TSP-1调控腹膜透析患者腹膜纤维化的机制研究

Long-term success of peritoneal dialysis (PD) depends on the structural and functional integrity of the peritoneal membrane. Ultrafiltration failure resulted from peritoneal fibrosis is a common reason for PD withdrawal. Transforming growth factor-β1 (TGF-β1) plays a critical role during the development of peritoneal fibrosis. TGF-β1 is primary secreted as an inactive precursor and activation is required before it can induce the fibrotic responses. Thrombospondin 1 (TSP-1) is a physiological activator of TGF-β1 in vivo. Previous research indicated that TSP-1 blocking could decrease the activation of TGF-β1, thus ameliorate the lung fibrosis and kidney fibrosis, etc. Glucose and angiotensin II can induce the synthesis of TSP-1. The present study is performed in vitro and in vivo: (1)We first determine whether TSP-1 plays a role in the activation of TGF-β1, Smad2/3 phosphorylation and Smad7 synthesis in cultured human peritoneum mesothelial cells (HPMC). We further assess whether TSP-1 could induce the synthesis of mesothelial-to-mesenchymal transition (MMT) markers (fibronectin, collagen I、α-SMA、Snail) and down-regulate the expression of E-cadherin. To assess whether TSP-1 could induce MMT independent of TGF-β1, HPMC will be incubated with TSP-1 in the present or absent of a neutralizing antibody to TGF-β1. We also investigate the effects of glucose, angiotensin II, pro-inflammation cytokines, etc, on the synthesis of TSP-1, TGF-β1/Smad pathway activation, synthesis of fibronectin, collagen I, α-SMA, Snail and E-cadherin in HPMC and whether TSP-1 blocking could ameliorate the effects of the above stimulus. (2) PD rat models will be used to investigate the role of TSP-1 in TGF-β1 activation and synthesis, Smad2/3 phosphorylation and Smad7 expression, fibroblasts enrollment, as well as inflammatory and fibrotic cytokines expressions in the peritoneum in vivo. We use modified peritoneal equilibration test to assess whether TSP-1 wil affect the peritoneum abilities of solute clearance and water removal. Furthermore, the effects of PD duration and peritoneal stimulus of glucose and angiotensin II on TSP-1 synthesis, TGF-β1/Smad pathway activation as well as fibrotic markers expressions in peritoneum will also be determined. We will also investigate whether TSP-1 blocking could ameliorate the effects of the above stimulus. These data will allow us to gain further insight into the mechanism of peritoneal fibrosis, which is prerequisite to designing novel therapeutic strategies to preserve the structure and functional properties of the peritoneum.

腹膜纤维化所致超滤衰竭是患者退出腹透的常见原因。既往研究证实转化生长因子β1（TGF-β1）为触发腹膜纤维化的重要因子，其需活化后才能发挥作用，血小板反应蛋白1（TSP-1）为TGF-β1的活化因子。本研究通过人腹膜间皮细胞（HPMC）体外培养，探讨TSP-1对HPMC中TGF-β1表达和活性、Smad通路活化、间叶转化的影响，葡萄糖、血管紧张素II等对TSP-1表达、TGF-β1表达和活性、Smad通路活化及间叶转化的影响，TSP-1拮抗剂可否缓解上述刺激因子的作用。建立腹透大鼠模型进一步探讨TSP-1对腹膜TGF-β1表达和活性、Smad通路活化、成纤维细胞募集、纤维化因子表达及超滤功能的影响，葡萄糖、血管紧张素II对TSP-1表达、TGF-β1/Smad通路活化及纤维化的影响，TSP-1拮抗剂可否缓解上述刺激因子的作用。本研究结果可为腹膜纤维化的发生机制以及治疗靶点提供新的理论依据。

腹膜透析利用患者自身腹膜清除溶质毒素和多余的水分，高糖透析液可导致腹膜纤维化，腹膜纤维化引起的超滤衰竭是影响患者技术生存率的重要危险因素。腹膜间皮细胞MMT是腹膜纤维化的重要机制。转化生长因子β1（TGFβ1）在间皮细胞MMT和腹膜纤维化过程中的作用已被证实。血小板反应蛋白1（TSP1）是TGFβ1体内生理活化物。本研究旨在探讨TSP1对TGFβ1活性的调控在腹透腹膜纤维化发生发展中的作用和机制。.本研究首先探讨了TSP1对人间皮细胞（Met5A细胞）TGFβ1表达和活化以及MMT的影响。实验分为1. 正常对照组；2. TSP1刺激组；3. TSP1刺激+ TGFβ1中和组。发现TSP1上调了Met5A细胞中TGFβ1表达和活性，上调了MMT标志因子纤连蛋白（FN）、人III型胶原蛋白（Coll III）、α平滑肌肌动蛋白（αSMA）、Snail的表达；使用TGFβ1中和抗体阻断TGFβ1与细胞表面受体结合，下调TSP1引起的MMT；TSP1刺激Met5A细胞引起Smad2/3磷酸化，TGFβ1中和抗体可部分下调TSP1引起的TGF-β1/Smad信号通路活化。.继而课题研究了使用TSP1拮抗多肽LSKL阻断TSP1对TGFβ1的活化，对高糖诱导的人间皮细胞MMT的影响。实验分为1. 正常对照组；2. 高糖刺激组；3. 高渗对照组；4. 高糖+TSP1拮抗剂组；5. 高渗+TSP1拮抗剂组。发现TSP1拮抗多肽LSKL可显著降低高糖诱导的Smad2/3磷酸化及细胞核内转移，下调高糖诱导MTT。.最后，本研究使用TSP1拮抗多肽LSKL阻断TSP1对TGFβ1的活化，对高糖诱导的大鼠腹膜纤维化的影响。本部分建立了腹透大鼠模型，分为1. 正常对照组；2.尿毒症非腹透组；3. 腹透组；4.腹透+LSKL组。发现TSP1拮抗多肽LSKL降低了高糖诱发的腹膜组织FN、Coll III、αSMA和Snail的表达上调，减轻了高糖诱发的腹膜间皮下层胶原沉积。.综上，研究发现： TSP1通过活化TGFβ1/Smad通路，促进人间皮细胞MMT的发生；高糖刺激人间皮细胞中TSP1的表达，TSP1拮抗多肽通过阻断TSP1对TGFβ1的活化抑制高糖诱导的人间皮细胞MMT；TSP1拮抗多肽改善高糖所致的大鼠腹膜纤维化。本研究结果为腹透腹膜纤维化的发生机制提供了新的理论依据。