許多研究顯示肌肉收縮時所分泌的肌肉激素可能會影響骨組織。為了在體外誘導肌肉激素分泌以利於了解兩者之間的關係，我們將導電高分子聚吡咯(PPy)製備於聚二甲基矽氧烷(PDMS)表面而得到高拉伸性導電薄膜，以用於構建生物反應器，將成肌細胞C2C12培養於其上並進行分化成肌管，可對其進行電刺激以產生主動收縮及循環拉伸以產生被動形變。電刺激的研究中發現IL-6及CXCL-1的分泌可以維持24小時，且隨著電壓而上升，因此我們對肌管進行連續24小時循環拉伸或電刺激，結果顯示許多肌肉激素基因都能被這兩種刺激所上調，但有些則只能透過循環拉伸來促進表現。將這些條件培養基用於分化成骨及破骨細胞，發現肌管條件培養基可促進成骨細胞分化基因的上調，而刺激過的肌管條件培養基上調效果更佳。由茜紅素染色及鈣分析可以發現相同的趨勢，且拉伸刺激的促進效果優於電刺激。另一方面，肌管條件培養基會下調破骨細胞基因表現，抗酒石酸酸性磷酸酶(TRAP)染色結果也證實破骨細胞形成會因此被抑制。我們的結果顯示兩種刺激均能促進肌肉激素基因表現，且所分泌的肌肉激素可增進骨生成並抑制破骨細胞分化，因此肌肉激素對於骨相關疾病研究的治療極具潛力，我們所開發的反應器可做為一體外平台，為肌肉激素對骨代謝的影響及治療代謝性骨病的研究提供有用的訊息。;Myokine secretion during muscle contraction may influence bone tissue formation. In order to investigate their relationship, we would like to induce myokine secretion in in vitro plateform. Conductive polypyrrole (PPy) was deposited onto the surface of polydimethylsiloxane (PDMS) to obtain a highly stretchable conductive membrane, which was used to construct a bioreactor. C2C12 myoblasts were seeded and differentiated to form myotubes on the bioreactor, which can be electrically stimulated to induce active contraction or cyclically stretched to allow passive cellular deformation. Both IL-6 and CXCL-1 myokines can be secreted from myotubes under electrical stimulation, which be maintained for 24 hours and their secretion increased with voltage. As we performed continuous cyclic stretching or electrical stimulation to myotubes for 24 h, and the qPCR results showed that many myokines could be up-regulated by both stimuli, but some can only be boosted through cyclic stretching. These conditioned media were used to differentiate osteoblasts and osteoclasts. Osteogenic differentiation genes can be upregulated by conditioned media, which were further promoted when the conditioned media were collected from stimulated myotubes. These trends were found in alizarin red staining and calcium quanrification experiments, and the promotion effect of cyclic stretching was better than that of the electrical stimulation. On the other hand, conditioned media down-regulated osteoclastic differentiation genes, and the results of tartrate-resistant acid phosphatase (TRAP) staining also demonstrated that osteoclast formation was inhibited by these conditioned media. Our results showed that myokine genes can be upregulated by both cyclic stretching and electrical stimulation, and their secretions not only improved bone formation but also inhibited osteoclast differentiation. Therefore, myokines are potential for the treatment of bone disease, and our developed bioreactor can be used as an in vitro platform for relative research.
