乳牙牙髓干细胞CD146阳性/阴性细胞亚群生物学特性的比较  

Comparison of the properties of CD146 positive and CD146 negative subpopulations of stem cells from human exfoliated deciduous teeth

作　　者：汪晓彤[1] 饶南荃 方腾姣子 赵玉鸣[2] 葛立宏[2] WANG Xiao-tong;RAO Nan-quan;FANG Teng-jiao-zi;ZHAO Yu-ming;GE Li-hong(Department of Emergency Dentistry,Peking University School and Hospital of Stomatology,Beijing 100081,China;Department of Pediatric Dentistry,Peking University School and Hospital of Stomatology&National Engineering Laboratory for Digital and Material Technology of Stomatology&Beijing Key Laboratory of Digital Stomatology,Beijing 100081,China)

机构地区：[1]北京大学口腔医学院·口腔医院急诊科,北京100081 [2]北京大学口腔医学院·口腔医院儿童口腔科,口腔数字化医疗技术和材料国家工程实验室,口腔数字医学北京市重点实验室,北京100081

出　　处：《北京大学学报（医学版）》2018年第2期284-292,共9页Journal of Peking University:Health Sciences

基　　金：北京大学泰盛口腔医学发展基金(94000-6672-H78)资助

摘　　要：目的:通过磁激活分选法(magnetically activated cell sorting,MACS)技术对人乳牙牙髓干细胞(stem cells from human exfoliated teeth,SHED)进行分选,获得纯度较高的CD146阳性及阴性细胞亚群,比较其生物学特性。方法:用MACS技术对SHED进行CD146分选获得未筛选的混合细胞群、CD146阳性和阴性细胞亚群,采用细胞计数试剂盒(cell counting kit-8,CCK-8)和集落形成实验(colony-forming unit,CFU)检测增殖能力;成骨诱导后进行茜素红染色,实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,q PCR)检测成骨相关基因RUNX2、OCN、BSP表达;成脂诱导后进行油红O染色,q PCR检测成脂相关基因LPL、PPARG表达;成神经诱导后细胞免疫荧光检测神经细胞标志蛋白nestin、NeuN、GFAP,qPCR检测成神经相关基因nestin、MAP2表达。结果:CD146阳性和阴性细胞亚群的增殖能力都显著低于混合细胞群。混合细胞群、阳性细胞亚群、阴性细胞亚群的集落形成率分别为28.6%±3%、17.1%±2.3%和27.5%±2.5%。诱导后,阳性细胞亚群的矿化物沉积最多、成骨相关基因表达升高最显著;阴性细胞亚群的脂滴形成最多、成脂相关基因表达升高最显著;未分选混合细胞群和阳性细胞亚群胶质细胞标志蛋白阳性表达,而阴性细胞亚群的神经前体细胞和神经元标志蛋白和基因表达升高显著。结论:CD146阳性与阴性细胞亚群的增殖能力弱于混合细胞群;阳性细胞亚群的成骨潜能最佳,阴性细胞亚群具有更强的成脂分化潜能;混合细胞群与阳性细胞亚群倾向于胶质细胞分化,阴性细胞亚群神经前体细胞和神经元分化潜能更强。Objective:Stem cells from human exfoliated teeth(SHED)were sorted by magnetically activated cell sorting(MACS)technique to obtain the CD146 positive and negative cell subpopulation.Then the biological characteristics of these subpopulations were compared to explore their specific application potential in tissue engineering.Methods:In this study,freshly extracted deciduous teeth without any caries or dental pulp disease were obtained.SHED was isolated using enzyme digestion method and then sorted by MACS,CD146 positive cells and CD146 negative cells were obtained after cell sorting.The biological characteristics of the unsorted mixed cells,CD146 positive subpopulation and CD146 negative subpopulation were compared.The proliferation ability was detected through cell counting kit-8(CCK-8)and colony-forming unit(CFU).After osteogenic induction,alizarin red staining was performed and the gene expression of osteogenic related markers was detected by quantitative real-time polymerase chain reaction(qPCR).After adipogenic induction,oil-red O staining was performed and the gene expression of adipogenic related markers was detected.After neurogenic differentiation induction,the expression of neural markers was detected by immunofluorescence and the gene expression of neural markers was detected by qPCR.Results:SHED of the fifth passage was sorted by MACS.And the CD146 positive cell subpopulation and CD146 negative cell subpopulation were obtained.CCK8 assay showed that the proliferative tendency of the three cell groups was consistent,but the proliferation potential of CD146 positive and negative cell subpopulations was significantly lower than that of the unsorted cells.The colony forming rates of the unsorted mixed cell group,CD146 positive and negative populations were 28.6%±3%,17.1%±2.3%and 27.5%±2.5%,respectively.After 21 days of osteogenic induction,alizarin red staining and qPCR showed that the CD146 positive cell population had more mineralized nodule formation and expressed higher level of osteogenic related ge

关 键 词：乳牙牙髓干细胞 磁激活分选法 增殖 分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]