高糖诱导蛋白激酶PKCδ磷酸化整合素α2介导糖尿病创面血管再生障碍的机制研究

Clinically, the most challenging wounds to manage are chronic wounds such as diabetic foot ulcers, which represent a significant medical burden. The changing demographics of an aging population and the prevalence of diabetes mellitus have increased the incidence of these wounds in recent years. Such wounds are biochemically complex and fail to progress normally through the stages of healing..Therefore, chronic skin wounds healing of diabetes mellitus becomes one of the key scientific issues in clinical treatment and basic research. Pulsed radio frequency energy (PRFE) is a new therapeutic method based on electromagnetic signals. Despite promotion of fibroblast proliferation and collagen-I synthesis in diabetic mice, our previous studies indicate that PRFE has no significant effect on angiogenesis in skin wounds. A number of literatures have suggested that collagen-I can promote angiogenesis via integrin α1β1-α2β1 polymer under physiological conditions, but its mechanism of action under diabetic pathological conditions remains unclear. In this study, we established a high-glycemic model of endothelial cells and an animal model of diabetes to verify the mechanism by which PKCδ mediates diabetic skin wound angiogenesis disorder via integrin-α2 phosphorylation from multiple perspectives in vivo and in vitro. The penetrating intervention peptide drug, named TAT-PKCδ/integrin-α2, which was targeted inhibition of PKCδ phosphorylated integrin-α2 protein on T1177 site. The purpose of this study is to evaluate the effects of TAT-PKCδ/integrin-α2 combined with PREF on accelerating the healing of diabetic wounds, providing a new method and theoretical basis for clinical treatment of diabetic wounds.

随人口老龄化及社会经济发展，我国糖尿病发病率与日俱增。由此导致的糖尿病慢性创面严重影响患者生存质量并带来沉重医疗负担，已成为临床研究热点之一。课题组前期研究发现脉冲射频能量治疗可通过促进糖尿病小鼠皮肤创面成纤维细胞增殖、Ⅰ型胶原蛋白合成而改善愈合，但血管再生却并无明显增加。既往研究显示生理条件下I型胶原可通过整合素α1β1-α2β1聚合体促进血管再生，但其在糖尿病病理条件下的作用机理目前尚不清楚。本研究拟通过建立血管内皮细胞高糖模型及糖尿病动物模型，从体内外多角度验证蛋白激酶PKCδ通过磷酸化整合素α2（integrin-α2）介导糖尿病皮肤创口血管再生障碍的机制，并观察靶向PKCδ磷酸化整合素α2蛋白T1177位点穿膜干预肽TAT-PKCδ/integrin-α2促进新生血管形成作用，最后评估干预肽与脉冲射频二联疗法促进糖尿病创面愈合的动物试验效果，为改善其临床疗效提供理论依据和新策略。

背景：糖尿病皮肤溃疡发病率高，且皮肤创面难以治疗。人皮肤成纤维细胞（human dermal fibroblasts, HDF）、人真皮微血管内皮细胞（human dermal microvascular endothelial cells, HDMEC）和人表皮角质形成细胞（human epidermal keratinocytes, HEK）是与糖尿病伤口愈合密切相关的三种细胞，我们用高糖刺激HDF、HDMEC和HEK来模拟糖尿病模型，并对样品进行转录组学研究。我们发现高糖刺激下HEK组角蛋白17（keratin 17，KRT17）表达显著上调，并激活PKCδ，而HDF和HDEMC组在两种条件下KRT17未检测到表达。.目的：明确糖尿病病理条件下HEK表达增多的角蛋白 17（KRT17）对血管内皮细胞生物学作用的影响，包括细胞增殖、迁移以及血管形成，研究 KRT17 在糖尿病创面愈合中的作用及机制。.方法：首先对前期研究中发现高糖刺激下人皮肤三种主要细胞，以及糖尿病动物模型中的 KRT17mRNA 的表达增多进行了验证；随后建立体外不同浓度的 KRT17刺激内皮细胞模型，检测内皮细胞增殖、迁移的变化，并进行了转录组测序和生信分析。筛选下游关键分子，验证结果并通过干预下游分子影响糖尿病小鼠创面愈合。.结果：高糖刺激HEK细胞中 KRT17 表达上调，并激活PKCδ；糖尿病小鼠以及糖尿病患者皮肤组织中 KRT17 表达上调；外源KRT17 体外刺激内皮细胞后抑制细胞成管，但增殖和迁移无影响；KRT17 刺激内皮细胞后的 RNA-seq提示调节细胞成管的关键分子GNB3和MEF2C显著升高；体外干预KRT17抑制GNB3和MEF2C表达，促进糖尿病小鼠创面血管生成，促进创面愈合。 .结论：糖尿病病理条件下，皮肤角质形成细胞表达 KRT17 增加，通过旁分泌作用于血管内皮细胞，激活pkcδ/GNB3-MEF2C信号通路，抑制创面血管生成，延缓伤口愈合。在糖尿病创面中干扰KRT17的表达促进创面血管生成，促进伤口愈合。高糖病理下表达增多的 KRT17 可以通过调节皮肤细胞影响皮肤功能，尤其影响创面愈合。KRT17 可成为糖尿病创面愈合治疗的分子靶点。