M2型巨噬细胞外泌体转运miR135α-5p调控HIF1AN/HIF-1α通路促进跨区穿支皮瓣血管新生的机制研究

One-stage reconstruction of the very large soft tissue defect which is caused by high-energy trauma always is a challenging problem for the reconstructive surgeons. The multi-territory perforator flap was an ideal method for the reconstruction of complex soft tissue defect. However, partial flap loss due to inadequate blood flow in this flap often results in multiple operations and even surgery failure. Thus, improving the angiogenesis in the multi-territory perforator flap to increase the survival area of this flap is the critical role to successfully reconstruct the very large soft tissue defect. There is growing evidence that M2 macrophages have a significant impact on the process of angiogenesis in the flap, and recent emerging researches demonstrate that exosomes derived from Macrophage could be effectively up-taken by vascular endothelial cells and regulated its function. However, the effect of exosomes derived from M2 macrophage (M2-Exo) on angiogenesis in the multi-territory perforator flap and its potential mechanism still have not been elucidated. Our primary date showed that M2-Exo enriched miR135α-5p, and bioinformatics analysis also showed that HIF1AN is a target gene of miR135α-5p. Previously our studies also have identified that HIF-1α as a downstream molecule of HIF1AN could up-regulate expression of VEGF and SDF-1 to promote angiogenesis. Based on these data we hypothesize that M2-Exo shuttled miR135α-5p to promote angiogenesis in the multi-territory perforator flap by regulating HIF1AN/HIF-1α signaling pathway to induce the expression of VEGF and SDF-1. This study proposed to reveal the effect of M2-Exo on angiogenesis in the multi-territory perforator flap, and identity that miR135α-5p is shuttled by M2-Exo plays an important role in the process of angiogenesis; Furthermore, we will focus on identifying the consequences of manipulating miR135α-5p expression on key downstream signaling pathways including HIF1AN/HIF-1α signaling pathway using luciferase gene reporter experiment and gene silencing strategy. Additionally, we will further demonstrate that optimized M2-Exo function using over-expressing miRNA strategy for angiogenesis in the multi-territory perforator flap. We anticipate these studies will provide a new strategy for clinically improving the survival area of multi-territory perforator flaps and identify new therapeutic targets.

如何促进跨区穿支皮瓣血管新生进而实现皮瓣高质量成活是临床棘手难题。研究表明巨噬细胞外泌体可被血管内皮细胞有效吞噬并发挥调控作用，而M2型巨噬细胞在皮瓣血管新生过程中扮演关键角色，但M2巨噬细胞外泌体（M2-Exo）对跨区穿支皮瓣血管新生的作用及机制尚不明确。我们预实验发现M2-Exo富集miR135α-5p，生物信息学分析显示HIF1AN为miR135α-5p靶基因；前期研究表明HIF-1α作为HIF1AN下游分子可上调VEGF、SDF-1促血管新生。故推测“M2-Exo介导miR135α-5p调控HIF1AN/HIF-1α通路上调VEGF、SDF-1促进跨区穿支皮瓣血管新生”。本研究拟采用基因沉默等手段，揭示miR135α-5p在M2-Exo促血管新生过程中的作用及靶向机制；并用过表达miRNA策略进一步优化M2-Exo并验证其效果。旨在为临床探索出一种促进跨区穿支皮瓣血管新生的新策略。

如何促进跨区穿支皮瓣血管新生进而实现皮瓣高质量存活是临床棘手难题。研究表明巨噬细胞外泌体可被血管内皮细胞有效吞噬并发挥调控作用，并且M2型巨噬细胞在皮瓣血管新生过程中扮演关键角色，但M2巨噬细胞外泌体（M2-Exo）对跨区穿支皮瓣血管新生的作用及机制尚不明确。本项目是采用M2-Exo改善跨区穿支皮瓣局部坏死的原创性研究。为此，本项目率先通过微血管皮窗直视技术动态观测M2-Exo对跨区穿支皮瓣内的choke vessels的改变；与此同时，课题组运用分子生物学、细胞生物学、生物信息学等前沿技术手段阐明了M2-Exo促进跨区穿支皮瓣血管新生的机制；本课题率先证实运载有特定miRNA的M2-Exo具有促进跨区穿支皮瓣血管新生的能力这一创新性理论。首次深入明确M2-Exo具有对跨区穿支皮瓣内的choke vessels转变为真性吻合血管与毛细血管新生的能力；并首次证实M2-Exo通过运载miR135α-5p调控HIF1AN/HIF-1α信号轴诱导VEGF表达进而改善跨区穿支皮瓣内的血管新生。本课题的成功实施将为穿支皮瓣手术提供具有自主知识产权的外泌体治疗新策略。这项研究还将提供新的视角探讨M2-Exo促进跨区穿支皮瓣内血管新生的机制，有望为促进跨区穿支皮瓣血管新生提供新靶点和思路，为临床构建可靠的跨区穿支皮瓣提供一种新策略，具有潜在的临床应用价值。