T细胞源性微泡通过上调miR-224促进SLE相关TMA内皮细胞转分化的机制研究

。 Systemic lupus erythermatosus(SLE)-associated thrombotic microangiopathy (SLE-TMA) has been shown to be the key factor affecting the long-term renal survival for the patients with lupus nephritis. Our previous studies found the presence of endothelial to mesenchymal cell transition (EndMT) in the endothelium of active vascular lesions in lupus nephritis with TMA. Plasma T lymphocyte-derived microvesicles (TMVs) level and renal endothelial miR-224 expression were also markedly increased and correlated with degree of the renal damage. We postulate that TMVs could induce EndMT by up-regulating miR-224 and lead the development of SLE-TMA. In this study, we will explain our hypothesis based on the researches in three aspects:the clinico-pathological analysis,in vitro cell culture study and lupus nephritis animal model. We will investigate the histopathological features, endothelial cell phenotypes and miR-224 expression in the glomeruli and renal blood vessels of lupus nephritis patients with or without SLE-TMA. The level and miR-224 expression of plasma TMVs from the patients will be examined to explore the correlation between the vascular endothelial phenotypes and the plasma TMVs level and miR-224 expression. The effects of plasma TMVs from patients with SLE-TMA and up-regulating endothelial cell miR-224 expression in promoting endothelial cell damage and endothelial EndMT will be studied in endothelial cell culture in vitro. The pathogenic roles of the TMVs from the patients and the influence of inhibiting miR-224 expression on renal vascular EndMT and vascular lesions are to be evaluated in mouse lupus nephritis model. This study will explore the mechanisms of TMVs in inducing EndMT by up-regulating endothelial cell miR-224 expression and explain the roles of TMVs and miR-224 in the pathogenesis of SLE-TMA, which providing new targets for developing the effective methods in the treatment of SLE associated TMA.

SLE相关血栓性微血管病（SLE-TMA）已经成为影响狼疮性肾炎肾脏远期预后的主要因素。我们前期研究显示SLE-TMA的血管病变存在内皮细胞向间充质细胞转分化（EndMT），患者外周T细胞源性微泡（TMVs）和肾组织miR-224含量显著增高，TMVs可能通过上调miR-224促进EndMT和导致SLE-TMA。本研究将从狼疮性肾炎伴TMA病例的肾间质血管病理及内皮细胞表型和miR-224表达、血浆TMVs含量和miR-224表达等分析TMVs和miR-224与内皮细胞表型和肾血管病变的相关性；从体外细胞模型，研究患者来源TMVs对内皮细胞表型的影响以及上调miR-224对TMVs致病作用的影响，探讨TMVs是否通过上调miR-224促进EndMT，从而阐明TMVs和miR-224在SLE-TMA发病机制中的作用，为SLE-TMA的干预研究提供新的思路和靶点。

系统性红斑狼疮相关的血栓性微血管病（SLE-TMA）通常导致严重肾损伤，因发病机制不明及缺乏有效的治疗，导致该病预后非常差。本项目通过研究SLE-TMA患者肾间质血管内皮细胞-间充质细胞转分化（EndMT）、患者血浆T细胞来源外泌体及其miRNA表达与TMA的相关性及T细胞来源外泌体及miR-21对体外培养人脐静脉内皮细胞（HUVEC）EndMT的影响，探讨SLE-TMA的发生机制。采用激光共聚焦显微镜检测血管内皮细胞分子表达，发现TMA血管内皮CD31和V-cad表达减弱，而与a-SMA共表达的细胞显著增加，证实了TMA血管内皮细胞存在EndMT；采用计算机图像定量分析血管内皮分子表达，发现慢性期TMA血管内皮CD31和V-cad的表达强度显著低于急性期TMA，a-SMA表达则显著高于急性期TMA，表明EndMT与TMA进展相关。计算机图像定量分析间质血管内皮细胞分子表达和肾间质纤维化面积，发现血管内皮a-SMA表达强度与间质纤维化面积呈显著正相关，CD31表达与间质纤维化面积呈负相关；血管a-SMA表达量是影响SLE-TMA患者治疗反应的独立危险因素，抑制a-SMA的表达可能成为治疗的新靶点。流式细胞测定SLE-TMA患者血浆不同细胞来源外泌体，发现SLE-TMA血浆T细胞来源外泌体数量明显增多且表达miR-21显著升高。SLE-TMA患者T细胞来源外泌体与HUVEC共培养，发现可使内皮细胞通透性增加、CD31和V-cad表达减弱，a-SMA表达增强，表明患者T细胞来源外泌体可导致内皮细胞EndMT。转染miR-21的HUVEC通透性及内皮细胞标志的变化与T细胞来源外泌体处理的HUVEC相同，表明T细胞来源外泌体可通过其高表达的miR-21介导内皮细胞损伤和EndMT。研究还观察了双重血浆置换治疗SLE-TMA的疗效，发现双重血浆置换联合免疫抑制治疗能显著改善需要SLE-TMA患者肾功能和远期肾脏存活率。本项目为SLE-TMA发病机制的深入研究及治疗新靶点的探索奠定了良好基础。.本项目资助完成了论文7篇（SCI论文3篇，2篇已在线发表），6篇论文参加国内外会议交流，其中2篇获得全国年会优秀论文。培养了4名研究生，课题负责人获得第一批江苏“卫生领军人才”称号。获得了中华医学科技奖一等奖、军队医疗成果一等奖和江苏医学科技奖一等奖各一项。