FGF8辅助牙源性上皮诱导hDPSCs分化为成牙本质细胞及牙髓细胞

Role of fibroblast growth factor 8 in process of dental epithelium-induced directional differentiation of human postnatal dental pulp stem cells into odontoblasts and pulp cells

目的:研究成纤维细胞生长因子8(FGF8)对成人牙髓干细胞(hDPSCs)定向分化为成牙本质细胞及牙髓组织的影响。方法:首先分离、克隆培养hDPSCs,通过流式细胞术检测细胞表面标志物鉴定hDPSCs;矿化液中添加50μg/L的FGF8诱导hDPSCs分化,通过real-time PCR检测分化后的细胞中牙本质涎磷蛋白(DSPP)、碱性磷酸酶(ALP)、骨涎蛋白(BSP)和核心结合因子α1(Cbfa-1)在mRNA水平的表达;E11.5小鼠牙源性上皮联合FGF8与hDPSCs细胞团重组,再将组织块种植于裸鼠肾囊膜下培养,通过DNA原位杂交鉴定成牙本质细胞及牙髓细胞的来源。结果:成功分离培养hDPSCs,其表面标志物CD29和CD90呈阳性表达;经FGF8诱导的hDPSCs形成较明显的矿化结节,并且牙本质特异性蛋白DSPP、BSP及Cbfa-1表达量上调;E11.5小鼠牙源性上皮联合FGF8可以诱导hDPSCs分化为成牙本质细胞及牙髓细胞。结论:FGF8能够辅助牙源性上皮定向诱导hDPSCs分化为成牙本质细胞及牙髓细胞,并形成牙本质及牙髓腔结构。

AIM： To study the effects of fibroblast growth factor 8（ FGF8） on directional differentiation of human dental pulp stem cells（ hDPSCs） into odontoblasts and pulp tissue. METHODS： hDPSCs were isolated and cultured,and identified with flow cytometry by detecting cell surface markers of hDPSCs. FGF8 at concentration of 50 μg/L was added into the mineralization fluid to induce the differentiation of the hDPSCs. The mRNA expression of dentin sialophosphoprotein（ DSPP）,alkaline phosphatase（ ALP）,bone sialoprotein（ BSP） and core-binding factor alpha 1（ Cbfa-1）in differentiated cells was detected by real-time PCR. FGF8 and mouse E11. 5 dental epithelium formed restructuring cell group with hDPSCs,and then the restructuring cell group was transplanted under renal capsule membrane in nude mice for tissue culture. DNA in situ hybridization was used to identify the sources of odontoblasts and pulp cells. RESULTS： The surface markers of CD29 and CD90 showed positive in isolated hDPSCs. FGF8 induced hDPSCs to form a distinct mineralization nodule,and the expression of dentin-specific proteins,DSPP,BSP and Cbfa-1,was increased. hDPSCs were induced to differentiate into odontoblasts and pulp cells by E11. 5 dental epithelium and FGF8. CONCLUSION： FGF8 can assist dental epithelium to induce directional differetiation of hDPSCs into odontoblasts and pulp cells,and formation of dentin and dental pulp cavity structure.