改良法分离培养人牙周膜干细胞

Isolation and identification of human periodontal ligament stem cells in vitro

目的改良法体外分离培养人牙周膜干细胞(PDLSC),并进行鉴定。方法采用酶消化组织块法获得人牙周膜细胞,通过有限稀释法克隆化培养、分离得到PDLSC,用含10%FBS的α-MEM培养液培养并传代;测定克隆形成率;免疫组织化学检测角蛋白及波形蛋白表达;流式细胞术分析细胞周期及表面标志物STRO-1、CD146的表达;并进行成骨、成脂诱导实验。结果本研究所得细胞有较强的克隆形成能力,波形蛋白染色阳性,角蛋白阴性,细胞周期分析大多细胞处于G0/G1期,STRO-1及CD146高表达,成骨、成脂诱导实验证明所得细胞有多向分化能力。结论改良法克隆化培养可成功分离得到PDLSC,为进一步研究PDLSC奠定了基础。

Objective To isolate and identify human periodontal ligament stem cells（PDLSC） by improved methods and assess the characteristics of PDLSC ex vivo.Methods The periodontal ligament cells were obtained from the healthy impacted third molars and teeth extracted for orthodontic purposes and used to isolate PDLSC by limiting dilution assay.PDLSC were cultured and expanded in α-MEM supplemented with 10% FBS.Colony-forming assay,immunohistochemistry,flow cytometry,osteogenic and adipogenic induction were used to identify PDLSC.Results The obtained cells had high colony-forming efficiency and were positive staining for vimentin and negative for pan-cytokeratin.Flow cytometry revealed that the isolated cells were positive for STRO-1 and CD146 antibodies and most were in the G0/G1 phase of cell cycle.Under specific conditions,they could differentiate to the osteoblast and adipocyte lineages in vitro.Conclusion Limiting dilution assay is an effective method to isolate PDLSC and the single-cell-derived colonies demonstrate the properties of stem cells in vitro.