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目的探讨转化生长因子(transforming growth factor,TGF)-β_3联合肝素在体外诱导兔牙髓干细胞(dental pulp stem cells,DPSCs)成骨向分化中的能力。方法新西兰幼兔4只,采用酶解组织块法分离培养兔DPSCs,传代培养至第3代兔DPSCs,分为空白对照组、TGF-β_3组和TGF-β_3+肝素组;空白对照组正常培养,TGF-β_3组在细胞培养基中加入20μg/L TGF-β_3,TGF-β_3+肝素组在细胞培养基中同时加入20μg/L TGF-β_3和5u/mL肝素。采用碱性磷酸酶(alkailine phosphate,ALP)试剂盒检测各组兔DPSCs成骨向诱导后第7、14天细胞内ALP活性,细胞爬片采用免疫细胞化学法检测成骨细胞标记物RUNT相关转录因子2(runt related transcription factor 2,RUNX-2)和骨钙素(osteocalcin,OC)表达情况,采用茜素红染色观察矿化结节形成情况。结果兔DPSCs在诱导体系中生长状态良好;TGF-β_3+肝素组第7、14天ALP活性[(27.20±1.01)、(29.06±1.39)u/(mg·pro)]高于TGF-β_3组[(12.69±1.03)、(9.44±1.05)u/(mg·pro)]和对照组[(5.96±3.68)、(6.10±3.66)u/(mg·pro)](P<0.01),TGF-β_3组高于对照组(P<0.01);TGF-β_3组和TGF-β_3+肝素组成骨诱导第7天RUNX-2开始出现阳性表达,第14天OC有阳性表达,对照组均为阴性;第21天茜素红染色对照组为阴性,TGF-β_3组为阳性,TGF-β_3+肝素组为强阳性。结论肝素有促进TGF-β_3体外诱导兔DPSCs成骨向分化的能力。
Objective To investigate the ability of transforming growth factor (TGF) -β_3 and heparin to induce osteogenic differentiation of rabbit dental pulp stem cells (DPSCs) in vitro. Methods Four New Zealand rabbits were divided into blank control group, TGF-β_3 group and TGF-β_3 + heparin group. The DPSCs were isolated and cultured to the third generation of rabbit DPSCs by enzymatic digestion method. The blank control group was cultured normally, In the TGF-β_3 group, 20μg / L TGF-β_3 was added to the cell culture medium, and 20μg / L TGF-β_3 and 5u / mL heparin were added into the cell culture medium. Alkaline phosphatase (ALP) kit was used to detect the ALP activity of DPSCs in each group on the 7th and 14th day after osteogenic induction, and the immunocytochemical method was used to detect the osteoblastic marker RUNT-related transcription The expression of RUNX-2 and osteocalcin (OC) were detected by alizarin red staining to observe the formation of mineralized nodules. The results showed that the growth of DPSCs in the induction system was good. The ALP activity in the TGF-β 3 + heparin group was significantly higher than that in the TGF-β 3 group [(27.20 ± 1.01), (29.06 ± 1.39) u mg / (12.69 ± 1.03), (9.44 ± 1.05) u / (mg · pro)] and control group (5.96 ± 3.68) and (6.10 ± 3.66) u mg / β_3 group was higher than that of the control group (P <0.01). TGF-β_3 group and TGF-β_3 + heparin group showed a positive expression of RUNX-2 on the 7th day, OC was positive on the 14th day, 21 days alizarin red staining negative for the control group, TGF-β_3 group was positive, TGF-β_3 heparin group was strongly positive. Conclusion Heparin can promote the osteogenic differentiation of DPSCs induced by TGF-β 3 in vitro.