间充质干细胞治疗铜绿假单胞菌所致肺部感染时调节PGE2表达水平的机制研究

Pseudomonas aeruginosa (PA) is one of the most common pathogens for hospital acquired lower respiratory tract infection and drug-resistant PA is related to the higher mortality, which lacks of effective treatment options. It has been demonstrated that multi-potent mesenchymal stem cells (MSC) have a function of lung repair and exhibit protective effects by inhibiting overproduction of prostaglandin E2 (PGE2), which subsequently restored impaired phagocytosis. More recently, upstream and downstream factors (miR-21 and 15-PGDH respectively) have been found to play a key role in regulating PGE2 expression. However, the mechanisms underlying these factors and microvesicles (MV), anuclear particles containing numerous proteins, mRNAs, microRNAs, organelles and lipids similar to those present in the cells from which they originate, remain incompletely understood. Therefore, our first aim is to observe whether MV released by MSC have the similar therapeutic effect among several pulmonary indices as MSC themselves in PA induced lung injury mice model. In the following steps, we plan to investigate whether the underlying mechanism of MSC MV is mediated in part through the transfer of miR-21 and 15-PGDH mRNA, probably to macrophages, with subsequent expression of the protein. This study is expected to figure out the optimal choice between MSC and MV treatment as well as to provide a potential new way for the intervention of life-threatening lower respiratory infection caused by drug-resistant pathogens.

铜绿假单胞菌(PA)是医院获得性肺炎的常见病原体，易致严重肺损伤。我们已证实，间充质干细胞(MSC)有潜在修复作用，通过调节前列腺素E2(PGE2)上下游因子COX2、15PGDH来抑制其过表达，修复巨噬细胞功能。且MSC能释放一种包涵众多来自本体细胞成分的微粒(MV)，通过传递各类可溶因子的mRNA/miRNA来调节其表达，发挥类似MSC的修复作用。最新研究提示，miR-21能有效抑制PGE2及其上游因子COX2的表达。据此推测，MSC是否通过释放携有PGE2相关调节因子的MV至靶细胞，调节其表达，实现类似修复作用。本课题拟1）验证MV是否具有类似于MSC本身的治疗PA所致肺部感染及损伤的保护作用；2）探讨MSC抑制PGE2过表达的机制是否部分通过其释放的MV携带miR-21及15-PGDH mRNA，并传递至巨噬细胞来实现。从而为选择理想的治疗靶点，调节炎症反应及清除细菌定植提供依据。

铜绿假单胞菌（pseudomonas aeruginosa, PA）极易耐药且合并肺损伤后死亡率极高。许多国内外课题组研究发现，间充质干细胞（mesenchymal stem cells, MSC）释放的细胞外囊泡（extrovesicles, EVs）对对急性肺损伤有明确的治疗作用，但MSC EVs在多重耐药铜绿假单胞菌（MDR-PA）肺炎中是否具有治疗作用及作用机制尚不清楚。我们首先经气管插管滴入MDR-PA构建小鼠急性肺部感染模型，通过生存分析、炎症水平验证MSC及EVs在MDR-PA肺部感染中具有治疗作用。我们通过microRNA array发现MSC EVs中miR-466表达水平最高。并进一步给予miR-466过表达的MSC EVs治疗，探讨MSC EVs在MDR-PA肺炎中的治疗作用部分是通过所携带的miR-466来实现。我们利用GW4869及CD44分别处理MSC及MSC EVs后与PA刺激后的巨噬细胞共培养，发现MSC及MSC EVs对炎症因子的抑制作用消失，巨噬细胞中miR-466的表达较空白组无差异。进一步将巨噬细胞与MSC共培养或转染miR-466-m后，巨噬细胞倾向于M2型极化。通过软件预测并结合文献阅读选定miR-466的靶分子之一为TIRAP。本研究主要发现MSC及MSC EVs对MDR-PA肺炎均有治疗作用；MSC EVs对MDR-PA肺炎的治疗作用与其携带的miR-466有关；miR-466通过影响巨噬细胞极化在宿主抗MDR-PA的固有免疫应答中发挥作用。