PI3K/Akt信号调节腹膜间皮细胞上皮-间充质转分化  

PI3K/Akt signaling regulates epithelial-mesenchymal transition of peritoneal mesothelial cells inperitoneal dialysis

作　　者：彭翔[1,2] 刘伏友[1] 孙林[1] 肖力[1] 王铭[1] 李军[1] 

机构地区：[1]中南大学湘雅二医院肾内科中南大学.肾脏病研究所湖南省血液净化与.肾脏病重点实验室长沙,410011 [2]广东省暨南大学附属清远人民医院肾内科

出　　处：《中华肾脏病杂志》2014年第5期363-369,共7页Chinese Journal of Nephrology

基　　金：国家自然科学基金青年基金（81100541）;国家自然科学基金面上项目（81370832）

摘　　要：目的通过体内外实验探讨P13K/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）B1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P13K/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-1mRNA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．B1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HPMCsZO-1mRNA和蛋白下调表达有明显回复作用，并抑制TGF-β1引起的VimentinmRNA和蛋白的高表达（均P〈0．01）。结论P13K/Akt信号参与调节PD腹膜间皮细胞EMT，以P13K/Akt信号为靶点可望为腹膜纤维化的防治提供一种新途径。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- I 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 notableincreased in the expression of mesenchymal markers Vimentin and FN were observed in PD mice （all P 〈 0.0l）. Moreover, the phospho1~clation of Akt also significantly increased under above condition （P 〈 0.01）. In vitro study, with the stimulation of TGF-~β1, the expression of Zo-1 was down-regulated, while the expression of Vimentin increased （all P 〈 0.01）. In addition, TGF-[31 remarkably increased pAkt in 1-IPMCs （all P 〈 0.01） in dose-dependent. However, LY294002 and DN-Akt dramatically inhibited the vimentin expression in HPMCs induced by TGF-131 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.

关 键 词：磷脂酰肌醇3-激酶 蛋白激酶 腹膜透析 腹膜间皮细胞 上皮-间充 质转分化 

分 类 号：R459.5[医药卫生—治疗学]