| 摘要: | 在二十世紀初，有許多輔助性的物理式治療骨癒合的醫療器材被發明。如:低能量的超音波，脈衝式電磁場。其中磁場醫學更被廣泛應用在身體全身部位，尤其在骨關節疾病上。然而這些物理性刺激所造成的細胞生理改變機制，到目前為止仍然不明，使得其終被歸類於替代性醫療。近年來從新興的生物物理(mechanobiology)學中，有許多細胞或者動物實驗指出力學傳遞路徑(mechanotransduction) 在骨母細胞的生長中扮演重要的角色。力學傳遞路徑有兩大類:直接式與間接式的力學傳遞路徑。本研究假設靜磁場影響骨母細胞之細胞膜流動性或者改變細胞膜上integrin的數量後，可能啟動三磷酸肌醇反應路徑(inositol triphosphate pathway,IP3 pathway )影響攜鈣素(calmodulin)的表現，calmodulin一方面會調控磷酸二酯酶(phosphodiesterase-1C,PDE-1C)的表現去抑制cAMP的作用。限制前驅骨母細胞的增殖，同時會促使Ca2+自內質網釋放，啟動分化機制並使其走向分化成熟。
本研究計畫利用MG63類骨母細胞暴露在靜磁場中，透過偏極化螢光的方法分析細胞膜流動性改變與免疫螢光染色的方式來標定integrin數量，並利用MTT法監測靜磁場影響MG63類骨母細胞的生長，而MG63細胞成熟化標誌以鹼性磷酸酶活性為代表。此外，並在實驗中分別加入三磷酸肌醇反應路徑上的抑制劑:W-7觀察靜磁場影響MG63類骨母細胞的生理表現是否有所改變。進而利用即時聚合酵素連鎖法來比較受靜磁場暴露與對照組的類骨母細胞其PDE-1C的表現。
本研究結果發現，隨著暴露靜磁場時間增加，類骨母細胞的細胞膜流動性相對於對照組有顯著下降趨勢。並且PDE-1C基因的表現上，隨著靜磁場暴露的時間增加，在第二小時有暴露組與對照組的PDE-1C基因表現量有統計上的差別。但加入W-7抑制劑可降低原受靜磁場暴露所升高類骨母細胞的鹼性磷酸酶活性PDE-1C基因表現量。而在靜磁場影響骨母細胞成熟化標誌的鹼性磷酸酶的實驗中，當暴露靜磁場的天數於第二天起，暴露4000高斯靜磁場的細胞相對於對照組鹼性磷酸酶活性有明顯的上升。但加入W-7抑制劑可降低原受靜磁場暴露所升高的類骨母細胞的鹼性磷酸酶活性。此結果顯示，三磷酸肌醇反應路徑在靜磁場誘導類骨母細胞分化之機轉中扮演一重要的角色。
For years, several clinical strategies based on physical principles, for example, pulsed electromagnetic fields (PEMF) and low intensity ultrasound, were carried out for orthopedic diseases. Although the clinical outcomes of these treatments were verified, the mechanisms of how the cells sensing and response to the physical stimulations were not clarified until the last decade. Recently, results from cellular and animal studies demonstrated that mechanotransduction, the signal transduction pathwaies and biological processes triggered by various mechanical factors, play an important role in the osteoblastic growth. The rational of this study proposal is that static magnetic fields (SMFs) may affect the cellular membrane fluidity or integrin expression, and then up-regulate calmodulin. Calcium is an important secondary massager in the cascade of inositol triphosphate pathway. It can increase the level of phosphodiesterase-1C (PED-1C), an inhibitor of the cAMP, and reduce the proliferation activity of the cells. Our study hypothesis was that SMF affect the properties of cellular membranes, reducing proliferation and promoting osteoblastic differentiation.
Therefore, the effects of SMFs on MG63 cells in terms of the changes in the physical properties of the cellular membrane post SMF exposure were initially assessed via analysis of cellular-membrane fluidity and intergrin expression by FACS analysis. MTT assay was carried out for monitoring the viability of the cells. ALPase activity of the cells, a differentiation marker of osteoblastic cells, was detected under SMF exposure. W-7 was added in the cultured system as inhibitors of calmodulin , respectively. Real-time PCR was used for comparing the alternation in PED-1C expressions between SMF exposed and shamed exposed cells.
Our results showed that SMFs exposure decreased the level of cell membrane fluidity. On the other hand, the inductive effects of SMFs on ALPase activity could be inhibited by W-7. Moverover, SMFs tured on the PDE-1C mRNA expression, but the inhibitor, W-7, depressed the gene expression by SMFs. Our results indicated that IP3-cascade may play an important role in the mechanisms of SMF-induced mechanobiology. |
