MSC旁分泌肝细胞生长因子诱导树突状细胞免疫耐受调控ARDS炎症反应的机制研究

Mesenchymal stem cells (MSCs) can inhibit severely uncontrolled inflammation in acute respiratory distress syndrome (ARDS), but the mechanism is still unclear. Dendritic cells (DCs), as one of the most important immunocytes, were proved in our previous study to be able to stimulate early inflammation and tissue impair in ARDS. Inhibition the maturation and proliferation of DCs can reduce inflammation in ARDS. MSCs have the capacity to induced DCs transformate to one specific subset, immune-tolerated DC (tDCs), which can induce immune tolerance and inhibit uncontrolled inflammation in ARDS. However, the mechanism for it has not yet been clarified. We and other researchers find that MSCs paracrines hepatocyte growth factor (HGF), which was found to be able to induce the transformation from immune-enhanced DC to immune-tolerated DC, is evolved in the therapeutic mechanisms of MSCs in treatment of ARDS. According to our preliminary trials, down-regulation the secretion of HGF from MSC results in decreased inhibition effect of inflammation. Simultaneously, HGF can upregulate the expression of Notch receptors and ligands, which has been demonstrated indispensable in DC differentiation process. Therefore, we hypothesize that MSCs inhibits inflammation of ARDS is partly mediated through the secretion of HGF, which promote the pulmonary tDCs generation by upregulate Notch pathway. The purpose of this study are as below: ① To explore the role of HGF secreted by MSC in the transformation from immunity enchanced DCs to tDCs by co-culture of mature DCs and MSC with over/under expression of HGF；② To determine the important role of Notch signaling pathway activated by HGF in the process of tDCs transformation from immunity enchanced DCs; ③ To demonstrated the effect of MSCs over/under expressing HGF on pulmonary DCs and inflammation, and the influence of Notch signaling pathway activated by HGF in wild type and RBP-J gene knockout ARDS mice model. This study may contribute to the new and effective treatment of ARDS.

MSC能有效抑制ARDS炎症，但机制仍不清楚。我们发现抑制DC增殖和成熟可减轻ARDS炎症，MSC能促进DC向免疫耐受的tDCs转化，可能是其抑制ARDS炎症的重要机制，但具体途径亟待阐明。MSC可分泌诱导tDCs生成的重要调节因子HGF，我们发现沉默HGF基因后MSC的抑炎作用明显下降。同时HGF可调节Notch通路受体及配体表达，而Notch通路在DC分化过程中不可或缺。本研究假设：MSC旁分泌HGF通过调控Notch通路诱导tDCs的生成，进而抑制ARDS炎症。本研究拟①采用慢病毒转染调控MSC对HGF的分泌，体外证实MSC分泌HGF诱导tDCs生成；②探讨Notch信号在HGF诱导tDC生成中的作用；③构建Notch信号基因敲除及野生型ARDS小鼠模型，证实MSC分泌HGF对肺部炎症的影响及Notch信号在HGF促进tDC生成中的作用，为改善MSC对ARDS的疗效提供新的靶点。

项目背景：急性肺损伤（ALI）及ARDS是ICU常见临床危重症，死亡率达40%。不同原因诱发免疫细胞激活引起炎症风暴是介导肺损伤的关键。连接固有免疫和适应性免疫的树突状细胞（DCs），是启动失控炎症反应的始动环节，而间充质干细胞（MSC）具有低免疫原性、向肺组织归巢等特点，被认为是ALI损伤修复的理想细胞，HGF被证明是MSC分泌改善ARDS的重要多功能因子；但MSC是否通过旁分泌HGF调控树突状细胞改善ARDS过度炎症反应仍不明确。.主要研究内容：提取C57BL/6小鼠骨髓细胞体外刺激诱导为不成熟 DCs；通过慢病毒转染构建高、低表达HGF的MSCs。LPS刺激DCs为成熟 DC(mDC)后与高、低表达HGF的MSCs共培养，此基础上干预Akt通路，评估MSCs旁分泌HGF对mDC的表型、细胞因子分泌谱及免疫调节能力的影响以及Akt通路在其中的作用。用高、低表达HGF的MSCs治疗ALI小鼠，检测肺内DCs、CD4+T数量和免疫状态及肺病理损伤，明确MSC旁分泌HGF对ALI时肺部DC及炎症损伤的影响。.重要结果、关键数据.本研究发现：⑴ ALI发生2小时后，小鼠肺部DCs数量及成熟比率增高，且与肺病理损伤程度成正相关。⑵MSCs降低肺常规DCs（CD11c+CD11b+DCs）的比例，原因可能是MSC降低血中DC的数量导致向肺内迁移DC数量减少。⑶MSC诱导肺部已经成熟的DC向DCreg分化，抑制T细胞（CD69+CD4+TC）活化、降低ALI小鼠肺病理损伤评分，减轻肺水肿（LWW/BW）。⑷高表达HGF-MSC治疗ALI小鼠后，能够显著促进肺部DCreg生成，降低肺部CD4+T细胞比例及活化，减轻肺病理损伤，低表达则减弱MSC上述作用。⑸MSC通过抑制常规DC向支气管旁淋巴结(PBLN)迁移，但促进DCreg（低表达CD86）的迁移，该过程可能通过旁分泌HGF实现。⑹细胞实验发现，MSC旁分泌HGF使Akt磷酸化诱导DCreg生成，该DCreg具有较强的吞噬、较弱的刺激淋巴细胞增殖能力、分泌更多IL-10、TGF-β及较少的促炎因子IL-12等。.科学意义.ALI时肺部DCs聚集及成熟与肺损伤程度密切相关，MSC旁分泌HGF诱导ALI时DCreg生成以及调控其迁移为临床ARDS的治疗提供理论依据；同时调控DC的功能为ARDS免疫治疗提供参考依据。