去分化来源表皮干细胞的分离与鉴定

Isolation,cultivation and identification of dedifferentiation derived epithelial stem cells

目的分离培养和鉴定去分化来源的表皮干细胞。方法采用碱性成纤维细胞生长因子(bFGF)诱导表皮细胞去分化形成表皮干细胞,采用Ⅳ型胶原黏附法分离获得去分化来源的表皮干细胞。观察去分化来源表皮干细胞的形态及增殖特点,并通过免疫细胞化学、免疫荧光及流式细胞技术对其形态和表型进行鉴定。结果去分化来源的表皮干细胞能够表达表皮干细胞及其亚群特异性的表面标志蛋白,如β1整合素、CK19、CK14等,而CK10的表达为阴性。激光共聚焦检测显示α6整合素和CD71在去分化来源表皮干细胞及其亚群中的分布均存在着区域性差异。此外,流式细胞检测结果显示去分化来源的表皮干细胞中主要包含α6+CD71-和α6+CD71+的两种细胞群落,分别占被检测细胞总数的24.52%±7.88%及66.97%±5.04%。结论去分化来源的表皮干细胞与机体自身来源的表皮干细胞在形态和表型上具有相似性,可以作为表皮干细胞的替代细胞应用于皮肤重建与再生的相关研究。

Objective To introduce an efficient method to isolate and identify the dedifferentiation derived epidermal stem cells. Methods HEKa cells obtained from Casacade （USA） were induced to reverse their differentiated process and produce immature, stem-like cells-the dedifferentiation derived epidermal stem cells （dHEK cells）-by basic fibroblasts growth factors （bFGF） in vitro. Improved collagen Ⅳ-coated adhesion method was used to isolate and cultivate the dHEK cells. Subsequently, morphological features and phenotypic changes of these immature dHEK cells were immunochemically stained and studied with eonfocal microscopy and flow FACS array, Results Immunocytochemical analysis proved that dHEK cells and their subunits were positive for 181 integrin, keratin 19 and 14, yet negative for the expression of keratin 10, which was considered as an established marker of differentiated cells. Double staining immunofluorescence demonstrated that markers such as α6 integrin and CD71 were co-expressed in dHEK cells, and it was shown that there was important regional differences in the distribution of α6 integrin and CD71 in dHEK cells and their subpopulations. More importantly, two-color flow cytometric analysis of α6 integrin and CD71 consistently revealed two phenotypically discrete populations of cells in the isolated dHEK cells, i. e a major subpopulation exhibiting high levels of both α6 and CDT1 expression （α6＋CD71＋ representing 66. 97%±5. 04% of the total cells） and a minor population characterized by high level of α6 expression and low level of CD71 expression （α6＋CD71- representing 24. 52%±7. 88% of the total cells） （n=3）. Conclusion The dHEK cells isolated by collagen Ⅳ-coated adhesion method have some characteristics of native epidermal stem cells, and perhaps it may act as a new vehicle for non-genetic manipulation and therapy for both skin disorders and systemic deficiencies. Additionally, although viewed as an ideal substitute of ESCs, the safety of dHEK cells and their functional difference with native epidermal stem cells are still unclear, which need further research.