溶血磷脂酸通过肺内源性MSCs的迁移促进急性肺损伤修复的作用及机理研究

The injury of alveolar epithelial cell and capillary endothelial cell are key points of acute lung injury(ALI).Lung resident mesenchymal stem cells (LR - MSC) repair the injury in lung through paracrine or differentiation into lung epithelial cells. However, LR-MSCs in adult lung are very low numbers and its mechanism remains to be determined. The study found that LPA levels were increased in bronchoalveolar lavage (BAL) of human lung allografts with increasing numbers of LR-MSC. lysophosphatidic acid (LPA) plays a principal role in the migration of human LR-MSCs through a signaling pathway involving LPA1-induced b-catenin activation. LR-MSCs migration is considered to be an important first step in lung injury. LPA is bioactive lipid, which promote migration and proliferation activity of a variety of cells through G protein coupled receptors on the membranes. In previous research, we also found that LPA promote the proliferation of human umbilical cord mesenchymal stem cells and endothelial progenitor cells derived from rat bone marrow, and increase the migration of cancer cells.Therefore, we are intending to study the functions of LPA on LR-MSCs survival rate, migration ability and the effects of ALI repair. A rat ALI model will be used, and the study will be present at cell, tissue and animal model levels.The detail signaling pathways and mechanisms will be researched.As an active phospholipids without immunocompetence, LPA can be used in a wide range of applications on LR - MSC migration and treating ALI. The objective of the study is exploring and expounding this application prospect.

急性肺损伤(ALI)主要发病原因是肺泡上皮细胞及毛细血管内皮细胞损伤。肺内源间充质干细胞(LR-MSC)可通过旁分泌或分化为上皮细胞修复肺组织。但LR-MSC数量少，其修复机制仍有待确定。研究发现，肺移植后支气管肺泡灌洗内溶血磷脂酸（LPA）与LR-MSC的数量都提高，体外LPA/ LPAR1通过β-catenin信号通路诱导LR-MSC迁移，而LR-MSC迁移对肺组织修复至关重要。LPA是活性磷脂，通过膜上的G蛋白偶联受体提高多种细胞增殖迁移活性。前期研究中我们也发现，LPA促进人脐带间充质干细胞，大鼠骨髓分离的内皮祖细胞的增殖，并促进肿瘤细胞的迁移。因此，该课题中拟复制大鼠ALI模型，从细胞-组织-动物层次研究LPA对LR-MSC存活，迁移活性及修复ALI的影响，并阐明其详细的信号转导机制。LPA在促进病体内源性LR-MSC迁移进而治疗ALI中显示出广泛的应用前景。

急性肺损伤(ALI)主要发病原因是肺泡上皮细胞及毛细血管内皮细胞损伤。肺内源间充质干细胞(LR-MSC)可通过旁分泌或分化为上皮细胞修复肺组织。研究发现，肺移植后支气管肺泡灌洗液内溶血磷脂酸(LPA)与LR-MSC的数量都提高，体外LPA/LPAR1通过β-catenin信号通路诱导LR-MSC迁移，而LR-MSC迁移对肺组织修复至关重要。LPA是活性磷脂，通过膜上的G蛋白偶联受体提高多种细胞增殖迁移活性。前期研究中我们也发现，LPA促进人脐带间充质干细胞，大鼠骨髓分离的内皮祖细胞的增殖，并促进肿瘤细胞的迁移。本课题的目的是通过大鼠ALI模型建立，从细胞-组织-动物层次研究LPA对LR-MSC存活、迁移活性及修复ALI的影响，并阐明其详细的信号转导机制。我们发现脂多糖诱导ALI大鼠肺组织出现严重的水肿及炎症反应，损伤显著模；更有趣且的是，质谱法分析发现ALI大鼠肺泡灌洗液中LPA的含量显著降低，而对外周血中LPA含量没有影响。因此，为了证明ALI大鼠肺泡灌洗液中LPA的减少与组织修复相关，雾化吸入LPA治疗了ALI大鼠。结果显示，雾化吸入LPA可以明显降低ALI大鼠的肺损伤，恢复肺泡灌洗液中LPA含量，1:1Ki16425(LPA1/3受体特异性抑制剂)组中未观察到此现象；经相关性分析发现，泡灌洗液中LPA的含量与肺损伤程度正相关(p<0.05)。进一步利用免疫荧光法检测肺组织中LR-MSCs和AECΠ数量，结果显示，ALI大鼠肺中CD73+/CD90+标记的LR-MSCs和SP-C+标记的肺泡Π型上皮细胞(AECΠ)数量显着降低；雾化吸入LPA可以显着增加24小时后ALI大鼠肺中LR-MSCs和AECΠ数量，该值甚至高于对照组，这表明LPA促进了LR-MSCs的增殖，并直接或间接的促进了AECΠ的存活；Ki16425完全抑制了这种作用。我们的结果表明，雾化吸入LPA可恢复ALI大鼠肺泡灌洗液中LPS诱导的LPA减少，同时增加肺组织中的LR-MSC和AECΠ的数量，肺损伤及炎症反应也显著降低，显示出对ALI大鼠有明显的治疗作用。通过利用LPA受体抑制剂Ki16425，我们初步判断LPA可能通过Lpar1或3受体介导促进LR-MSC增殖、迁移及分化,其机制还需进一步研究。我们的研究表明LPA在治疗ARDS过程中显示出干细胞保护剂的作用，具有很好的应用价值。