1. Department of Hematology, Tarbiat Modares University, Tehran, Iran.
Cell J. 2012 Summer;14(2):90-7. Epub 2012 Aug 31.
Mechanism of zoledronic acid on osteoblastic differentiation of mesenchymal stem cells (MSCs) has not fully understood. With the knowledge of some drugs mechanism that alter methylation pattern of some genes, the present research sets out to evaluate osterix (OSX) promoter methylation pattern during zoledronic acid-induced osteoblastic differentiation of MSCs.
In this experimental study, MSCs were isolated from human bone marrow. For osteogenic differentiation, MSCs were pulse treated with 5ìM Zoledronic acid for 3 hours and incubated after a medium change in osteogenic differentiation medium for 3 weeks. DNA and RNA were extracted on days 0, 7, 14 and 21 of MSCs differentiating to osteoblast. After cDNA synthesis, OSX expression was evaluated by RT-PCR and quantitative Real-Time PCR. After multiplicity of infection (MOI) treatment, gene specific methylation of OSX was analyzed by methylation specific PCR (MSP).
The mRNA expression of OSX was increased in osteoblast differentiated cells induced by zoledronic acid, especially on days 14 and 21 of differentiation (p<0.05), but expression of OSX didn't change in undifferentiated MSCs. MSP revealed that, on day 0, undifferentiated MSCs are totally methylated. But, on day 7 of differentiation, MSCs treated by zoledronic acid were totally unmethylated. OSX promoter remained unmethylated, afterwards.
MSP revealed that OSX had a dynamic pattern in methylation, while MSCs gradually differentiated to osteoblasts. Our finding showed that promoter region of OSX is hypomethylated independently from zoledronic acid treatment during osteoblastic differentiation. This knowledge is important to understand drug mechanisms and can be useful for developing new therapies to combat against bone diseases.
唑来膦酸对间充质干细胞（MSCs）成骨分化的作用机制尚未完全阐明。由于了解一些药物可以改变某些基因的甲基化模式，本研究旨在评估唑来膦酸诱导 MSCs 成骨分化过程中osterix（OSX）启动子的甲基化模式。
在这项实验研究中，我们从人骨髓中分离出 MSCs。为了进行成骨分化，我们用 5μM 唑来膦酸脉冲处理 MSCs 3 小时，然后在成骨分化培养基中孵育 3 周。在 MSCs 分化为成骨细胞的第 0、7、14 和 21 天提取 DNA 和 RNA。在 cDNA 合成后，通过 RT-PCR 和定量实时 PCR 评估 OSX 的表达。在感染复数（MOI）处理后，通过甲基化特异性 PCR（MSP）分析 OSX 的基因特异性甲基化。
唑来膦酸诱导的成骨分化细胞中 OSX 的 mRNA 表达增加，尤其是在分化的第 14 和 21 天（p<0.05），但未分化的 MSCs 中 OSX 的表达没有变化。MSP 显示，在第 0 天，未分化的 MSCs 完全甲基化。但在分化的第 7 天，用唑来膦酸处理的 MSCs 完全去甲基化。此后，OSX 启动子保持非甲基化状态。
MSP 显示 OSX 的甲基化呈动态模式，而 MSCs 逐渐分化为成骨细胞。我们的发现表明，在成骨分化过程中，OSX 的启动子区域在没有唑来膦酸处理的情况下呈低甲基化状态。这一知识对于理解药物机制非常重要，并且可以为开发治疗骨疾病的新疗法提供帮助。
