人牙周膜干细胞分离培养及生物学特性探究.docVIP

人牙周膜干细胞分离培养及生物学特性探究[摘要]目的：分离筛选人牙周膜干细胞，研究其生物学特性并进行初步鉴定。方法：采用STRO-1为标记物以免疫磁珠分离筛选人牙周膜干细胞，观察细胞生长及克隆形成情况，流式细胞仪细胞周期、细胞表型分析，免疫细胞化学染色技术检测STRO-1、Vimentin表达，并测定细胞体外多向分化能力。结果：免疫磁珠法可获得人牙周膜干细胞，细胞具有克隆形成能力，增殖速度低。细胞周期分析大多数细胞处于G0/G1期,为慢周期性;细胞表型分析证实CD146、CD44高表达，CD34、CD45低表达。STRO-1及Vimentin均为阳性染色，矿化诱导和成脂诱导证实该细胞具有多向分化能力。结论：免疫磁珠法是有效的分离纯化牙周膜干细胞的方法。所分离细胞具有干细胞的细胞周期、表型特点及多向分化能力。 [关键词]干细胞；人牙周膜干细胞；免疫磁珠；流式细胞计量术；免疫细胞化学 [中图分类号]R783.5 [文献标识码]A [文章编号]1008-6455（2008）05-04 Isolation, identification and bionomics of human periodontal ligament stem cells PAN Feng1,DING Yin1,WANG Guang1,ZHAO Yu2,NI Hua2 (1.Department of Orthodontics,Stomatology Hospital,The Fourth Military Medical University,Xi’an 710032,Shaanxi,China; 2.Center of Tissue Engineering, Stomatology Hospital,The Fourth Military Medical University) Abstract:ObjectiveTo isolate and identify the periodontal ligament stem cells and demonstrate its bionomics. MethodsPDLSCs were isolated by immunomagnetic method. The flow cytometry and immunocytochemistry procedure were used to disclose the cell cycle and surface marker of the PDLSCs.Osteoinduction and adipoinduction were done to conform the multidirectional differentiation of PDLSCs. ResultsThe acquired cells had clonality and low proliferation. Most of the cells were in phase G0 /G1 and there were high expression of CD146 and CD44 on these cells, meanwhile, CD34 and CD45 were of low expression. The cells were Vimentin and STRO-1 positive while their multidirectional differentiation ability was confirmed in vitro. Conclusionimmunomagnetic method was an effective way to isolate and purify the PDLSCs. The acquired cells showed the characteristics of stem cells. Key words:stem cell;human periodontal ligament stem cell;immunomagnetic beads;flow cytometry;immunocytochemistry 研究证实牙周膜内存在具有横向分化能力的干细胞(牙周膜干细胞)，在体外微环境作用下还可分化为成牙骨质细胞或成骨细胞[1-2]。因此，牙周膜干细胞（PDLSCs）作为一种新发现的干细胞，在牙周组织工程研究中有较大的应用前景。但是牙周膜组织量小、分离困难，一直成为制约牙周膜干细胞研究的重要原因。已有的克隆筛选法操作程序繁琐，周期较长[1]，因此需要寻找简便、快捷的筛选方法来分离牙周膜干细胞。本研究应用免疫磁珠