大鼠表皮基底层干细胞体外分离与培养

Isolation and cultivation of rat epidermal stem cells in basal layer

目的建立简单、可靠的大鼠表皮基底层干细胞体外分离培养方法。方法取新生大鼠背部皮肤,采用胰酶两步消化法获得单细胞悬液,Ⅳ型胶原差速贴壁法富集干细胞,将慢黏附细胞作为对照组细胞,均以角质形成细胞无血清培养基培养。以β1-整合素和角蛋白19(K19)双重荧光染色鉴定细胞表型,克隆形成实验检测细胞体外增殖能力。结果分离培养的细胞为多角形、呈铺路石样排列,倍增时间约为24h,细胞形态和生长规律符合基底层干细胞的特征。免疫荧光鉴定显示细胞共表达β1-整合素和K19,基底层干细胞克隆形成能力显著强于对照细胞。结论两步消化联合Ⅳ型胶原差速贴壁法获取基底层干细胞简易可行,培养的细胞活力好、表型可靠。

Objective To establish a simple and reliable method for isolation and cultivation of epidermal stem cells from neo- natal rat skin basal layer. Methods The single cells were dissociated with twice trypsinization form neonatal rat skin. Thereafter we purified the basal layer stem cells with differential velocity adherent technique with collagen IV, and the slow adherent cells were cultured as negative control cells. Both basal layer stem cells and control cells were cultivated with keratinocyte serum-free medium （K-sfm）. Stem cells were identified with β1-integrin and Keratin 19 by co-immunofluorescence assay, and colony forming assay was executed to evaluate the proliferation potential of stem cells. Results The polygonal cells grew like flagstones, with doubling time of approximately 24 hours. Both the morphology and growth properties of cells were in accordance with the character of basal layer stem cells. Co-immunofluorescence identification showed the cells were positive for the expression of β1-integrin and Keratin 19. Basal layer stem cells had stronger clone forming ability in vitro compare with control group. Conclusion The results indicate that two-procedure trypsinization plus differential velocity adhesion is an ideal method for basal layer stem cells separation followed with vigorous vitality and reliable phenotype.