胎盘干细胞调控巨噬细胞极性改善糖尿病下肢缺血的机制研究

Lower limb ischemic disease has high mortality and morbidity. There is no treatment currently available . Recently, stem cell based therapy has brought breakthroughs for developing effective treatment of this disease, however, the underling molecular mechanism remains unknown. Previous studies have shown that the activation of inflammatory cascade after lower extremity arterial ischemia plays an essential role in tissue injury and/or repair. As major acting cells of this inflammatory response, M1 macrophages have been shown to be involved more in inflammation, while activation of M2 macrophages promote tissue repair. Our previous studies found that: Placenta-Derived Mesenchymal Stem Cells (PMSCs) therapy could decrease macrophage infiltration of muscle tissue after ischemic injury, reduce the release of inflammatory cytokines and promote tissue repair. PMSCs can induce macrophage differentiate to M2 subsets through secretion of PGE2. The recent study also revealed that stem cells can modulate the functions of these immune cells. Based on these exciting findings, we hypothesize that: PMSCs based therapy might be an effective means for the treatment of ischemic lower extremity vascular disease. The molecular mechanisms underlying the beneficial effects of PMSCs treatment may be associated with the regulation of macrophage polarization. Thus, this application is designed to study the effects of lower limb ischemia on activation of macrophage and to elucidate how PMSCs regulate the macrophage subset phenotype following lower limb ischemia. Dissect the possible molecular mechanisms. This study will use in vitro and in vivo experiments. We will also use TALEN mediated genomic editing and lentiviral infection to manipulate expression of target genes. We hope these studies will provide novel evidences for targeting the regulation of macrophage polarization for the treatment of ischemic diseases.

糖尿病血管并发症是糖尿病致死致残的主要原因之一。本课题组前期应用胎盘干细胞（PMSCs）移植治疗，发现PMSCs显著降低下肢缺血动物的致死致残率。我们还发现：PMSCs显著降低小鼠下肢缺血肌肉组织巨噬细胞浸润；在体外共培养体系中，PMSCs分泌PGE2促进巨噬细胞向M2型分化。研究显示缺血后炎症级联反应是损伤的关键因素，而巨噬细胞是炎症反应的主要效应细胞。因此我们推测：PMSCs促进缺血下肢组织修复可能与调控巨噬细胞极性有关。但下肢缺血后巨噬细胞亚群的变化，PMSCs调控机制如何，目前均无相关报道。因此本项旨在:1)通过对组织巨噬细胞表型分析，明确下肢缺血发生后巨噬细胞亚群变化。2)探讨PMSCs是否通过调控巨噬极性，促进糖尿病小鼠下肢缺血组织修复。3)运用TALEN及慢病毒等技术干扰PGE2表达，探讨PMSCs治疗的信号分子机制，为以巨噬细胞为调控靶点的缺血性疾病的治疗提供新的理论依据。

缺血性下肢血管病致死致残率高，干细胞治疗为缺血性下肢血管病带来了新的希望。我们前期研究发现胎盘间充质干细胞（PMSCs）具有多向分化潜能可在缺氧条件下向内皮细胞分化，改善下肢缺血。缺血后期损伤的关键因素之一是炎症连锁反应，巨噬细胞（macrophages）是炎症连锁反应的主要效应细胞，巨噬细胞具有很高的表型可塑性，可分为促炎（M1）或抗炎（M2）亚型。CD4+和CD8+ T细胞是T淋巴细胞的主要亚群，参与这些过程中，但CD4+ T淋巴细胞更加活跃。研究显示CD4+ T淋巴细胞很可能通过调控巨噬细胞的功能，促进血管生成。本研究推测PMSCs调控巨噬细胞M2表型改善下肢缺血的机制与CD4+ T 淋巴细胞有关。本项目通过构建构建C57BL/6小鼠下肢缺血模型，将PMSCs移植到氯膦酸二钠脂质体处理组及未经氯膦酸二钠脂质体处理组的小鼠缺血下肢，并建立磷酸盐缓冲盐水（PBS）对照组。进一步，构建野生型C57BL/6小鼠与BALB/c裸鼠（无T淋巴细胞）下肢缺血模型，同时通过抗CD4抗体建立CD4+ T淋巴细胞缺乏的野生型C57BL/6小鼠下肢缺血模型，将PMSCs移植到小鼠的缺血下肢，同时通过分离野生型C57BL/6小鼠CD4+ T淋巴细胞，静脉移植于BALB/c裸鼠。研究发现PMSCs升高小鼠缺血组织M2型巨噬细胞水平，促进血管新生，增加缺血组织血管密度，改善下肢血流。当巨噬细胞被氯膦酸二钠脂质体耗尽时，C57BL/6小鼠的血管生成受损。通过抗CD4抗体干预CD4+ T淋巴细胞，野生型C57BL/6小鼠血管生成受损。缺血组织中M2巨噬细胞减少，后肢血流恢复延迟，肌肉萎缩/纤维化增加。纯化CD4+ T淋巴细胞输注到PMSCs移植的无胸腺BALB/c裸鼠，增加缺血组织中血流量和M2巨噬细胞的数量。本项目发现PMSCs通过CD4+ T淋巴细胞依赖性调控巨噬细胞M2极性，改善小鼠下肢缺血。