PDF(Pubmed)
Abstract：
Diabetes often results in chronic ulcers that fail to heal. Effective treatment for diabetic wounds has not been achieved, although stem-cell-treatment has shown promise. Hair-follicle-associated-pluripotent (HAP)-stem-cells from bulge area of mouse hair follicle have been shown to differentiate into keratinocytes, vascular endothelial cells, smooth muscle cells, and some other types of cells. In the present study, we developed HAP-cell-sheets to determine their effects on wound healing in type-2 diabetes mellitus (db/db) C57BL/6 mouse model. Flow cytometry analysis showed cytokeratin 15 expression in 64% of cells and macrophage expression in 3.6% of cells in HAP-cell-sheets. A scratch cell migration assay in vitro showed the ability of fibroblasts to migrate and proliferate was enhanced when co-cultured with HAP-cell-sheets. To investigate in vivo effects of the HAP-cell-sheets, they were implanted into 10 mm circular full-thickness resection wounds made on the back of db/db mice. Wound closure was facilitated in the implanted group until day 16. The thickness of epithelium and granulation tissue volume at day 7 were significantly increased by the implantation. CD68 positive area and TGF-β1 positive area were significantly increased; meanwhile, iNOS positive area was reduced at day 7 in the HAP-cell-sheets implanted group. After 21 days, CD68 positive areas in the implanted group were reduced to under the control group level, and TGF-β1 positive area had no difference between the two groups. These observations strongly suggest that the HAP-cell-sheets implantation is efficient to facilitate early macrophage activity and to suppress inflammation level. Using immuno-double-staining against CD34 and α-SMA, we found more vigorous angiogenesis in the implanted wound tissue. The present results suggest autologous HAP-cell-sheets can be used to heal refractory diabetic ulcers and have clinical promise.
摘要：
糖尿病通常导致无法治愈的慢性溃疡。尚未实现对糖尿病伤口的有效治疗，尽管干细胞治疗已经显示出了希望。毛囊相关的多能（HAP）干细胞从小鼠毛囊的凸起区域已被证明分化成角质形成细胞，血管内皮细胞,平滑肌细胞，和一些其他类型的细胞。在本研究中,在2型糖尿病(db/db)C57BL/6小鼠模型中,我们开发了HAP细胞片,以确定其对伤口愈合的影响.流式细胞术分析显示,在HAP-细胞片中,64%的细胞中表达细胞角蛋白15,在3.6%的细胞中表达巨噬细胞。体外划痕细胞迁移试验显示，与HAP细胞片共培养时，成纤维细胞的迁移和增殖能力得到增强。为了研究HAP细胞片的体内作用，将它们植入db/db小鼠背部的10毫米圆形全层切除伤口中。在植入组中促进伤口闭合直到第16天。通过植入，第7天的上皮厚度和肉芽组织体积显着增加。CD68阳性面积和TGF-β1阳性面积显著增加;在HAP细胞片植入组中，iNOS阳性面积在第7天减少。21天后,植入组CD68阳性面积降低至对照组水平以下,TGF-β1阳性面积两组间无差异。这些观察结果强烈表明，HAP-细胞片植入可有效促进早期巨噬细胞活性并抑制炎症水平。使用针对CD34和α-SMA的免疫双重染色，我们在植入的伤口组织中发现了更强烈的血管生成。目前的结果表明，自体HAP细胞片可用于治愈难治性糖尿病溃疡，并具有临床前景。
