本研究旨在建立一套可同時施加電刺激與機械拉伸刺激之體外生物反應器系統，以探討不同刺激模式對肌管分泌肌肉激素(myokine)與基因調控之影響。為達成可拉伸且具導電性之培養基材，本研究以導電高分子聚吡咯(PPy)塗佈於聚二甲基矽氧烷(PDMS)表面而得到高拉伸性導電薄膜，並用於構建生物反應器。將C2C12成肌細胞培養於薄膜表面並分化為肌管後，首先建立電刺激條件，分別評估定電壓定電場刺激(DC)與電脈衝刺激(EPS)對肌肉激素分泌之影響，於確認DC刺激可有效誘導反應後，再進一步探討其與循環拉伸刺激(1 Hz 、10 %拉伸量)結合之複合刺激效果。利用酵素免疫分析法(ELISA)量測肌肉激素蛋白質分泌量，並以即時定量聚合酶連鎖反應(qPCR)分析肌肉激素相關基因表現。 結果顯示，電刺激與機械拉伸刺激皆能顯著誘導C2C12肌管分泌肌肉激素，其中以IL-6之反應最為明顯。在電刺激模式比較中，DC在誘導IL-6分泌之效果優於EPS，顯示即便非典型的生理放電型態，定電場仍能有效促進肌肉激素分泌，突顯電性環境在肌肉激素調控中的重要性。進一步比較複合刺激與單一刺激後發現，複合刺激雖然可促進肌管之肌肉激素基因轉錄及分泌，但相較於對應之單一刺激，整體反應未呈現加成效應，顯示電刺激與機械刺激之調控可能受訊號整合機制限制。 綜合而言，本研究成功建立一套可同時施加電刺激與機械拉伸刺激之體外肌肉模型，並證實不同刺激模式可差異性調控肌肉激素之分泌與基因轉錄，其中發現定電壓定電場刺激亦能促進肌肉激素分泌為本研究的重要貢獻之一。本研究所建立之平台可作為未來探討運動模擬、肌肉代謝及肌肉相關疾病研究之實驗工具與基礎。;This study aimed to establish an in vitro bioreactor system capable of simultaneously applying electrical stimulation and mechanical cyclic stretch to investigate how different stimulation modes regulate myokine secretion and gene expression in myotubes. To achieve a stretchable and conductive culture substrate, a highly extensible conductive film was fabricated by depositing the conductive polymer polypyrrole (PPy) onto the surface of polydimethylsiloxane (PDMS) and integrated into the bioreactor platform. C2C12 myoblasts were cultured on the conductive film and differentiated into myotubes. We first established the electrical stimulation conditions by comparing constant-voltage direct-current electric field stimulation (DC) with electrical pulse stimulation (EPS) in terms of their effects on myokine secretion. After confirming that DC stimulation elicited a robust response, we further evaluated the effects of DC combined with cyclic stretch (1 Hz, 10% strain). Myokine protein secretion was quantified using enzyme-linked immunosorbent assay (ELISA), while myokine-related gene expression was analyzed by quantitative real-time polymerase chain reaction (qPCR). The results demonstrated that both electrical stimulation and mechanical stretch significantly promoted IL-6 myokine expression in C2C12 myotubes. Among the electrical stimulation modalities, DC stimulation induced a greater increase in IL-6 secretion than EPS, indicating that even non-physiological stimulation patterns, such as constant electric fields, can effectively enhance myokine secretion and highlight the critical role of the electrical microenvironment in myokine regulation. Further comparison between combined and single stimulation conditions revealed that although cyclic stretch promoted myokine gene transcription and secretion, the combined stimulation did not produce a synergistic effect, suggesting that the regulatory effects of electrical and mechanical stimulation may be constrained by intracellular signal integration mechanisms. In summary, this study successfully established an in vitro platform that allows simultaneous application of electrical and mechanical stimulation to myotubes and demonstrated that different stimulation modalities differentially regulate myokine secretion and gene transcription. Notably, the finding that DC stimulation can promote myokine secretion represents a key contribution of this work. The developed platform provides a valuable experimental tool for future studies on exercise simulation, muscle metabolism, and skeletal muscle-related diseases.
