JAM-A对糖尿病慢性难愈性创面免疫调节的机制研究

Nonhealing wounds of Type Ⅱ diabetes mellitus (DM) have always been a clinical difficulty and research focus. Recent studies have shown that vascular endothelial cell (EC) dysfunction is one of the important mechanisms of that. Our team previously found that junctional adhesion molecule A (JAM-A) was up-regulated in vascular EC from DM wounds, and might positively regulate interleukin-16 (IL-16). Therefore, combined with domestic and foreign researches, we come up with the hypothesis: in type Ⅱ DM, damaged vascular ECs induce overexpression of JAM-A, followed by direct interact with IL-16; up-regulated IL-16 can affect the proliferation and migration of vascular ECs, deteriorating the inflammatory environment and delaying the wound healing. In this proposed study, we will (1) collect wound samples from DM patients to determine whether JAM-A and IL-16 are significantly up-regulated and whether they can combine with each other; (2) increasing or silencing the expression of JAM-A to observe changes of IL-16 and alterations in the proliferation and migration of vascular ECs;(3) Specifically antagonize JAM-A or IL-16 in db/db mouse model to observe whether wound healing is promoted and inflammation is inhibited; (4) comprehensively screen for differentially expressed genes in vascular ECs from DM patients by Gene chips, and construct co-expression networks to explore the underlying mechanisms. This study starts from clinical samples to primary cells, genes, proteins, in vitro layers, in vivo layers steps by steps and attempts to clarify the effects and mechanisms of JAM-A on the functions of vascular ECs in DM wounds, and to provide a new biological target, which is of great theoretical importance and application value.

Ⅱ型糖尿病（DM）创面难愈是临床和基础研究热点，而血管内皮细胞（EC）功能异常是其主要机制之一。我团队前期发现，连接黏附分子A（JAM-A）在DM创面血管EC中上调且正性调控IL-16。结合国内外研究，我们假设：DM创面的血管EC过表达JAM-A，直接结合并上调IL-16，影响血管EC增殖迁移，恶化炎症环境，延迟创面愈合。本研究拟行：①明确DM血管EC的JAM-A和IL-16直接结合并表达上调；②验证过表达/沉默JAM-A，IL-16相应改变，影响血管EC增殖迁移分泌；③证实拮抗JAM-A或IL-16能恢复EC功能，减轻炎症，促进愈合；④基因芯片筛选影响创面愈合的差异基因，构建共表达网络探索潜在机制。本研究从临床标本、原代细胞、基因、蛋白、体外、体内层层深入，全面阐释JAM-A在DM中对血管EC增殖迁移分泌功能的影响及机制，为新药研发提供全新可靠的理论依据，增添创面修复新的药物靶点。

糖尿病创面难愈是临床和基础研究热点，而血管内皮细胞功能异常是其主要机制之一。本研究我们发现连接黏附分子A（JAM-A）在糖尿病患者及小鼠皮肤的血管内皮细胞中低表达，体外高糖环境下培养的血管内皮细胞中JAM-A亦低表达。进一步体外实验发现高糖环境能抑制血管内皮细胞增殖、迁移及粘附功能，并增加血管内皮细胞渗透功能及促炎因子表达，而过表达JAM-A后能逆转高糖环境对血管内皮细胞的功能抑制。RNA-seq测序分析血管内皮细胞过表达JAM-A后差异基因，发现其富集于PI3K-AKT通路。进一步Western Blot验证PI3K-AKT通路关键蛋白表达，发现血管内皮细胞过表达JAM-A后PI3K-AKT通路激活。以PI3K-AKT通路的小分子抑制剂作用于血管内皮细胞，发现其能抑制JAM-A过表达引起的血管内皮细胞功能增强。最后，以过表达JAM-A的慢病毒修复糖尿病小鼠全层皮肤缺损创面，发现JAM-A过表达能加速创面愈合，增加创面新生血管数目，调控创面巨噬细胞向M2型表型转化。本研究系统性探索了JAM-A在糖尿病创面血管内皮细胞中的表达、功能及关键作用通路，为糖尿病创面难愈发生的机制及可能的治疗靶点提供了参考。