以间充质干细胞为主的多因素干预治疗耐药铜绿假单胞菌所致肺部重症感染及其机制研究

Pseudomonas aeruginosa (PA),with a high rate of multi-drug resistance, is a common Gram-negative pathogen isolated from nosocomial infection and critically ill patients, leading to extremely high mortality if accompanied with lung injury. An innovative non-antibiotic therapeutic strategy is, therefore, urgently required. The crucial role of type III secretion system (TTSS) of PA has been demonstrated as a key initiating factor during the pulmonary infection, not only because of the activation of innate immune system and excessive inflammation but also leading to pulmonary epithelial and endothelial cells damage and successive lung injury. We have previously investigated that mesenchymal stem cells (MSC) were capable of treating lung injury through releasing antimicrobial peptide LL-37, depressing Nod-like receptor mediated stimulation on macrophages, regulating coagulation and fibrinolysis system and promoting endothelial cell repair. To demonstrate the therapeutic value of MSC in treating multi-drug resistant PA pulmonary infection in this study, we aim to trace MSC colonization and differentiation in vivo by labeling with Luciferase, to show the mechanism of the release of LL-37 and the regulation of innate immune system and excessive inflammation by MSC, and to observe the influence on macrophages phagocytic capacity and MSC colonization by adjusting localized redox balance. We anticipate that the findings, in this study, would help offer the preclinical theoretical basis on the non-antibiotic treatment strategy of multi-drug resistant PA pulmonary infection.

铜绿假单胞菌（PA）是院内感染和呼吸危重病患者常分离到的革兰阴性杆菌，极易耐药且一旦合并肺损伤后死亡率极高，因此急需开发新的非抗生素治疗策略。PA的III型分泌毒素系统（TTSS）作为关键始动因素不仅造成固有免疫系统激活和过度炎症反应，也导致肺上皮及内皮细胞破坏从而诱发肺损伤发生。我们发现，间充质干细胞（MSC）能通过释放抗微生物多肽LL-37；下调巨噬细胞表面Nod样受体介导的固有免疫激活；调控凝血纤溶系统并促进血管内皮细胞修复等多种途径治疗肺损伤。因此，为明确MSC在耐药PA肺部感染中的治疗价值，本研究拟通过萤火虫荧光蛋白标记示踪MSC在体内的定植及分化；探讨MSC促LL-37释放、调控固有免疫细胞功能及抑制过度炎症反应的机制；以及观察调节局部氧化还原平衡对于巨噬细胞吞噬能力及MSC定植后促进血管内皮细胞修复能力的影响。本研究预期为临床耐药PA肺部感染的非抗生素防治策略奠定理论基础。

铜绿假单胞菌（PA）是院内感染和呼吸危重病患者常分离到的革兰阴性杆菌，极易耐药且一旦合并肺损伤后死亡率极高，因此急需开发新的非抗生素治疗策略。PA的III型分泌毒素系统（TTSS）作为关键始动因素不仅造成固有免疫系统激活和过度炎症反应，也导致肺上皮及内皮细胞破坏从而诱发肺损伤发生。我们成功构建耐药PA所致重症肺部感染模型，同时阐明间充质干细胞治疗感染所致急性肺损伤中参与调控固有淋巴细胞（ILCs）的机制。此外发现间充质干细胞可表达C型凝集素受体CD69，以识别结合PA，促进胞内ERK1磷酸化，进而调节GM-CSF的分泌，最终发挥治疗PA肺部感染的作用。我们成功构建过表达LL-37的成年小鼠及人终末气道干细胞（LL-37-DASC）及制备LL-37蛋白纳米颗粒（LL-37 PNP），以优化其抗菌能力。发现并明确肺内源性间充质干细胞（LRMSC）在感染所致急性肺损伤治疗中的作用及机制。探讨了血管内皮细胞中BMP4通过调控FoxM1表达参与肺损伤中血管内皮再生和修复过程的机制。发现迷走神经及其受体α7通过调控成纤维细胞生长因子10表达促进肺干细胞再生参与肺损伤修复。证实雾化吸入间充质干细胞释放的细胞外囊泡（MSC-EVs）在PA所致肺部感染模型中的有效性及安全性，并率先证实经雾化吸入MSC-EVs在健康志愿者中的临床应用安全性。本研究为临床耐药PA肺部感染的非抗生素防治策略奠定理论基础。