT,이PI3K/Akt신호위파점가망위복막섬유화적방치제공일충신도경.
Objective To investigate the role of PI3K/Akt signaling in the regulation of epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) in peritoneal dialysis in vitro and in vivo.Methods The level of Phosphorylated serine/threonine kinase Akt and the expression of EMT associated gene and protein,including ZO-1,Vimentin and FN,were measured in mice EMT model.In vitro study,phosphorylation level and nuclear translocation of Akt,ZO-1 and Vimentin expression induced by TGF-β1 in human peritoneal mesothelial cells (HPMCs) were also observed.Moreover,HPMCs were pre-treated by one of PI3K/Akt inhibitor,LY294002,or transfected with dominant-negative Akt plasmid to inhibit PI3K/Akt signaling,then analyzed its effect on Zo-1 and Vimentin expression induced by TGF-β1.Results Compared with the control,thickened parietal peritoneum and remarkable decrease in mRNA and protein of the epithelial marker ZO-1,and notable increased in the expression of mesenchymal markers Vimentin and FN were observed in PD mice (all P ＜ 0.01).Moreover,the phosphorylation of Akt also significantly increased under above condition (P ＜ 0.01).In vitro study,with the stimulation of TGF-β1,the expression of Zo-l was down-regulated,while the expression of Vimentin increased (all P ＜ 0.01).In addition,TGF-β1 remarkably increased pAkt in HPMCs (all P ＜ 0.01) in dose-dependent.However,LY294002 and DN-Akt dramatically inhibited the vimentin expression in HPMCs induced by TGF-β1 after inhibition of pAkt.On the other hand,the expression of ZO-1 also was restored (P ＜ 0.01).Conclusion PI3K/Akt signaling is involved in EMT of peritoneal mesothelial cells in peritoneal dialysis,and may be a new target for the prevention and treatment of peritoneal fibrosis during PD.