Mage-D1与活化后的p75神经营养因子受体结合可正向调节大鼠外胚间充质干细胞的矿化

目的 检测Mage-D1与活化后p75NTR的结合及对外胚间充质干细胞（EMSCs）矿化的调控。方法 取孕19.5 d SD大鼠牙胚，组织贴壁法获取EMSCs，流式细胞术检测细胞表面抗原。设置实验分组：空白对照组（NC），100 ng/mL NGF刺激组（100 ng/mL NGF）、慢病毒干扰Mage-D1组（SH-Mage-D1）。邻位连接技术检测Mage-D1与经NGF活化后p75NTR结合的变化，并比较不同分组间的结合强度差异。茜素红与ALP染色观察染色，RT-PCR与Western blot检测ALP、Runx2、OCN、BSP、OPN、Col1、Msx1及Dlx1的表达水平。结果 组织块贴壁法获得孕19.5 dEMSCs，流式细胞术检测细胞表面抗原结果为CD44、CD90、CD29、CD146，CD105高表达，CD45低表达；邻位连接法检测Mage-D1与经NGF活化后p75NTR的结合随着时间的延长而增多，且实验组与对照组相比结合强度差异具有统计学意义（P<0.05）；茜素红与ALP染色结果显示矿化结节与碱性磷酸酶与强度变化一致；RT-PCR与Western blot检测ALP、Runx2、OCN、BSP、OPN、Col1、Msx1及Dlx1在100 ng/mL NGF组最高（P<0.05）。结论 Mage-D1在外胚层间充质细胞中可与经NGF活化后的p75NTR直接结合，且二者的结合对EMSCs的矿化有正向调节作用。

Mage-D1 binding to activated p75NTR positively regulates mineralization of rat ectomesenchymal stem cells in vitro

Objective To detect the binding of Mage-D1 with activated p75NTR and explore their role in regulating mineralization of ectomesenchymal stem cells (EMSCs). Methods EMSCs were isolated from the tooth germs of embryonic SD rats (19.5 days of gestation) by tissue explant culture and were identified for surface markers using flow cytometry. The cultured cells were divided into blank control group, 100 ng/mL nerve growth factor (NGF) stimulation group, and lentivirus-mediated Mage-D1 interference (SH-Mage-D1) group. Proximity ligation assay was used to detect the binding of Mage-D1 with activated p75NTR in the EMSCs, and the binding strength was compared among the 3 groups. Alizarin red staining and ALP staining were used to observe mineralization of the induced cells. The expressions of ALP, Runx2, OCN, BSP, OPN, Msx1 and Dlx1 at both the mRNA and protein levels were detected using RT-PCR and Western blotting. Results The isolated EMSCs expressed high levels of cell surface markers CD44, CD90, CD29, CD146, and CD105 with a low expression of CD45. The results of proximity ligation assay showed that the binding of Mage-D1 with activated p75NTR in the cells increased over time, and the binding strength was significantly greater in NFG-treated cells than in the cells in the other two groups (P<0.05). Alizarin red staining and ALP staining of the induced cells showed that the changes in the mineralization nodules were consistent with those of ALP activity. The cells treated with 100 ng/mL NGF exhibited significantly increased expressions of ALP, Runx2, OCN, BSP, OPN, Col1, Msx1 and Dlx1 as compared with the cells in the other two groups (P<0.05). Conclusion Mage-D1 directly binds to activated p75NTR in embryonic rat EMSCs to positively regulate the mineralization of the EMSCs.