P75NTR通过调节自噬促进表皮干细胞在糖尿病创面生存的研究

The viability of epidermal stem cells (ESCs) in diabetic wound which is anoxia and ischemic is one of the key points of the research on diabetic wound healing. Neurotrophic factor receptor P75NTR has proven be very important to promote stem cell survival in anoxia and ischemic condition .Our previous studies have found that P75 significantly promoted ESCs biological activity and accelerated wound healing. But P75 expression in diabetic wound was obviously decreased. Recent studies have shown that autophagy is one of the important means for a variety of stem cell remaining active in anoxic conditions. So we put forward the hypothesis that P75 promote ESCs survive in diabetic wound by regulating autophagy and promote wound healing. This study intends to apply molecular imaging techniques to observe the survival of ESCs in diabetic wound , to assess the protection of P75 for ESCs survival in diabetes, and to explore the autophagy pathway. This study will provide new ideas for the basic research of diabetic wound, offer a new target for healing of refractory wounds .

表皮干细胞（ESCs）在糖尿病创面缺血缺氧环境中的生存能力、干细胞特征的保持是研究糖尿病创面愈合的关键点之一，神经营养因子受体P75NTR已被证明缺血缺氧条件下对促进干细胞生存十分重要。我们前期研究发现P75明显促进ESCs生物活性，加速创面愈合，而糖尿病创面P75表达明显降低。最近的研究表明自噬是多种干细胞缺氧条件下保持活性的重要手段之一，因此我们提出P75通过调节自噬促进表皮干细胞在糖尿病创面存活并促进创面愈合。本研究拟应用分子影像学技术观察ESCs在糖尿病创面的生存与转归，评估P75对糖尿病ESCs生存所起的保护作用，深入探讨P75通过自噬促进ESCs存活的通路机制。本研究将为糖尿病创面的基础研究提供新思路，为难愈性创面的修复提供新的靶向机制。

糖尿病创面缺血缺氧等微环境变化对创面修复的种子细胞发挥功能存在影响。自噬是多种干细胞缺氧条件下调控方式之一。P75神经生长因子受体能促进表皮干细胞（ESCs）的生物活性，并且在糖尿病和非糖尿病组织中被发现存在差异表达。本项目构建了P75敲基因小鼠，提取了原代表皮干细胞，通过电子显微镜观察、流式细胞术、CCK8、qPCR、Western blot、免疫荧光等实验，检测了细胞增殖、凋亡、自噬及其信号通路，结果显示：体外实验中在缺氧模型状态下，p75+ ESCs组的增殖水平明显高于p75- ESCs组，p75- ESCs组的凋亡比例高于p75+ ESCs组。Western blot 和qPCR实验发现，与p75+ ESCs组相比，p75- ESCs组Lc3，Beclin1、ULK1，AMPK表达水平更高，证明自噬存在差异，P75可通过AMPK- ULK1调节表皮干细胞的自噬。最后用敲基因鼠建立糖尿病创面模型，发现敲基因组小鼠创面发生延迟愈合。因此，P75是促进表皮干细胞增殖，抵抗凋亡，调节自噬的重要因子之一。本项目初步阐明了P75神经营养因子受体在糖尿病创面愈合中的作用，对于糖尿病创面的理论提供了新的思路，为临床难愈性创面的修复提供了新的治疗靶标。