电针促进皮肤创伤修复血管新生的PDGF-B/PDGFR-β信号通路调控机制研究

Angiogenesis, including migration buds of endothelial cells, proliferation and recruitment of pericytes, was considerate as pivotal procedure in repair of pathophysiology of skin wound healing, our previous study indicates that electropuncture can improve skin wound healing, wound angiogenesis, migration of endothelial cells, proliferation and recruitment of pericytes, some results hint that all these functions are related with the expression of platelet derived growth factor type B(PDGF-B), but the precise mechanism of electropuncture in skin wound healing still remain unclear. To confirm the function of electropuncture in skin wound healing, angiogenesis and change of pericytes, we first use local skin excision injury mouse model to mimic skin wound healing, mice were treated with optimization of electropuncture parameters, skin wound healing, angiogenesis, function of pericytes and PDGF-B/PDGFR-β cell signal of pericytes were measured after electropuncture in vivo.To confirm the mechanism of electropuncture, pericytes-endothelial cells co-culture cell model will be used in vitro, local microdialysate near puncture point after electropuncture will be added as intervention factor.Combined with in vivo and in vitro, we try to demonstrate that electropuncture can improve skin wound healing, this function may be based on angiogenesis, proliferation and recruitment of pericytes, PDGF-B/PDGFR-β cell signal of pericytes may be involved as precise mechanism

血管新生是皮肤创伤修复病理生理过程中的关键环节，包括内皮细胞的迁移出芽和周细胞的增殖募集等过程，课题组前期研究中发现电针可促进皮肤创伤修复，以及创面血管新生和内皮细胞生存迁移。除此之外，电针还可促进周细胞的增殖和募集，且该作用和电针提高PDGF-B蛋白表达量有关。基于此，本课题拟从体内体外两方面开展研究：体内实验，应用电针干预小鼠局部皮肤切除伤模型，优选电针参数，研究电针对皮肤创伤修复、血管新生及周细胞的影响，以及周细胞PDGF-B/PDGFR-β信号通路在其中的作用。体外实验，首先，采集电针针刺点局部微透析液，模拟电针对针刺局部微环境的改变；其次，用其干预周细胞-内皮细胞共培养模型，然后，研究其干预作用是否与周细胞PDGF-B/PDGFR-β信号通路被激活有关。以探索电针加速创伤修复血管新生，促进周细胞增殖迁移募集的功能，以及周细胞PDGF-B/PDGFR-β信号通路在其中的调控作用。

血管新生是皮肤创伤修复病理生理过程中的关键环节，包括内皮细胞的迁移出芽和周细胞的增殖募集等过程，课题组前期研究中发现电针可促进皮肤创伤修复，以及创面血管新生和内皮细胞生存迁移。除此之外，电针还可促进周细胞的增殖和募集，且该作用和电针提高PDGF-B蛋白表达量有关。本课题研究发现，皮肤创面修复肉眼观察可见电针治疗后伤口收缩较快，渗出液较少，肉芽组织新鲜红润，伤口愈合时间较短，而模型组有大量的血性和脓血分泌液，结痂较硬，愈合时间较长。除造模第1d，电针组在各时间点的创面收缩率明显高于模型组，创面平均愈合时间明显短于模型组。与正常组相比，造模后模型组和电针组创面即刻血流量明显升高，随着愈合修复呈现先降低后上升至正常水平的趋势。模型组干预前后血流无显著差异，电针组针刺后即刻血流明显高于针刺前。HE染色结果发现，电针组的炎性细胞浸润时间、皮肤组织细胞增殖速度和瘢痕修复程度均早于或好于模型组，电镜超微结构可见内皮细胞有丝分裂出芽成管和周细胞增殖稳固管壁的时间明显早于模型组。CD34+免疫组化的结果发现，与正常组相比，除造模1d外，模型组和电针组在各时间点的微血管计数明显增多，在创面愈合过程中呈现先升高后降低的趋势，在造模第7d达到高峰，且电针组微血管增多的倍数明显高于模型组。免疫荧光结果显示，α-SMA+PDGFR-β+双标的周细胞共定位水平与创面组织微血管计数一样呈现先上升后下降的趋势，除造模1d外，电针组在各时间点荧光双标的共定位程度明显高于模型组，在7d时两组的共定位差距达到最大，且电针组α-SMA+PDGFR-β+共定位程度的峰值出现在第7d早于模型组的第10d。皮肤创伤后创面组织PDGF-B、PDGFR-β和ERK1/2的平均荧光强度呈现先升高后降低的趋势，除造模1d外，电针组和模型组的PDGF-B、PDGFR-β和ERK1/2平均荧光强度明显高于正常组，且电针组在各时间点的表达量明显高于模型组。Western Blot结果显示，与模型组相比，抑制剂组创面组织内PDGFR-β和ERK1/2蛋白的磷酸化水平明显降低，而电针+抑制剂组PDGFR-β和ERK1/2蛋白磷酸化水平较抑制剂组显著升高.