两种培养条件对牙周膜干细胞生物学特性影响的对比研究  

Effects of two kinds culture conditions on the biological characteristics of periodontal ligament stem cells

作　　者：王飞翔[1] 贺慧霞[1] 刘嘉婧[2] 贾雅丽[2] 

机构地区：[1]解放军总医院口腔科,硕士生北京100853 [2]军事医学科学院野战输血医学研究所干细胞与再生医学研究室,北京100850

出　　处：《中华老年口腔医学杂志》2016年第2期65-71,共7页Chinese Journal of Geriatric Dentistry

基　　金：全军后勤科研计划重大项目(项目编号:AWS14C007);国家高技术研究发展计划(863计划)(课题编号:2015AA033502);国家自然科学基金(项目编号:81070833)

摘　　要：目的:对比无血清培养基和含血清培养基对原代培养、分离的人牙周膜干细胞(Human periodontal ligament stem cells,hPDLSCs)生物学特性的影响,为基于hPDLSCs的临床应用研究和药物试验奠定基础。方法:分别用含血清和无血清培养基培养分离hPDLSCs,倒置显微镜下观察细胞形态,CCK-8检测细胞增殖能力;流式细胞仪分析细胞表型;免疫荧光染色、real-time PCR检测PDLSCs成骨、成脂分化诱导后相关特异性蛋白及基因表达。结果:采用无血清与含血清培养条件原代培养分离的h PDLSCs形态相似;但无血清培养基组细胞增殖力更强;两种培养条件培养的hPDLSCs表型相似,均表达CD105、CD44、CD166、CD73和CD90,不表达CD45、CD3、CD14、CD38、HLA-DR、CD31和CD34;且阳性表达vimentin、α-SMA和N-cadherin,阴性表达CK18和E-cadherin。无血清培养条件培养的hPDLSCs具有成骨、成脂潜能,且诱导效能强于含血清培养者。结论:无血清培养基能够在体外培养扩增hPDLSCs,并维持其干细胞特性。采用无血清培养条件培养hPDLSCs可能更适合其在细胞治疗和实验研究中应用。Objective： To compare the biological characteristics of human periodontal ligament stem cells in primary culture with serum free medium or serum containing medium and provide the foundation for h PDLSCs based clinical application research and drug test.Methods： The hPDLSCs were isolated and cultured by using serum free medium or serum containing medium and purified by monoclonal method. Then observed and compared the differences of cell morphology, cell proliferation curves, surface markers expressions, osteogenic and adipogenic differentiation potential by using inverted microscope, CCK-8 assay, flow cytometry, immunofluorescence staining and real-time PCR, respectively.Results：h PDLSCs showed a similar morphology in two kinds medium. The cells proliferation curve of hPDLSCs cultured in serum free medium was significantly higher than that of in serum containing medium. Flow cytometry analysis showed that the surface markers of hPDLSCs cultured in two kinds of culture medium were similar, that of all positive expression of CD105, CD44, CD166, CD73 and CD90, negative expressing CD45, CD3, CD14, CD38, HLA- DR, CD31 and CD34.Immunofluorescence staining of hPDLSCs cultured in two kinds of culture medium indicated similar in expression of mesenchymal stem cell associated cell surface antigens： vimentin, α-SMA, N-cadherin. Differentiation experiment and real-time PCR detection showed that hPDLSCs cultured in serum free medium have osteogenic and adipogenic potential,and the differentiation abilities were higher than hPDLSCs cultured in serum containing medium. Conclusion：Serum free medium can amplify hPDLSCs in vitro, and maintain the stem cells characteristics. The hPDLSCs culture in serum free medium may be more suitable for its application in cell therapy and experimental study.

关 键 词：牙周膜干细胞 培养基 无血清 体外培养 

分 类 号：R781[医药卫生—口腔医学]