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目的:探讨转化生长因子-β3(transforming growth factor-β3,TGF-β3)诱导体外培养的兔牙髓干细胞(dental pulp stem cells,DPSCs)成骨向分化的情况。方法:采用酶消化法分离并培养兔DPSCs,光镜下进行形态学观察;并将体外培养的第4代DPSCs分为空白对照组,矿化液阳性对照组与TGF-β3实验组。1、3、5、7 d后采用碱性磷酸酶(Alkaline phosphatase,ALP)试剂盒检测各组细胞内ALP活性变化情况;1、3、5、7 d后行细胞免疫化学法检测成骨细胞标记物相关转录因子2(RUNX-2)和骨涎蛋白(BSP)的表达情况;14 d后观察各组细胞的矿化结节情况;7 d后使用Western blot检测各组细胞成骨分化指标BSP变化情况。结果:兔DPSCs在诱导体系中生长状态良好;实验组较两对照组上调细胞内ALP活性明显;免疫化学检测TGF-β3组与矿化液组RUNX-2第5 d呈阳性表达,BSP第7d阳性表达,空白对照组均呈弱阳性;14 d茜素红染色显示TGF-β3组矿化结节较矿化组明显,空白对照组阴性;与对照组相比,TGF-β3可增强兔DPSCs内BSP相关蛋白表达。结论:TGF-β3能够一定程度上促进兔DPSCs成骨向分化。
Objective: To investigate the osteogenic differentiation of rabbit dental pulp stem cells (DPSCs) induced by transforming growth factor-β3 (TGF-β3) in vitro. METHODS: Rabbit DPSCs were isolated and cultured by enzymatic digestion. Morphological changes were observed under light microscope. DPSCs of the fourth passage were divided into blank control group, mineralized fluid positive control group and TGF-β3 experimental group. The changes of intracellular ALP activity in each group were detected by Alkaline phosphatase (ALP) kit after 1, 3, 5, and 7 days. On the 1st, 5th, 5th and 7th days, the cell immunocytochemistry was used to detect the osteoblast (RUNX-2) and bone sialoprotein (BSP). The mineralized nodules were observed on the 14th day and the osteoblastic differentiation index BSP changes. RESULTS: The DPSCs were well grown in the induction system. The ALP activity in the experimental group was significantly up-regulated compared with the two control groups. The expression of RUNX-2 in the TGF-β3 group and the mineralized group was positively expressed on the 5th day after immunostaining. The expression of TGF-β3 in the group of TGF-β3 was significantly lower than that of the mineralized group and negative in the blank control group on 14th day. Compared with the control group, TGF-β3 enhanced the expression of DPSCs BSP-related protein expression. Conclusion: TGF-β3 can promote the osteogenic differentiation of DPSCs to a certain extent.