血管生成素1在间充质干细胞释放的微粒治疗急性肺损伤中的作用

Acute lung injury is an acute respiratory failure characterized by hypoxemia and mainly caused by any stimulus of local or systemic inflammation. ALI remains a major cause of morbidity and mortality in hospitalized patients, which lacks of effective treatment options. Multi-potent mesenchymal stem cells have a function of lung repair and Angiopoietin-1, secreted by MSC, is a known endothelial survival and vascular stabilization factor that prevents the increase in lung protein permeability. However, the mechanisms underlying the therapeutic benefit of MSC remain incompletely understood. Recently, MSCs have been found to release circular membrane fragments called microvesicles. These anuclear particles, contain numerous proteins, mRNAs, microRNAs, organelles and lipids similar to those present in the cells from which they originate, have already been determined to be involved in cell-cell communication and the transfer of cellular material. Based on the previous works, we first aim to observe the effect of Ang-1 siRNA pretreatment of MSCs on the therapeutic effect of MVs among several pulmonary indices such as inflammatory cell influx, inflammatory cytokine level and pulmonary edema. In the following steps, we plan to obtain Ang-1 mRNA high-expression MVs by means of the transduction of Ang-1 gene into MSCs using a lentivirus vector to discuss the feasibility of clinical use with interventional MVs. This study is expected to figure out the partial mechanism of MVs in treating ALI as well as to investigate a potential new way to improve the therapeutic benefit of MVs by increasing the transfer of specific mRNAs into target cells.

急性肺损伤（ALI）是一种高病死率的由各种致伤因素导致的急性低氧性呼吸功能不全，临床上缺乏有效治疗手段。间充质干细胞（MSC）具有潜在修复作用，其分泌的血管生成素1（Ang-1）能降低血管通透性并维持肺泡毛细血管膜的稳定性，但是关于干细胞的治疗机制仍存在较大争议。最新发现MSC能够释放一种同样具有修复作用的包涵众多来自于本体细胞成分的膜微粒（MV）。已经证实这些类球型无核颗粒在参与细胞间信号转导的过程中起关键作用。本研究拟首先利用特异性siRNA干扰微粒所携带的Ang-1 mRNA，观察其对于肺部炎症损伤程度的影响；其次利用携带Ang-1基因的质粒经病毒载体系统包装，转染至干细胞，制备高表达Ang-1的微粒，探讨通过干预微粒以强化其损伤修复作用的可行性。本研究不仅明确微粒治疗急性肺损伤的部分作用机制，而且有助于为干预改造微粒，发挥更有效的调节炎症反应及修复肺损伤提供新途径。

急性肺损伤（ALI）是一种高病死率的由各种致伤因素导致的急性低氧性呼吸功能不全，临床上缺乏有效治疗手段。间充质干细胞（MSC）具有潜在修复作用，其分泌的血管生成素1（Ang-1）能降低血管通透性并维持肺泡毛细血管膜的稳定性，但是关于干细胞的治疗机制仍存在较大争议。最新发现MSC能够释放一种同样具有修复作用的包涵众多来自于本体细胞成分的膜微粒（MV）。已经证实这些类球型无核颗粒在参与细胞间信号转导的过程中起关键作用。本研究首先，利用特异性siRNA干扰微粒所携带的Ang-1 mRNA，并证实了提取的干扰MV中Ang-1 mRNA显著下调。其次，体内实验证实LPS刺激后，小鼠BALF中Ang-1蛋白水平明显升高，经气管给予MSC MV、Neg SiRNA MSC MV或MSC后，BALF中Ang-1蛋白水平进一步显著升高，然而Ang-1 SiRNA MSC MV组的Ang-1蛋白水平与LPS组相似，无进一步升高；同时，经气管给予Neg SiRNA MSC MV、 MSC MV或MSC能明显减轻肺部炎症，同时减轻肺水肿。而Ang-1 SiRNA MSC MV的抗炎症及减轻肺水肿作用明显减弱。以上动物实验结果提示MSC MV部分通过传递Ang-1 mRNA至受损靶细胞并促进Ang-1蛋白的合成而参与治疗急性肺损伤。最后，体外试验亦证实MSC MV或者Negative SiRNA MSC MV能抑制小鼠巨噬细胞RAW264.7释放促炎症因子TNF-α，同时促进其释放抗炎因子IL-10。而Ang-1 SiRNA MSC MV对TNF-α释放无明显抑制作用；对IL-10释放虽有部分促进作用，但作用较Negative SiRNA MSC MV减弱。以上体外实验结果提示微粒部分通过传递Ang-1 mRNA至巨噬细胞发挥免疫调节作用。本研究对于阐明MSC MV的作用机制提供了新数据，为未来基于干细胞的“非细胞”治疗提供理论依据。