转化生长因子β1对人牙髓干细胞成骨分化作用研究

Effects of transforming growth factor-β1 on the osteogenic differentiation of human dental pulp stem cells

目的研究不同浓度的转化生长因子β1(transforming growth factor-β1,TGF-β1)对人牙髓干细胞(human dental pulp stem cells,hDPSCs)成骨分化的影响。方法采用酶消化法提取hDPSCs,通过流式细胞术鉴定干细胞的表面标记物。实验组利用含有不同浓度TGF-β1(1、5、10、20 ng/mL)的α-MEM培养细胞,对照组以不含TGF-β1的α-MEM培养细胞,分别在第7天和14天时,采用实时荧光定量PCR(qRT-PCR)方法检测成骨相关因子的表达情况。在第21天时,通过茜素红染色观察矿化结节形成情况。结果显微镜下可见hDPSCs呈长梭形,形态类似成纤维细胞。流式细胞术检测结果显示细胞呈CD73和CD90阳性表达,CD31、CD34和CD45阴性表达,具有典型的干细胞表面标记物。q RT-PCR结果表明,含有TGF-β1的各实验组均能明显促进成骨相关因子Runt相关转录因子2(Runx-2)、骨涎蛋白(BSP)和骨钙素(OCN)m RNA的表达。第7天时,TGF-β1浓度为1 ng/mL组的Runx-2、BSP和OCN的表达水平分别为对照组的11.3倍、8.7倍和11.7倍,明显高于其他各浓度组(P <0.05);第14天时,1 ng/mL组的Runx-2、BSP和OCN表达水平分别为对照组的5.08倍、7.17倍和3.03倍(P <0.05),20 ng/mL组BSP表达水平与对照组差异无统计学意义(P> 0.05)。茜素红染色显示各浓度的TGF-β1均能促进hDPSCs矿化结节形成。结论TGF-β1可以促进h DPSCs的成骨分化。

Objective To investigate the effects of different concentrations of transforming growth factor β1（TGF-β1）on the osteogenic differentiation of human dental pulp stem cells（hDPSCs）. Methods hDPSCs were isolated by enzymatic digestion and identified by flow cytometry. The cells were treated by cell culture medium containing different concentrations of TGF-β1（1 ng/mL,5 ng/mL,10 ng/mL,20 ng/mL）and the mRNA expression of bone-related factors at 7 and 14 days,respectively,were assessed by RT-PCR. The formation of mineralized nodules was observed by alizarin red staining at 21 days. Results h DPSCs were spindled in shape and similar to fibroblasts under microscope. Flow cytometry results showed that CD73 and CD90 were positively expressed and CD31,CD34 and CD45 were negatively expressed. RT-PCR results showed that all the experimental groups containing different concentrations of TGF-β1 significantly promoted the mRNA expression of osteogenesis-related factors,such as Runt-related transcription factor 2（Runx-2）,bone sialoprotein（BSP）and osteocalcin（OCN）. For the 1 ng/mL group,Runx-2,BSP and OCN expressionlevels were 11.3,8.7 and 11.7 times,respectively,higher than the control group at 7 days,and higher than the other experiment groups（P〈0.05）. At 14 days,Runx-2,BSP and OCN expression levels of 1 ng/mL group were 5.08,7.17 and 3.03 times,respectively,higher than the control group（P〈0.05）. BSP expression in the 20 ng/mL groupshowed no significant difference compared with the control group（P〉0.05）. Alizarin red staining showed that the various concentrations of TGF-β1 could promote the formation of mineralized nodules in hDPSCs. Conclusion TGF-β1 can promote the osteogenic differentiation of human dental pulp stem cells.