血管紧张素II促进骨髓间充质干细胞跨肺微血管内皮迁移的机制研究

Mesenchymal stem cells (MSC) based cell therapy is a hopeful treatment of acute lung injury（ALI）.However,the rate of engraftment is very low. The mechanisms determining MSC recruitment to the lung remain unclear,but the transendothelial migration of MSC may play the most important role in the process of engraftment because MSC are large cells and this may will cause the cells to be held up in the capillary beds of lungs for a while.The process of transendothelial migration will be determined by the functions of migration and adhesion of MSC which will be influenced by the local microenvironment of injuried lungs.Recently,A number of studies have showed that Angiotensin Ⅱ(AngⅡ) which is highly expressed in the injuried lung tissues and acts as an pro-inflammatory cytokine plays an important role in the process of cancer metastasis by increasing transendothelial migration. Interestingly, in our previous study, we found that AngⅡ can promote the migration of MSC by Transwell migration assay.Besides,there was expression of angiotensin Ⅱ receptor 1 (AT1R) on MSC.So it can be hypothesized that AngⅡ particpate in the transendothelial migration of MSC in the lung. In this study, We investigate the role of AngII and its receptor AT1R by using AT1R's blocker in the homing process especially the transendothelial migration of MSC and identify its mechinism whether it is associated with the regulation of focal adhesion comeplexes and the rearrangement of actin cytoskeleton in vitro study. In addition,we will demonstrate that focal adhesion kinase (FAK) is the key regulator of focal adhesion comeplexes and can also activate its downstream signaling protein especially the RhoA and Rac1 pathway to make the actin remode and contract,and then to increase the transendothelial migration of MSC.What's more, in vivo mouse model, the LPS-induced lung injury, we quantify the migration and engraftment of MSC carrying AT1R to the injured lung and the expression of AngⅡ and also evaluate the injuried lung restoration to demostrate the protective effect of AT1R pretreated MSC for ALI.

间充质干细胞（MSC）促进肺损伤细胞修复是急性肺损伤（ALI）重要治疗方向，但MSC向损伤肺组织归巢率低，跨内皮迁移是其向肺实质归巢的关键。研究发现血管紧张素Ⅱ（AngⅡ）可促进肿瘤细胞跨内皮迁移，ALI时肺组织内AngⅡ水平明显高于血液；预实验结果表明AngⅡ可增强MSC纵向迁移，且MSC表达AngⅡ的受体AT1R，推测AngⅡ可能通过AT1R促进MSC跨内皮迁移。本研究拟①用氯沙坦阻断MSC的AT1R，观察AngⅡ对MSC跨内皮迁移、黏着斑的形成和细胞骨架排列的影响；②对AT1R胞内信号途径进行干预，证实黏着斑激酶是AngⅡ促进MSC跨内皮迁移的重要分子，并可调控RhoA和Rac1参与细胞骨架的重排；③体内试验明确高表达AT1R的MSC移植可促进MSC向损伤肺组织归巢，修复损伤细胞的同时降低肺内AngⅡ的水平，抑制炎症反应。本研究通过探讨MSC归巢机制，为ALI修复提供新的治疗途径。

弥漫性肺泡损伤是急性呼吸窘迫综合征（ARDS）的基本病理改变，有效修复损伤肺泡对治疗ARDS及改善预后具有关键作用。外源性间充质干细胞（MSC）向损伤肺组织归巢并发挥其作用，为ARDS治疗提供了新途径，但MSC的低归巢率严重阻碍了MSC的应用。间充质干细胞（Mesenchymal stem cells，MSCs）在通过血液迁移到损伤部位过程中可能受到炎症介质的趋化。研究表明促炎症介质血管紧张素II（Angiotensin II，Ang II）在体外可以通过血管紧张素II受体介导促进多种类型细胞的迁移，但Ang II对MSCs迁移的影响及其作用机制尚不明确。本研究在体外使用外源性Ang II 刺激MSCs，再联合AT1R拮抗剂和/或AT2R拮抗剂预处理，结果发现Ang II主要通过AT2R促进MSCs的迁移。并且该作用不是由Ang II促进的细胞增殖所介导的。然后分别使用粘着斑激酶（focal adhesion kinase, FAK）抑制剂PF-573228，RhoA抑制剂C3转移酶，Rac1抑制剂NSC23766或Cdc42抑制剂ML141来研究细胞粘附蛋白和Rho-GTPase蛋白家族（RhoA，Rac1和Cdc42）在Ang II介导MSCs迁移中的作用。结果发现Ang II 激活FAK后促进RhoA和Cdc42激活从而增加细胞骨架重排，进而促进细胞收缩，共同影响MSCs的迁移。共同表明Ang II-AT2R主要通过FAK和RhoA/Cdc42途径的信号传导调节人骨髓MSCs迁移。本研究进一步通过慢病毒介导AT2R过表达和干扰进行骨髓MSCs的转染，在LPS诱导的ARDS小鼠模型的基础上，通过观察AT2R过表达和干扰的MSCs在ARDS小鼠肺内的存留以及肺损伤的修复，明确过表达AT2R的MSCs移植入急性肺损伤小鼠体内对其向损伤肺组织的迁移以及对肺损伤修复的影响。结果表明过表达AT2R可显著增强MSCs的迁移能力并增加其在ALI损伤肺组织内的存留，从而更好的促进肺内屏障功能的恢复，抑制肺内的炎症反应，促进肺损伤的修复。本研究揭示了促进MSCs有效归巢至损伤肺组织的新靶点，从而为优化MSCs治疗ALI/ARDS的效果提供了新的方向。