源于人躯干皮肤真皮的成纤维样细胞的间充质干细胞分化潜能

Potential to differentiation of human dermis-derived fibroblast-like cells into mesenchymal stem cells in vitro

目的:研究从人皮肤真皮组织中分离培养的成纤维样细胞向中胚层细胞分化的潜能,初步探讨其替代自体骨髓间充质干细胞(BMSCs)用于组织损伤和缺损修复的可行性。方法:将一定大小的成人腹部全层皮肤组织分别用分散酶和Ⅰ型胶原酶消化,分离获得真皮来源总细胞。单层培养扩增的细胞在含有一定浓度N2、B27、碱性成纤维细胞生长因子(bFGF)和表皮细胞生长因子(EGF)的无血清培养液中进行悬浮培养,并收集培养液中悬浮存在的球状细胞聚集体(SDDSs),用含胎牛血清的培养液进一步单层扩增细胞。以免疫细胞荧光法分析细胞和鉴定细胞的分化特性。结果:真皮中分离的一些单个细胞在无血清培养液中分裂增殖形成SDDSs;在含血清的单层培养条件下,从SDDSs分离培养的细胞增殖活跃,细胞表达间充质干细胞标志物CD90、CD105和波形蛋白;分别向骨细胞、软骨细胞以及脂肪细胞诱导分化后,呈现类似于BMSCs分化特点,分化的细胞呈茜素红、番红O和油红O特殊染色阳性反应,但真皮细胞分化形成的软骨细胞番红O染色弱于骨髓干细胞来源的软骨细胞。结论:结合无血清培养和有血清培养法,可从人真皮组织中分离培养具有向软骨细胞、骨细胞和脂肪细胞分化潜能的细胞,此细胞将有望替代BMSCs用于临床组织损伤修复的再生治疗。

Objective.. To study the differentiation capacity of the fibroblast-like cells isolated from human skin dermis into mesenchymal stem cells, and to explore the feasibility to use these cells as alternative cell source of autologus bone marrow mesenchymal stem cells （BMSCs） for regeneration of tissue injuries and defects. Methods.. Full thickness skin samples were obtained from the abdomen of surgical patients, then digested with dispase and collagenase t subsequently. Thereafter, the digested cells were collected and cultured, followed by suspension with serum free medium containing N2, B27, basic fibroblast growth factor （bFGF）, and epidermal growth factor （EGF）. The skin dermis derived spheroids （SDDSs） were collected and monolayer cultured in serum-containing medium. Finally, the cells were characterized by immunofluorescence staining and differentiation assays. Results： The dermis derived cells proliferated and formed SDDSs in the suspension of serum-free medium. After monolayer cultivation in serum-containing medium, the cells from spheroids were successfully expanded to large number. The cells expressed mesenchymal stem cells markers CD90, CD105 and vimentin. Under osteogenic, chondrogenic and adipogenic differentiation conditions, these cells were differentiated into the alizarin red, safranin O, and oil red 0 staining positive cells, displayed similar differentiation traits with BMSCs. However, safranin 0 staining was weaker in the dermis derived cells than BMSCs. Oonclusion： A kind of fibroblast-like cells exist in human skin dermis, and have osteocytic, chondrogenic and adipogenic differentiation potentials, demonstrating that these cells will be utilized as a novel cell source for repairing the tissue injury and defect in clinic.