STC-1修饰的脂肪干细胞来源外泌体促进血管损伤后再内皮化的作用及机制研究

Chronic inflammation and dysfunction of endothelial cells (EC) mediated by inflammasome plays an important role in the restenosis after endovascular therapy (RET). As the new “cell free” stem cell therapeutic strategy, exosome secreted by ADSC can transfer homologous vascular endothelial growth factor (VEGF) and other proteins, however our pre-experiment showed that the exosome can not inhibit the activation of inflammasome efficiently. Recently, it was reported that gene modification of ADSC can promote related abilities of its exosome. As an important secretive glycoprotein that directly acts on mitochondrion, stanniocalcin (STC)-1 can inhibit the generation of reactive oxygen species, which inhibits inflammasome activation and up-regulate VEGF, which promots proliferation of EC. We propose the hypothesis that modification of ADSC by STC-1 to enhance the STC-1 level in its exosome will protect EC function and promote re-endothelialization to a better extent. This project aims to transfer STC-1 gene to ADSC using nuclear transfection technology, leading to the overexpression of STC-1, and study the effect of its exsome (S-ADSC-Exo) on EC inflammasome activation and re-endothelialization through transfer STC-1 in vitro and vivo, use STC-1 receptor related antagonist to study the changes of signal pathways and elucidate the mechanism of S-ADSC-Exo promotes the re-endothelialization of the injured blood vessel, which will bring new insights into the prevention and treatment of RET.

炎症小体介导内皮细胞（EC）慢性炎症/功能障碍在血管腔内治疗术后再狭窄（RET）中有重要作用。作为“无细胞”的干细胞治疗新策略，ADSC分泌的外泌体可传递同源VEGF等蛋白，但我们预实验发现其并不能有效抑制炎症小体激活。最近发现，利用基因修饰干细胞可增强其外泌体的相关能力。作为少数可直接作用于线粒体的分泌型糖蛋白，斯钙素（STC）-1可直接减少活性氧产生抑制炎症小体激活，上调VEGF等因子促进EC增殖。我们假设：利用STC-1修饰ADSC，提高其外泌体中STC-1水平，会更好地保护EC功能、促进血管损伤后再内皮化。本项目拟利用核转染使ADSC过表达STC-1，在体内外研究其外泌体（S-ADSC-Exo）传递STC-1等对EC炎症小体激活、再内皮化的影响，结合STC-1相关受体拮抗剂探讨信号通路变化，阐明S-ADSC-Exo促进血管损伤后再内皮化的机制，为临床上防治RET提供新的思路。

炎症小体介导内皮细胞慢性炎症/功能障碍在血管腔内治疗术后再狭窄中有重要作用。本项目以“外泌体是干细胞（MSC）调控靶细胞炎症，促进组织修复的重要媒介”为视点，通过斯钙素-1（STC-1）转染脂肪干细胞（ADSC），以增强其外泌体抑制内皮细胞炎症小体活化的能力，从而更好地促进血管损伤后再内皮化。本项目成功建立过表达STC-1的ADSC外泌体（S-ADSC-Exo）体系并进行鉴定，体外研究发现S-ADSC-Exo可被小鼠主动脉内皮细胞（MAECs）摄取，显著提高MAECs中STC-1表达水平。S-ADSC-Exo与MAECs共培养后，MAECs迁移能力及其成管能力显著增强。S-ADSC-Exo处理组MAECs中活性氧簇（ROS）水平显著降低，且NLRP3、Caspase-1 p10以及IL-1β p17蛋白及mRNA表达水平显著低于ADSC-Exo组和Control组。以上体外研究结果提示S-ADSC-Exo可能通过降低ROS水平抑制炎症小体激活，从而促进MAECs再内皮化。体内实验中，建立小鼠颈动脉导丝损伤模型，我们发现S-ADSC-Exo组小鼠颈动脉组织中CD68+巨噬细胞数量显著低于ADSC-Exo组和Control组，同时S-ADSC-Exo处理组小鼠颈动脉组织中ROS水平显著下降、NLRP3、Caspase-1 p10、IL-1β p17蛋白及mRNA表达水平显著降低。HE染色结果显示，S-ADSC-Exo处理组小鼠颈动脉厚度显著低于对照组、ADSC-Exo处理组。Even’s blue染色结果表明，S-ADSC-Exo处理组颈动脉再内皮化水平显著高于对照组、ADSC-Exo处理组。以上结果提示，鼠尾静脉注射S-ADSC-Exo可能通过降低小鼠颈动脉组织中ROS水平，抑制炎症小体激活从而促进血管再内皮化。