人毛囊干细胞的分离培养鉴定与生物学特性的初步研究

Biological Characteristics study of huaman hair follicle stem cells

目的探讨人毛囊干细胞分离获取的方法及其相关生物学特性,作为组织工程研究的种子细胞的可行性。方法整形美容手术中废弃的人头皮组织,通过单细胞培养法和组织块培养法获得人毛囊干细胞,体外传代培养及生物学特性研究:细胞形态学观察,细胞生长曲线测定,免疫组化染色鉴定与流式细胞仪检测其细胞表型,将HFSCs进行成骨,成脂肪,成软骨多向分化诱导检测。结果 HFSCs生长旺盛,呈成纤维细胞样生长,多角形或梭形;免疫组化染色显示K19表达阳性,流式细胞仪检测显示:K15,K19,CD200和整合素β1为阳性,CD31,CD45,HLA-DR为阴性;成骨诱导von Kossa染色显示钙化基质沉积表达;成脂肪诱导油红-O染色阳性,胞浆内见脂滴形成;成软骨诱导细胞聚集成小团块,Alcian Blue染色显示硫酸蛋白聚糖表达阳性。结论 HFSCs体外生长能力旺盛,高表达干细胞相关抗原,具有向软骨,骨,脂肪多向分化潜能。

Objective To investigate the separation, cultivation and identification of human hair follicle stem cells, and to observe biological characteristics of HFSCs. Methods The HFSCs were digested with Trypsin, cultured and subcultured in vitro. Growth curves and cumulative population doubling were determined to evaluate the proliferation potential of HFSCs. The phenotypes of HFSCs were identified by immunohistochemistry and flow cytometry. The multilineage potential of HFSCs was confirmed by inducing them into osteogenic, chondrogenic, and adipogenic differentiation. Results The results of flow cytometry and immunocytochemistry showed tbat the expressions of stem cell-related antigens （ K15 ,K19, CD200, ntegrinjl l ） were positive, and those of hematopoiesis-related antigens （CD31） and other antigens （CD45, HLA-DR） were negative. The calcified nodules could be observed by Von Kossa staining in osteogenic HFSCs. The intracellular lipid droplets could be observed by Oil Red staining in adipogenic HFSCs. Cells aggregation and glycosaminoglycan could be detected by Alcian Blue staining in chondrogenic HFSCs. Conclusion HFSCs can be abundantly harvested and have stable proliferation in vitro. HFSCs have multi-lineage potential and can be the suitable seed cells for tissue engineering.