PEDF调节内皮祖细胞抑制糖尿病创伤愈合的作用及机制

Impaired neovascularization is the major risk factor for diabetic wound healing， endothelial progenitor cells(EPCs) play vital role in vasculogenesis of wound healing. Recent studies have reported that the plasma pigment epithelium-derived factor(PEDF) of type I and type II diabetic patients was significantly elevated, while circulating EPCs numbers decreased . Our previous study found that PEDF induced endothelial cell apoptosis, whereas the effect of PEDF on EPCs has less reports. Our preliminary data showed that intraperitoneal injection of PEDF significantly slowed down the wound healing process and reduced the circulating EPCs numbers in normal mice. Thus, we speculate that high PEDF level in case of diabetes decreases peripheral neovascularization and impairs wound healing by down-regulating EPCs. This study was aimed to illustrate the role and mechanism of PEDF in diabetic would healing. Firstly, we will investigate whether PEDF in serum and the main metabolic organs were increased in diabetic wound healing mice model, then the possibility of improving wound healing when antagonizing PEDF. Secondly, we will identify the effects and the underlying mechanism of PEDF on the mobilization, homing and differentiation of EPCs. Finally, we will elucidate how PEDF expression is regualted by high glucose and AGEs. Overall, this study will elucidate the possible reason of neovascularization impairment in diabetic foot ulcer and identify the key molecules, therefore provides novel target molecules for the treatment of diabetic foot ulcer.

血管新生受阻是糖尿病创伤愈合的主要危险因素，内皮祖细胞（EPC）在糖尿病创伤愈合血管新生中起重要作用。最近报道 I 型、II 型糖尿病患者血中色素上皮衍生因子（PEDF）显著上调，EPC减少，我们以往研究发现 PEDF 诱导血管内皮细胞凋亡，但PEDF对EPC的调节尚待深入研究。预实验结果表明腹腔注射PEDF显著减缓小鼠的伤口愈合并降低血中EPC,由此我们的科学假设是糖尿病情况下上调的PEDF通过抑制EPC阻碍血管新生、抑制创伤愈合。本研究拟设计下列内容：①建立糖尿病创伤动物模型，检测血清和主要代谢器官中PEDF是否高表达及创伤愈合功能变化，拮抗PEDF可否改善创伤愈合；②明确PEDF抑制EPC释放、伤口归巢及向内皮细胞分化等功能的作用和机制；③观察高糖及其终产物上调PEDF 表达的作用及机制。旨在明确糖尿病创伤修复过程中血管新生能力下降的原因和关键调控分子，为治疗糖尿病足提供干预新靶点。

血管新生受阻是影响糖尿病创伤愈合的主要危险因素，内皮祖细胞在糖尿病创伤愈合血管生成中起重要作用。色素上皮衍生因子（PEDF）是目前发现的活性较强的血管新生抑制因子。包括我们在内的研究表明PEDF通过诱导血管内皮细胞（ECs）凋亡抑制肿瘤和视网膜血管新生，但PEDF对EPCs的调控尚未见文献报道。最近证实I 型、II 型糖尿病患者血浆PEDF显著上调，全血EPCs数量减少，功能下降，但原因尚不明确。本项目旨在探讨糖尿病创伤修复过程中血管新生能力下降的原因并证明PEDF是介导血管生成抑制的关键分子；明确PEDF是否通过减少EPCs数量、抑制EPCs功能阻碍血管新生从而抑制糖尿病创伤愈合并阐明其机制。为此本项目设计相关内容并取得以下重要结果：（1）临床资料提示合并糖尿病足的糖尿病人血浆PEDF含量明显增高，全血EPCs数量明显降低。相关性分析结果提示PEDF含量和EPCs数量存在负相关。（2）高脂饮食联合STZ诱导的糖尿病小鼠和db/db小鼠血浆PEDF含量显著升高。（3）PEDF中和抗体和PEDF基因敲除促进糖尿病小鼠伤口愈合，外源注射PEDF重组蛋白减缓C57BL/6小鼠伤口愈合。（4）PEDF抑制伤口处血管新生，PEDF单克隆抗体增加伤口处血管新生。同时多普勒血流检测仪和H&E染色的结果显示PEDF基因敲除使小鼠皮肤血流加快，皮肤厚度增加。（5）PEDF降低全血EPCs的数量，PEDF单克隆抗体和PEDF基因敲除显著增加糖尿病小鼠全血中EPCs的数量。（6）PEDF使EPCs的功能受损，PEDF抗体和PEDF基因敲除显著改善糖尿病小鼠EPCs的功能。（7）PEDF抑制EPCs的动员和募集能力。（8）PEDF通过抑制Wnt信号通路影响伤口愈合过程。（9）Wnt通路下游的激动剂改善糖尿病高水平PEDF导致的伤口愈合受损。该项目首次证明合并糖尿病足病人血浆PEDF水平上调，全血中EPCs数量下降。体内试验证明PEDF通过阻断Wnt信号通路下调VEGF和SDF-1的表达，从而抑制EPCs的动员和募集过程最终影响EPCs的数量和功能，使创伤愈合过程中血管新生受抑制。本研究为寻找糖尿病足干预新靶点提供科学依据。