大鼠牙囊细胞成骨分化过程中Notch信号表达情况

Expression of Notch signal during osteogenic differentiation process of rat dental follicle cells

目的探讨在大鼠牙囊细胞(DFC)成骨分化时Notch信号的表达情况。方法分离、培养及纯化DFC,在光镜下观察原代及第3代DFC的形态。对第3代DFC进行波形蛋白(Vimentin)、角蛋白-14(CK-14)免疫荧光染色鉴定其组织来源。将第3代DFC分为对照组(基础培养液进行培养)和成骨诱导组(成骨诱导液进行培养),28 d后进行茜素红染色,鉴定DFC的成骨分化能力。应用定量逆转录聚合酶链反应检测两组DFC的Notch信号表达情况。结果成功分离、培养及纯化DFC。DFC的胞体呈长梭形,呈典型成纤维细胞表型,Vimentin阳性表达,CK-14阴性表达,表明DFC来源于间充质且无上皮污染。培养28 d后成骨诱导组DFC较对照组茜素红染色更加明显,表明DFC在成骨诱导液诱导下可向成骨方向分化。成骨诱导组DFC经28 d培养后Notch信号表达量呈下降趋势,且明显低于对照组,差异有统计学意义(P<0.05)。结论DFC具有间充质干细胞的特性,在成骨分化过程中Notch信号通过负反馈回路调节DFC的成骨分化。

Objective To investigate the expression of Notch signal during osteogenic differentiation of rat dental follicle cells(DFC).Methods DFC were isolated,cultured and purified.The morphologies of primary and the third generation of DFC were observed under light microscope.The immunofluorescent staining of Vimentin and CK-14 was used to identify the origin of the third generation of DFC.The third generation of DFC was divided into the control group(cultured in the basal medium)and osteogenic induction group(cultured in the osteogenic induction medium).After 28 d,the osteogenic differentiation ability of DFC was identified by alizarin red staining.Notch signal expression in the control group and osteogenic induction group were detected by qRT-PCR,respectively.Results DFC were isolated,cultured and purified successfully.The cell body of DFC showed the long spindle type,presenting the typical fibroblast phenotype,Vimentin was positively expressed,CK-14 was negatively expressed,which showed the DFC was derived from mesenchyma without epithelial contamination.After 28 d culture,the alizarin red staining in the osteogenic induction group was more obvious than that in the control group,which showed that DFC could differentiated into the osteogenesis direction under the induction of osteogenic induction solution.The Notch signal expression levels of DFC after 28 d culture in the osteogenic induction group showed the decreasing trend,moreover which was significantly lower than that in the control group,and the differences were statistically significant(P<0.05).Conclusion DFC have the characteristics of mesenchymal stem cells.The Notch signal regulates the osteogenic differentiation of DFC during the osteogenic differentiation process by negative feedback loop.