Electron microscopy and immunocytochemistry on endothelial progenitor cells during healing process of burn injury in adult rats

血管生成被定义为血管形成间充质细胞转化为内皮祖细胞的新生血管形成过程。尽管最近的研究证实骨髓来源的内皮细胞通过外周血管主要参与成人组织创伤愈合过程中的血管生成，但我们已经报道了位于多个成人组织中的组织成纤维细胞来源的多潜能间充质细胞转化为内皮祖细胞，并参与血管生长的加速。本研究对烧伤愈合过程中内皮祖细胞的超微结构和免疫细胞化学进行了研究。采用深度麻醉成年Wistar大鼠背部皮肤烫伤模型，以10 0℃加热圆柱体照射1min，取伤后组织进行光镜、免疫细胞化学和电子显微镜观察。烧伤后3d，组织成纤维细胞从完整真皮向受损真皮迁移较多。这些细胞偶尔与生长中的毛细血管的内皮衬里有关，或与邻近的毛细血管接触。它们通常形成坚固的细胞索，最终与不断生长的毛细血管接触。由于这些细胞对von Willebrand因子呈免疫反应，并具有大量的Webel-Palade(WP)小体，我们将其定义为EPC的表型。在生长中的毛细血管内皮细胞和内皮祖细胞中可见血管内皮生长因子的免疫反应，而这些血管内皮细胞中的血管内皮生长因子受体阳性，提示内皮细胞产生的血管内皮细胞生长因子通过受体介导的方式激活组织成纤维细胞向内皮祖细胞的转化。此外，在这些内皮祖细胞中可见内皮素1(ET)-1、BIG-1和ET转换酶(ECEs)-1的免疫反应。由于人们普遍认为ET-1和ECE1存在于WP小体中，我们现在认为具有WP小体特征的EPC具有合成ET-1的能力。

Vasculogenesis is defined as a neovascularization process by transformation of vasoformative mesenchymal cells to endothelial progenitor cells (EPCs). Although recent researches insisted that bone marrow-derived EPCs via peripheral blood vessels are mainly involved in vasculogenesis during wound healing process of adult tissues, we have reported that multipotent mesenchymal cells derived from tissue fibroblasts located in several adult tissues transform to EPCs and participate in the acceleration of the vascular growth. This study deals with ultrastructural and immunocytochemical analysis on EPCs during healing process of burn injury. Deeply anesthetized adult Wistar rats received burn injury in their back skin by exposure to heated cylinder for 1 min at 100℃, and then injured tissue were prepared for light microscopic immunocytochemistry and electron microscopy, respectively. At 3 days after receiving burn injury, migration of tissue fibroblasts from intact dermis to injured dermis was often seen. These cells were occasionally associated with endothelial lining of growing capillaries, or in contact with the neighboring ones. They often formed solid cell cords that eventually make contact with the growing capillaries. Since these cells show immunoreactivities for von Willebrand factor and possess many Weibel-Palade (WP) bodies, we defined them as phenotypes of EPCs. Immunoreactivities for VEGF are seen along endothelial lining of growing capillaries and in such EPCs and these for VEGF receptors are detected in the EPCs suggesting that VEGF produced by the endothelial cells activates transformation of tissue fibroblasts to EPCs via receptor mediated fashion. In addition, immunoreactivities for endothelia (ET)-1, big ET-1 and ET converting enzyme (ECE)-1 are seen in such EPCs. Since it is widely accepted that ET-1 and ECE-1 are stored in WP bodies, we consider now that EPCs which are characterized by possessing WP bodies have ability to synthesize ET-1.