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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。
BACKGROUND: There are reports on the effect of transforming growth factor β superfamily on the osteogenesis and mineralization of various stem cells. However, the effect of transforming growth factor β3 and heparin on the proliferation and mineralization ability of dental pulp stem cells remains to be determined the study. Objective: To observe the effect of transforming growth factor β3 on the proliferation and mineralization of human dental pulp stem cells. Methods: The dental pulp of human deciduous teeth was isolated and cultured by enzymatic digestion method, the colony formation rate of the colony was determined, the cell surface marker CD146 was identified by flow cytometry, the Vimentin and STRO1 immunochemical staining was used to identify the dental pulp stem cells of human deciduous teeth. The 3rd generation human dental pulp dental pulp stem cells cultured in vitro were treated with heparin and 1, 25μg / L transforming growth factor β3. MTS assay cell growth curve; alizarin red staining; alkaline phosphatase kit alkaline phosphatase activity changes. RESULTS AND CONCLUSION: The colony forming rate of colonies was high, CD146 positive cell surface markers, Vimentin and STRO1 immunochemistry showed strong positive, identified human deciduous dental pulp stem cells. MTS results showed that TGF-β3 stimulation of human deciduous dental pulp stem cells did not significantly promote proliferation. Alkaline phosphatase activity test results showed that the combination of transforming growth factor β3-heparin enhanced the alkaline phosphatase activity of dental pulp stem cells of human deciduous teeth with the increase of concentration, in which the concentration of 5,25μg / L transforming growth factor β3 and heparin combined with the group of transforming growth factor β3 alone, heparin alone and the control group significantly increased (P <0.01). The alizarin red staining of TGF - β3 - heparin combined group showed positive staining, of which 5μg / L transforming growth factor β3 - heparin combined group showed the strongest staining. The results confirmed that the combination of transforming growth factor β3 and heparin can promote human deciduous dental pulp cell mineralization ability.