山羊表皮干细胞分离纯化的研究

Isolation and Purification of Goat Epidermal Stem Cells

表皮干细胞(epidermalstemcells,ESCs)干细胞特性的维持是干细胞内在属性和微环境共同作用的结果,体外长期培养ESC的关键是维稳定的微环境。实验将组织块脱出的细胞经分次消化和型胶原快速贴附分离出表皮干细胞,在含20%条件培养基的无血清培养液进行培养。原代细胞表现K19阳性,并且能形成PGC集落样细胞团。从表皮和真皮组织中均可得到上皮样细胞,经纯化后均表现出明显的表皮干细胞特性:呈片状生长,铺路石样形态。将两类细胞分别传至5代和8代,后代细胞β1整合素染色呈阳性。对5代和8代细胞分别进行克隆分析实验,其克隆形成率分别为18.5%和8.5%。实验证明组织块法能够得到较好的维持ESC的微环境,所分离的ESC具有干细胞特性并能稳定传代。

Stemness of epidermal stem cells(ESCs) is the result of the combination of intrinsic characteristics and extrinsic microenvironment. The key point of in vitro culturing ESC in long term is maintaining the elements of microenvironment. This experiment isolates ESC from the cells originating from skin explant cultures through respective trypsinization and type collagen rapid adhering. And ESCs are cultured in serum-free medium containing 20% conditioned medium. The original cells show K19 positive immunofluorescence staining, and form PGCs colony-like cell groups. Cells originating from both epidermis and dermal tissue show typical characteristics, colony forming and road metal-like shape. They are respectively passaged to 5 and 8 passage. Passaged cells showβ1 integrin positive immunofluorescence staining. The colony-forming efficiency (CFE) of passage 5 and passage 8 are 18.5% and 8.5%. All these results demonstrate that skin explant culturing method can maintain the microenvironment of ESC, and the harvested ESC show characteristics of stemness and can be stably passaged.