人牙周膜干细胞与人牙髓干细胞的表型及生长特性比较

摘要

目的:比较人牙周膜干细胞(human Periodontal ligament stem cells,hPDLSCs)和牙髓干细胞(humanDental pulp stem cells,hDPSCs)的表型及生长特性,为深入研究这两种细胞生物学特性提供依据.方法:组织块法培养获得原代人牙周膜细胞和牙髓细胞,采用有限稀释法分别对两者克隆化培养、分离纯化得到hPDLSCs和hDPSCs,采用倒置相差显微镜观察细胞形态、CCK8法检测细胞生长活性并绘制两者生长曲线、流式细胞技术检测干细胞表面标志物,分析比较两种细胞集落形成率(colony formation ratio,CFR).结果:hPDLSCs和hDPSCs镜下形态相似,生长曲线均呈“S”形,牙周膜细胞中STRO-1表达hPDLSCs阳性率为15.88±0.48％,牙髓细胞中STRO-1表达hDPSCs阳性率为11.86±0.43％,两者无显著性差异.两种干细胞均阳性表达间充质干细胞(Mesenchymal stem cells,MSCs)表面标志物STRO-1、CD29、CD44、CD90、CD73和血管内皮标志物CD105,其中STRO-l、CD29、CD90、CD73、CD105及CD166百分率达90％以上,阴性表达造血干细胞表面标志物CD34和CD45.hDPSCs的集落形成率(4.31±0.08％)显著高于hPDLSCs的集落形成率(2.68±0.06％)(P＜0.05).结论:hPDLSCs和hDPSCs细胞的形态相似,均高表达MSCs表面特异性标志物,hDPSCs的集落形成率显著高于hPDLSCs,说明hDPSC自我更新能力较hPDLSCs强,可为深入研究这两种干细胞提供实验依据.%Objective:We aim to compare the biological characteristics and phenotypic after cultivating and separating of the human Periodontal ligament stem cells (hPDLSCs) and human Dental pulp stem cells (hDPSCs).Methods:HPDLSCs and hDPSCs were respectively separated and amplificated ~om the original generation of cultures by magnetic-activated cell selection system (MACSS).Inverted phase contrast microscope was employed to observe the morphology.What is more,we make use of CCK-8 method to check and draw up the growth curves.Moreover,the expression of stem cell surface markers were analysized by flow cytometry instrument (FCM).Finally,we analyse and compare the colony formation ratio (CFR).Results:Fusiform or triangle,the morphology of hPDLSCs and hDPSCs is similar.The growth curvesare "S" shape.The expression ratio of STRO-1 in hPDLSCs and hDPSCs is respectively 15.88± 0.48％ and 11.86± 0.43％.There is no significant difference between the two cells.But the hDPSCs colony formation rate (4.31 ± 0.08％) is significantly higher than that of the hPDLSCs (2.68± 0.06％)(P＜0.05).Both of the two stem cells are positive for the CD29,CD44,CD90 and CD105 which are characterized by the employed to distinguish hPDLSCs and hDPSCs,and we can conclude that the separated stem cells share similar morphology.HPDLSCs and hDPSCs that can refresh and differentiate multi-directionally are characterized by stem cells due to the expression of stem cell markers.But the hDPSCs owns a significantly stronger colony formation rate than that ofhPDLSCs,then we can make a conclusion that hDPSCs is a promising stem cells resource.