大鼠真皮多能干细胞分离培养及其多分化潜能特性

Culture and pluripotency of skin-derived precursors isolate from neonatal rat dermis

目的建立分离培养大鼠真皮来源多能干细胞(SKPs)的方法并探讨其生物学特性。方法0.25%中性蛋白酶(protease)选择性的消化表皮与真皮的细胞连接,剥离真皮,采用0.25%的胰蛋白酶消化真皮层细胞于超低黏附培养板中进行悬浮无血清培养,机械法传代,免疫荧光染色法检测细胞表面分子的表达,体外诱导其向脂肪细胞分化,研究其多分化潜能。结果体外培养2～3d后真皮多能干细胞聚集成球悬浮生长,培养4代后去除生长因子后并添加血清培养,细胞帖壁生长,呈长梭形成纤维样,免疫荧光染色显示,体外培养的真皮多能干细胞可表达CD29和Nes- tin,不表达CD34与vimentin。体外诱导实验结果显示体外培养的真皮多能干细胞能够被诱导成脂肪细胞,油红染色阳性。结论采用悬浮培养方法可成功分离培养大鼠真皮多能干细胞,培养的细胞可表达神经干细胞标志并具有多分化潜能。

Objective To isolate muhipotent skin-derived precursors（SKPs） from adult rat dermis and study their functional properties. Methods Dermis of neonatal rat were digested with 0.25% trypsin and suspension culture in ultra-low cluster flat bottom with serum-free medium after protease selectively digested the dermo-epidermal junctions. A mechanical method was chosen to passage these cells and they were identified by the immunofluorescence staining. The cells were induced to differentiate into adipocytes. Results Floating spheres appeared in the plate 2-3 days later. After 4 times passaging, SKPs could adhere if cultured in the medium containing serum but not growth factor. The morphology of SKPs changed into longer shape, and showed fibroblast-like shape. Immunofluorescence staining revealed that these cells were positive for nestin and CD29 ,yet negative for CD34 and vimentin expression. The positive of Oil Red staining was demonstrated that SKPs could differentiate into adipocytes. Conclusion SKPs that expressed nerve stem cells markers could be isolated from neonatal rat dermis successfully by suspension culture.