转化生长因子β3对人乳牙牙髓干细胞增殖和矿化能力的影响

摘要

BACKGROUND:The role of transforming growth factorβsuperfamily has been reported in bone mineralization of various types of stem cells, but the effects of transforming growth factorβ3 (TGF-β3) combined with heparin on proliferation and mineralization of dental pulp stem cells from human deciduous teeth remains to be studied. OBJECTIVE:To evaluate the effects of TGF-β3 on the proliferation and mineralization of dental pulp stem cells from human deciduous teeth. METHODS:The enzyme digestion method was utilized to separately culture dental pulp stem cells from human deciduous teeth. The cellcolony forming efficiency was determined. Flow cytometry was utilized to identify cellsurface marker CD146. Immunochemistry for Vimentin and STRO1 was performed to measure dental pulp stem cells from human deciduous teeth. The third passage dental pulp stem cells from human deciduous teeth cultured in vitro were intervened with heparin and TGF-β3 of 1, 5, 25μg/L mass concentration. The MTS method was applied to measure cellgrowth curves. Alizarin red staining was carried out. The changes in alkaline phosphatase activity were determined with alkaline phosphatase kit. RESULTS AND CONCLUSION:The cellcolony forming efficiency was high. cells were positive for CD146, and strongly positive for Vimentin and STRO1. Dental pulp stem cells from human deciduous teeth were identified. MTS assay indicated that there was no obvious effect on promoting proliferation of dental pulp stem cells from human deciduous teeth after stimulation of TGF-β3. Detection results of alkaline phosphatase activity demonstrated that the combination of TGF-β3 and heparin could strengthen the alkaline phosphatase activity of dental pulp stem cells from human deciduous teeth with increased concentration. Alkaline phosphatase activities were significantly higher in the TGF-β3+heparin group, TGF-β3 group and heparin group than in the control group (P<0.01). Alizarin red staining was positive in the TGF-β3+heparin group, and the staining was strongest in the 5μg/L TGF-β3+heparin group. Results indicated that TGF-β3 combined with heparin promoted mineralization of dental pulp stem cells from human deciduous teeth.%背景：目前已有研究报道转化生长因子β超家族对各类干细胞成骨矿化的作用，但转化生长因子β3与肝素联合作用后对人乳牙牙髓干细胞的增殖和矿化能力的作用仍有待研究。目的：观察转化生长因子β3对人乳牙牙髓干细胞增殖和矿化能力的影响。方法：采用酶消化法将人乳牙牙髓分离培养，测定细胞集落克隆形成率，流式细胞术鉴定细胞表面标记物CD146，细胞爬片行Vimentin和STRO1免疫化学染色进行人乳牙牙髓干细胞鉴定；对体外培养的第3代人乳牙牙髓干细胞给予肝素和质量浓度1，5，25μg/L的转化生长因子β3进行干预。MTS法测细胞生长曲线；茜素红染色；碱性磷酸酶试剂盒检测碱性磷酸酶活性的改变。结果与结论：实验所得细胞集落克隆形成率高，细胞表面标记物 CD146呈阳性， Vimentin和STRO1免疫化学呈强阳性，鉴定获得人乳牙牙髓干细胞。MTS结果显示，给予转化生长因子β3刺激人乳牙牙髓干细胞后并无明显的促增殖的作用。碱性磷酸酶活性检测结果显示，转化生长因子β3-肝素联合作用可随着浓度的增加而增强人乳牙牙髓干细胞的碱性磷酸酶活性，其中质量浓度分别为5，25μg/L 转化生长因子β3和肝素联合作用组与转化生长因子β3单独作用组、肝素单独作用组以及对照组相比显著性升高(P<0.01)。转化生长因子β3-肝素联合作用组的茜素红染色均呈阳性，其中以5μg/L转化生长因子β3-肝素联合作用组染色最强。结果证实，转化生长因子β3与肝素联合作用可促进人乳牙牙髓干细胞矿化能力。