犬牙髓干细胞的分离培养与鉴定

Isolation,cultivation and identification of dental pulp stem cells from canine

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c DPSCs。

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 immunofluoreseence. 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 15.17% ±2.79%. FC observasion showed that the CD90, STRO-1 and CD24 positive cells were 24.43%±7.10%, 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.