过表达E-cadherin抑制腹膜间皮细胞转分化的作用及机制研究

Peritoneal fibrosis (PF) induced by high glucose would lead to peritoneal filtration failure. Peritoneal mesothelial mesenchymal transition (EMT) is the key event in PF. Loss of E-cadherin (ECAD) is the early event in EMT. But recent data shows that over expressed ECAD promotes the up-regulation of TGFβ1 via inhibiting Smad3/2 phosphorylation by recruiting RhoA. Our unpressed data shows that increased ECAD level inhibits EMT in human peritoneal mesothelial cells (HPMCs), which suggests that ECAD might play a role in EMT regulation. But the mechanism is remained unclear. Our investigation is aimed to determine the role and possible mechanism of ECAD inhibiting HPMCs EMT. Firstly, the level of ECAD, p120ctn and RhoA in HPMCs isolated from peritoneal dialysis solution (PDS) were detected. Co-immunoprecipitation is used to confirm that p120ctn binds to ECAD. The various EMT markers including ZO-1, vimentin, FN, Col-I and α-SMA are evaluated by realtime PCR and western blot. The transepithelial electrical resistance (TER) is detected to evaluate the permeability of HPMCs. Secondly, whether ECAD inhibits Smad3/2 phosphorylation via binding to p120ctn and then switching the RhoA activity, by which inhibits EMT in HPMCs, are investigated.1.HPMC line (HMrSV5) transfected ECAD-pcDNA3d are treated with 4.25% PDS for 24h. The level of TGFβ1, p120ctn and RhoA are evaluated. The distribution of p120ctn in HMrSV5 cells is detected by immunofluorescence. RhoA activity is detected by spectrophotometry; p-Smad2/3 and Smad7 are detected by immuno- fluorescence and western blot. And the EMT markers are detected. 2.The HMrSV5 cells over expressed ECAD are pretreated with p120ctn siRNA or Rock inhibitor Y-27632 to inhibit p120ctn and Rho/Rock signal, after that the cells are treated with 4.25% PDS for 24h, TGFβ1 and EMT markers are evaluated by realtime PCR and western blot, p120ctn distribution and RhoA activity are detected respectively.p-Smad2/3 and Smad7 level are detected to investigate the effects of ECAD on TGFβ1/Smad signal with silence of p120ctn or RhoA/Rock signal. Lastly, ultrasound-mediated microbubble destruction in enhancing ECAD is used in PF rat model. Pathology of peritoneum and peritoneal equilibrium test are investigated to confirm that PF in rat model is attenuated by overexpressed ECAD. Our investigation of understanding about overexpression of ECAD attenuates the EMT in HPMC via Rho/Rock signal might lead to the development of more effective therapies for peritoneal fibrosis in PD patients.

腹膜纤维化是影响腹膜透析(PD)远期疗效的重要因素，腹膜间皮细胞转分化（EMT）在其中发挥重要作用。E-cadherin（ECAD）下调是EMT的起始事件。近期研究和我们的前期结果均提示ECAD在EMT中发挥保护作用，而并非仅是EMT效应分子。本研究拟通过体内外实验，以PD患者腹透液脱落细胞、腹膜间皮细胞株为研究对象，以p120ctn和RhoA/Rock信号为切入点，应用realtime PCR、western blot、免疫共沉淀、免疫荧光等方法，观察过表达ECAD对腹膜间皮细胞EMT关键分子、p120ctn表达量和分布的影响、RhoA活性的变化以及对Smad2/3磷酸化和Smad7表达的影响，探讨过表达ECAD是否通过p120ctn影响RhoA活性继而影响Smad通路从而抑制腹膜间皮细胞EMT；进行动物试验检测过表达ECAD对腹膜纤维化大鼠腹膜功能和腹膜纤维化的影响，证实其治疗效应。

E-cadherin 是主要的细胞粘附分子之一，维持正常黏附功能需要与连环蛋白（catenin）结合形成E-cadherin/catenin 复合体，调控细胞粘附和信号转导。p120ctn是catenin家族的重要成员，可以在胞膜处通过与E-cadherin 的JMD 区结合。我们前期研究发现上调腹膜间皮细胞E-cadherin的表达水平可在一定程度上逆转TGFβ1 诱导的腹膜间皮细胞α-SMA的表达增高，但具体机制未明确。在本项目资助下，我们进一步开展E-cadherin和p120ctn在腹膜间皮细胞转分化中的作用和可能的机制研究。我们对不同透析龄患者腹透液脱落细胞原代培养，发现在长期腹透患者中E-cadherin和P120ctn的表达明显降低，新PD患者组脱落细胞p120ctn主要表达在细胞膜，在细胞质中仅有少量表达，而腹透一年以上组脱落细胞p120ctn在细胞膜上表达明显减少，在细胞质中表达明显增加，提示随着腹膜透析时间延长，腹膜间皮细胞EMT的发生，p120ctn由细胞膜转移至胞质中，提示P120ctn可能通过钙粘蛋白-连环蛋白复合体，参与了腹膜纤维化过程。我们进一步应用腹膜间皮细胞株进行体外实验，发现：TGFβ1刺激后腹膜间皮细胞p120ctn可由细胞膜转移到细胞质中，细胞膜E-cadherin明显减少，与原代培养改变趋势一致，慢病毒转染过表达E-cadherin后，提取总蛋白检测E-cadherin、α-SMA，vimentin、p120ctn，发现过表达E-cadherin可在一定程度上减少TGFβ1导致的腹膜间皮细胞α-SMA、vimentin、E-cadherin的表达变化，提取膜蛋白检测E-cadherin和p120cth发现，发现过表达E-cadherin削弱TGFβ1诱导的腹膜间皮细胞E-cadherin下调，同时削弱TGFβ1导致的腹膜间皮细胞p120ctn由细胞膜向细胞质转位作用。提示过表达E-cadherin可通过与p120ctn作用维持钙黏蛋白-连环蛋白复合体而调节腹膜间皮细胞EMT。