Phenamil 促进BMP2 诱导骨髓间充质干细胞成骨信号通路的研究

Bone deficiency is a key factor to restrict the widely use of dental implants in restoring lost teeth for patients. How to improve bone quantity and quality in order to achieve faster and better osseointegration is still a problem to solve in dentistry. . As we know, bone is continuously remodeled throughout life by a tightly coupled process involving absorption by osteoclasts and formation by osteoblasts. Dysregulation of this coupled remodeling can lead to diseases relating to bone. Bone mesenchymal stem cells (BMSCs) are precursors of osteoblasts, and can be regulated by multiple signaling pathways, including transforming growth factor-1, Hedgehog, Wnt, fibroblast growth factors, insulin-like growth factor 1, bone morphogenetic proteins (BMPs), et al. . Among these factors, BMPs have been successfully used in animal experiments and humans to regenerate bones. However, the high cost and large doses of BMPs necessary to achieve osteoinductive activity reduce its wide use in clinic. It seems that other methods are needed to facilitate its function in osteogenesis among patients.. Recent articles and our previous research showed that phenamil, a small molecule derivative of the diuretic amiloride, induces osteoblastic differentiation and mineralization of mouse MSCs. Phenamil and BMPs also show additive effects on the expression of BMP target osteogenic mineralization. These results open a door for repair bone deficiency by treatment through small molecules. The molecular formula of phenamil is C12H12ClN7O • CH3SO3H, with molecular weight 401. Diuretic amiloride and its derivatives can influence the epithelial Na+ channel and Na+/H＋ pump, and now is used to treat renal diseases. Whether phenamil improve osteogensis of BMSCs through Na+ channel or BMP signal pathway is still to be discussed. . Since the mechanisms of these effects are not completely understood, elucidation of molecular mechanisms underlying osteogenesis not only is important for our understanding of bone development but also may advance strategies for bone repair.. The aim of our research is to verify the effect of phenamil of improving bone repair by osteoinduction. And by technique of PCR chip and gene silencing, we aim to revolve the possible signal pathway of osteogenic inductive effect of phenamil in vivo. Identification of small molecules that enhance BMP signaling could open the door to new therapeutic strategies. The result will reveal the mechanisms of the phnamil’s facilitating effect on BMPs , reduce the cost needed in implant therapy, and provide a second avenue for potential therapeutic development.

骨形成蛋白-2（bone morphogenetic protein-2，BMP2）可以促进骨髓间充质干细胞成骨分化，已被成功运用于临床促进成骨，但BMP2蛋白临床应用时需采用很大剂量，使用成本高昂。申请者前期研究结果显示小分子化合物phenamil能够提高细胞碱性磷酸酶（ALP）活性，促进osterix等基因的表达，而且其能够明显促进BMP2对骨髓间充质干细胞成骨分化的诱导作用，但其作用原理和机制并不清楚。本研究在证实phenamil能够促进多种成骨相关因子基因、蛋白表达的同时，通过PCR芯片、基因沉默等方法，揭示phenamil促进BMP2诱导骨髓间充质干细胞成骨作用中涉及的信号通路。本研究将为提高BMP2的诱导成骨效能提供必要的理论基础，并为今后降低骨再生治疗的成本并提高其治疗效果提供新思路，具有重要的科学意义和社会效益。

骨形成蛋白-2（bone morphogenetic protein-2，BMP-2）可以促进骨髓间充质干细胞成骨分化，已被成功运用于临床促进成骨，但BMP-2蛋白临床应用时需采用超生理剂量，使用成本高昂且存在副作用。Park等2009年的研究报道显示利尿剂阿米洛利（amiloride）的一种衍生物——Phenamil，能在体外诱导鼠间充质干细胞向成骨细胞分化。. 目的：证实Phenamil联合BMP-2促进骨髓间充质干细胞成骨诱导分化的效果。. 方法：分别采用5μM、10μM、20μM Phenamil与BMP-2相混合，以单独使用BMP-2及相应浓度Phenamil为对照，诱导复苏的鼠骨髓间充质干细胞M2-10B4细胞成骨分化。测定M2细胞碱性磷酸酶（ALP）活性，筛选出Phenamil与BMP-2联合应用的适宜浓度，并以该浓度Phenamil作为实验浓度进行后续研究。M2细胞经空白对照、50ng/mL BMP-2、实验浓度Phenamil及50ng/mL BMP-2+实验浓度Phenamil诱导成骨后，进行RT-PCR、Western Blotting、茜素红及碱性磷酸酶染色观察M2细胞成骨及矿化效果。. 结果：与5μM、10μM浓度相比，20μM Phenamil与BMP-2联合应用时，从诱导分化第3天开始即能显著提高M2细胞的ALP活性。与单独使用BMP-2相比，50ng/ml BMP-2、20μM Phenamil联合应用促进Osterix、Osteocalcin、Alp、Runx2基因及蛋白水平的表达更显著。50ng/ml BMP-2、20μM Phenamil联合运用较单独运用，其促进M2细胞成骨矿化的程度更显著。. 结论：Phenamil可以促进BMP-2诱导M2细胞成骨分化。50ng/ml BMP-2、20μM Phenamil联合运用较单独运用，其促进骨髓间充质干细胞Osterix、Osteocalcin、Alp、Runx2基因及蛋白水平的表达更显著。50ng/ml BMP-2、20μM Phenamil联合运用较单独运用，其促进骨髓间充质干细胞成骨矿化的程度更显著。