犬牙髓干细胞的分离培养与鉴定

摘要

目的：分离培养犬牙髓干细胞（cDPSCs）。方法：取犬磨牙获取牙髓，采用酶消法和组织块法取原代牙髓细胞，观察计数；免疫荧光和流式法鉴定细胞蛋白表达；进行成牙本质、成脂诱导。结果：酶消法和组织块法均可获得贴壁、集落生长的梭形细胞并稳定增殖。细胞克隆形成率为（15．17％±2．79％）。流式细胞术观察：CD90（24．43％±7．10％）、STRO-1（20．67％±1．42％）、CD24（2．03％±0．06％）、CD34阴性。免疫荧光法观察：Nestin，Vimentin 表达阳性、ALP 表达弱阳性、DSP 表达阴性或微弱阳性。成牙本质诱导见矿化结节，ALP 与 DSP 阳性。成脂诱导后细胞质见大量脂滴。结论：犬健康年轻磨牙牙髓组织中可稳定分离培养出具有一定克隆能力的 cDPSCs。%Objective:To culture canine dental pulp stem cells(cDPSCs)in vitro.Methods:Canine pulp cells were isolated and cultured by enzyme digestion and explanted tissue culture respectively.Cell morphology was observed under phase-contrast micro-scope.The clone forming unit(CFU)of the cells was examined by plate clone formation assay.Cell markers and protein-expression were examined by flow cytometry(FC)and immunofluorescence.Odontogenic and adipogenic potential were evaluated by alizarin red staining and oil red O staining.Results:Short spindle fibroblast-like and steadily growing cells were obtained by both methods.The clone assay showed that CFU was 1 5.1 7% ±2.79%.FC observasion showed that the CD90,STRO-1 and CD24 positive cells were 24.43% ±7.1 0%,20.67% ±1 .42% and 2.03% ±0.06% respectively,but CD34 was negative.Immunofluorescence analysis showed positive expression of Nestin,Vimentin,weak expression of ALP and negative expression of DSP of the cells.Differentiation ex-periment confirmed the odontogenic and adipogenic differentiation potential of the cells.Conclusion:cDPSCs can be cultured in vitro.