人毛囊干细胞分离培养与鉴定方法的研究  

The study of solated culture and identification method of human hair follicle stem cells

作　　者：姜金豆[1] 胡葵葵[1] 陈容容[1] 夏学颖[1] 孙赛[1] 李丽燕[1] 张碧芳[1] 

机构地区：[1]广东省妇幼保健院医学美容科,广东广州510010

出　　处：《中国美容整形外科杂志》2015年第11期644-647,共4页Chinese Journal of Aesthetic and Plastic Surgery

摘　　要：目的实现毛囊干细胞的高效快速分离。方法将人头部皮肤标本显微分离毛囊隆突区组织由中性蛋白酶消化后，将其直接接种于Matrigel包被的培养板中培养。细胞迁出后，待细胞融合达到60％～70％时，传代培养。取传代培养4周的人毛囊干细胞进行流式细胞学分析及克隆形成能力测定，鉴定毛囊干细胞的表型及增殖潜能。结果分离培养所得毛囊干细胞呈铺路石样生长，细胞胞体较大，细胞核大而明显，具有较高的核质比，干细胞细胞学特征明显。流式细胞仪鉴定毛囊干细胞表面标记物Lgr5阳性率为84．02％，Lgr6阳性率为66．32％。细胞增殖能力及克隆形成能力测定结果显示，毛囊干细胞具有较强的增殖潜能，可由单细胞生成明显的克隆集落。结论本研究通过显微解剖结合消化法，实现了人头皮毛囊干细胞的高效快速分离培养。采用显微分离培养获得毛囊隆突区细胞，细胞表型分析证实其Lgr5及Lgr6阳性干细胞比率、细胞形态、细胞周期及克隆形成能力均符合干细胞特性。Objective Towards efficient expansion of human hair follicle stem cells in vitro. Methods Hair follicles derived from scalp skin were incubated in Dispase. Matrigel coated flasks were designed for culture of HFSCs. HFSC grown from enzymatically digested human hair follicles were passaged after they grew with 60% - 70% conflunce. Phenotype was evaluated by light microscopy and flow cytometry after 4 weeks in culture. The expression of stem cell markers, cell cycle , cell apoptosis and viability were investigated. Results Human hair follicles were cultured for a week, round and phase bright cells with big nucleus migrating from the primary culture of human hair follicle bulge areas. Cell surface marker expression was profiled by flow cytometry and about 84.02% of the sorted cells were positive for Lgr5 expression, 66.32% cells were positive for Lgr6. Grown at clonogenic density showed a high self renewal capacity in colony formation. Conclusion HFSC were efficiently expanded after micro dissection and enzymatically digestion. Round, phase bright human hair follicles cells migrated from the primary culture of human hair follicle bulge areas, cell cycle, cell apoptosis and viability indicate that HFSC represent a subpopulation of cells that are positive for Lgr6, LgrS.

关 键 词：人毛囊干细胞 LGR5 Lgr6 细胞增殖 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]