血管周细胞和内皮细胞Cross-talk调控腹膜通透性增高的机制研究

High peritoneal permeability develops with the duration of continuous peritoneal dialysis (PD), leading to deterioration of peritoneal function and ultimately, ultrafiltration failure (UFF), which accounts for the major cause of dropout of long-term PD therapy. However, the mechanisms of increased peritoneal permeability remain unclear. The present study: 1. Stable PD patients are enrolled and prospectively followed up to screen the early predictors for development of high peritoneal permeability; 2. Primary human omental tissue endothelial cells (HOMTECs) are cultured in vitro. After exposure to bioincompatibility factors of PD solution, inflammation cytokines and uremic serum, expression of pericyte/ endothelial cell cross-talk related molecules, tight junction proteins as well as permeability of HOMTECs are determined; 3. Uremic PD animal model is constructed. Data are collected on changes in peritoneal vascular caliber, pericyte number, coverage rate, phenotype, and expression of PDGF-B/PDGFR-β and Angpt/Tie2. Vascular leakness is measured and peritoneal equilibrium test is used to evaluate peritoneal permeability and function. Effects of interfering the pericyte/ endothelial cell cross-talk will be investigated by intraperitoneal administering DNA aptamer AX102, which is a selective PDGF-B-blocking oligonucleotide, and the angiopoietin 1 mimic COMP-Angpt1. This study explores the mechanism of high peritoneal permeability during long-term PD through pericyte/ endothelial cell cross-talk pathway, and might provide a novel target for prevention and treatment of peritoneal membrane failure.

腹膜通透性增高是导致腹透患者不良预后的关键因素。长期腹透患者发生腹膜通透性增高的确切机制仍不明了，是否与腹膜血管周细胞和内皮细胞的Cross-talk发生异常有关？目前国内外未见有关研究报道。为此，本研究拟开展前瞻性的临床研究随访腹透患者的腹膜功能，寻找腹膜通透性增高的早期预测因素；采用体外培养的腹膜微血管内皮细胞，研究炎症、非生理性腹透液和尿毒症血清对细胞表达内皮紧密连接蛋白和通透性的影响，以及血管周细胞和内皮细胞Cross-talk对其的作用；利用尿毒症腹透动物模型探讨血管周细胞和内皮细胞Cross-talk与其腹膜血管周细胞形态分布和覆盖率、微血管渗漏性及腹膜通透性之间的关系，并进一步研究干扰周细胞和内皮细胞间Cross-talk对腹膜形态、微血管渗漏及腹膜通透性的影响，以期阐述长期腹透后腹膜通透性增高的分子调控机制，为今后尿毒症腹透患者防治腹膜功能衰竭提供新的理论依据。

腹膜通透性增高是导致腹透患者不良预后的关键因素，本研究从腹膜血管周细胞和内皮细胞的Cross-talk的角度探讨了长期腹透患者腹膜通透性增高的机制：1.入选稳定的腹透患者，寻找腹膜通透性增高的早期预测因素；2. 体外培养的血管内皮细胞，研究非生理性腹透液对细胞紧密连接蛋白和细胞通透性的影响，以及血管周细胞和内皮细胞Cross-talk在其中的作用；3. 利用尿毒症腹透动物模型探讨血管周细胞和内皮细胞Cross-talk与腹膜血管周细胞形态分布和覆盖率、微血管渗漏性及腹膜通透性之间的关系，并进一步研究干扰周细胞和内皮细胞间Cross-talk对腹膜形态、微血管渗漏及腹膜通透性的影响。本研究共入选320例规律腹透患者，结果显示患者的BSA、既往葡萄糖暴露量、血清IL-6和Ang-1、透出液中的IL-6AR和Ang-1AR与腹膜小分子溶质转运率相关，而血清Ang-1和IL-6与腹膜大分子溶质转运功能相关；高浓度葡萄糖以及甲基乙二醛可上调内皮细胞Ang-2蛋白的表达，下调内皮细胞连结蛋白VE-cadherin、Occludin和Claudin5的表达，增加内皮细胞对FITC-BSA的通透性，进一步采用Ang1 拟似物COMP-Ang1与Ang2 竞争性结合Tie2 受体可降低内皮细胞的通透性；尿毒症腹透大鼠腹膜血管周细胞覆盖率显著降低，Ang-1蛋白和磷酸化Tie-2蛋白表达显著下调，大鼠腹膜血管内皮连接蛋白VE-cadherin和Occludin表达均显著下调，通过腺病毒转染Ang-1蛋白，可显著增加大鼠腹膜血管周细胞的覆盖率，改善腹膜的通透性及腹膜溶质转运功能。同时，下调PDGF-B蛋白水平能降低腹膜血管的周细胞覆盖率，增加新生血管的通透性和腹膜溶质转运功能。上述结果提示尿毒症和非生物相容性腹透液均会引起腹膜血管周细胞覆盖率下降，内皮间连接蛋白的表达减少，血管通透性增高，导致腹膜超滤减少，腹膜血管内皮细胞和周细胞间Cross-talk参与了腹膜通透性改变，其中Ang/Tie-2通路和PDGF/PDGFR通路在长期腹透时调节腹膜血管新生中起了重要的作用，上述通路可能是防治腹膜通透性增高的新的治疗靶点。