Notch信号通路参与BMP-6对人牙髓细胞增殖分化作用的研究

摘要

目的：本研究通过观察骨形成蛋白-6(BMP-6, bone morphogenetic protein-6)对人牙髓细胞(hDPCs, human dental pulp cells)增殖和成牙本质向分化的影响，以及Notch信号在其中可能的作用，初步探讨骨形成蛋白-6在牙髓损伤修复中的作用与机制。方法：采用组织块酶消化法体外培养hDPCs，分别应用BMP-6和γ-分泌酶抑制剂DAPT单独或共处理第3-5代hDPCs，采用CCK8法检测细胞增殖情况，碱性磷酸酶(ALP, alkaline phosphatase)试剂盒检测ALP活性，茜素红染色法检测矿化结节形成情况，观察牙髓细胞的增殖及成牙本质向分化能力。结果： CCK8结果显示， BMP-6促进hDPCs增殖， DAPT抑制hDPCs增殖，而经DAPT预处理的hDPCs， BMP-6对其促增殖作用减弱，与对照组相比均具有统计学差异(P＜0.05)； ALP结果显示， BMP-6可促进hDPCs的ALP活性， DAPT则明显抑制其ALP活性，与对照组相比均具有统计学差异(P＜0.05)，且DAPT可减弱BMP-6对细胞ALP活性的增强作用，与 BMP-6组比较具有统计学差异(P＜0.05)；茜素红染色结果显示， BMP-6处理组矿化结节数量最多， BMP-6和DAPT共处理组次之， DAPT处理组最少。结论： BMP-6可促进hDPCs增殖和成牙本质向分化能力，而阻断Notch信号，可抑制BMP-6对hDPCs的促增殖及促成牙本质向分化作用。%Objective:To investigate the involvement of the Notch pathway in bone morphogenetic protein-6(BMP-6)-induced proliferation and odontoblastic differentiation of human dental pulp cells (hDPCs) in vitro.Methods:The hDPCs were cultured by the methods of tissue explants digested with collagenase. Cells from the third-fifth passages were treated with BMP-6 or/and DAPT, aγ-secretase inhibitor. The proliferation and ALPase activity of hDPCs were tested by using CCK-8 assay and ALP kit, and mineralization nodules were observed by Alizarin Red Staining.Results:CCK8 and ALP results showed that BMP-6 induced proliferation and odontoblastic differentiation of hDPCs, while DAPT palyed an opposite role. Treatment with a combination of BMP-6 and DAPT reversed the increase of both proliferation and odontoblastic differentiation induced by BMP-6 alone. Mineralization was significantly increased in response to BMP-6 treatment, while DAPT attenuated mineralization induced by BMP-6 in hDPCs.Conclusion:BMP-6 can promote the proliferation and odontoblastic differentiation of hDPCs, and suppressed the Notch pathway and can significantly attenuate the effects of both proliferation and odontoblastic differentiation of hDPCs induced by BMP-6.