摘要: 目的 观察人甲状旁腺激素（PTH）的N端活性片段PTH（1-34）对人成骨肉瘤细胞MG63基质Gla蛋白（MGP）表达的影响，以及PTH通过Wnt/β-catenin信号通路介导MGP表达调控的作用，探讨PTH（1-34）在防治骨质疏松症作用中可能的分子机制。方法（1）用不同浓度PTH（1-34）（10-9、10-8、10-7 mol/L），单独或联合Wnt/β-catenin信号通路抑制剂DKK-1（200 ng/mL）干预MG63细胞；（2）检测碱性磷酸酶（ALP）染色及活力测定用于判断细胞分化情况；（3）MGP及Wnt/β-catenin信号通路各组分的 mRNA及蛋白的表达分别采用实时定量 RT-PCR及 Western blotting方法。结果 （1）PTH（1-34）上调 MGP基因的表达，MGP mRNA的表达分别是对照组的 2.56倍、4.14倍、7.81倍（P<0.05 或 P<0.01），且呈剂量依赖性增高；（2）PTH增强 MG63细胞 ALP活力，Dkk-1抑制 ALP活性，Dkk-1与 PTH联用部分阻断 PTH上调 ALP活力（P<0.05）；（3）PTH上调 MGP及 Wnt/β-catenin信号通路中相关因子 LRP5、β-catenin、Runx2 mRNA及蛋白水平，其 mRNA分别是对照组的 2.65、4.01、3.48倍（P<0.05或<0.01）；DKK-1对PTH（1-34）促MGP表达没有影响，但大部分阻断了Wnt/β-catenin信号通路中相关因子的表达。结论 PTH（1-34）能明显上调MGP及Wnt/β-catenin信号通路中相关因子的表达，Wnt/β-catenin信号通路和MGP在PTH调节骨代谢中起重要作用。
Abstract: Objective To observe the effect of parathyroid hormone (PTH)(1-34) on the expression of matrix Gla protein (MGP) and Wnt/β-catenin signaling pathway and elucidate the possible molecular mechanism of PTH (1-34) in the prevention and treatment of osteoporosis. Methods MG63 cells treated with PTH (1-34) at 10-9, 10-8, and 10-7 mol/L, alone or in combination with Wnt/beta-catenin signaling pathway inhibitors DKK-1 (200 ng/ml) were examined for mRNA and protein expressions related with Wnt/β-catenin signaling with real-time PCR and Western blotting. The cell differentiation after the treatment was assessed with alkaline phosphatase (ALP) staining and cell viability assay. Results PTH (1-34) significantly increased the expression of MGP in a dose-dependent manner in MG63 cells (P<0.05 or P<0.01). PTH treatment obviously enhanced ALP activity in the cells, and this effect was suppressed by DKK-1. Combined treatment with DKK-1 partially blocked PTH-induced enhancement of ALP activity (P<0.05). PTH promoted the expression of MGP and enhanced LRP5, β-catenin, and Runx2 expressions in Wnt/β-catenin signaling pathway at both protein and mRNA levels (P<0.05 or P<0.01). DKK-1 partially blocked the effect of PTH (1-34) on Wnt/β-catenin signaling pathway (P<0.05) without affecting MGP expression. Conclusion PTH (1-34) significantly increases the expressions of MGP and proteins in the Wnt/β-catenin signaling pathway. Wnt/β-catenin signaling pathway and MGP mediate the regulation of osteogenosis by PTH.
| [V1] | 2017-12-27 12:40:28 | ChinaXiv:201712.00910V1 | 下载全文 |
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