Identification of endothelial progenitor cells derived from cultured fibroblasts of the rat dermis

血管发生被定义为血管形成间充质细胞向内皮祖细胞（EPCs）转化的新生血管过程。虽然最近的研究者认为，在成人组织的伤口愈合过程中，骨髓来源的内皮祖细胞主要参与血管生成，但我们已经报道了几种成人组织中来自组织成纤维细胞的多能间充质细胞转化为内皮祖细胞并参与血管生长的加速。本研究对培养的大鼠真皮成纤维细胞内皮祖细胞进行超微结构和免疫细胞化学分析。深度麻醉的成年Wistar大鼠背部皮肤活检，然后在胶原包被细胞培养皿中培养成纤维细胞。将这些细胞置于含5% CO_2、95%空气、37℃的加湿培养箱中，用添加胎牛血清、10ng/ml碱性成纤维细胞生长因子和10ng/ml血管内皮生长因子的培养基中培养。分别对培养的细胞进行光镜免疫细胞化学和电镜观察。这些细胞首次达到单层融合，对血管性血友病因子无免疫反应。几天后，一些培养的细胞具有丰富的细胞质突起，偶尔与邻近的细胞接触，形成实细胞束。由于这些细胞对血管性血液病因子具有免疫反应性，并且具有许多webel - palade （WP）小体，我们将其定义为EPCs的表型。此外，内皮素(ET)-1、大ET-1和ET转化酶(ECE)-1在这些EPCs中具有免疫反应性。由于人们普遍认为ET-1和ECE-1储存在内皮细胞的WP小体中，我们现在认为具有WP小体特征的成纤维细胞来源的EPCs可能已经转化为内皮细胞并具有合成ET-1的能力。少

Vasculogenesis is defined as a neovascularization process by transformation of vasoformative mesenchymal cells to endothelial progenitor cells (EPCs). Although recent researchers 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 analyses on EPCs derived from cultured fibroblasts of the rat dermis. Deeply anesthetized adult Wistar rats received skin-biopsy in their back skin, and then fibroblasts were cultured on collagen-coated cell dishes. These cells were maintained in a humidified incubator containing a gas mixure of 5% CO_2 in 95% air at 37℃, and fed a cultured medium supplemented with fetal bovine serum, 10ng/ml basic fibroblast … More growth factor and 10ng/ml Vascular endothelial groeth factor. These cultured cells were prepared for light microscopic immunocytochemistry and electron microscopy, respectively. These cells, which reached monolayer confluence in the first time, showed no immunoreactivities for von Willebrand factor. After several days, some cultured cells possessed an abundance of cytoplasmic projections and were occasionally in contact with the neighboring ones forming solid cell cords. Since these cells showed immunoreactivities for von Willebrand factor and possessed many Weibel-Palade (WP) bodies, we defined them as phenotypes of EPCs. In addition, immunoreactivities for endothelin (ET)-1, big ET-1 and ET converting enzayme (ECE)-1 were seen in such EPCs. Since it is widely accepted that ET-1 and ECE-1 are stored in WP bodies of endothelial cells, we consider now that fibroblast-derived EPCs which are characterized by possessing WP bodies may be already transformed into endothelial cells and have ability to synthesize ET-1. Less