博碩士論文 109324029 詳細資訊




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姓名 田昀珊(Yun-Shan Tian)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 肌管分泌的肌肉激素對成骨細胞及破骨細胞的影響
(The effects of myotube-secreted myokines on osteoblasts and osteoclasts)
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摘要(中) 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。
摘要(英) 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.
關鍵字(中) ★ 肌肉激素
★ 電刺激
★ 機械刺激
★ 成骨細胞
★ 破骨細胞
★ 肌肉
關鍵字(英) ★ myokine
★ electrical stimulation
★ mechanical stimulation
★ osteoblast
★ osteoclast
★ muscle
論文目次 Abstract VI
致謝 VII
目錄 VIII
圖目錄 XI
表目錄 XIII
第一章 緒論 1
1-1 研究背景 1
1-2 研究目的 1
1-3 研究方法 2
第二章 文獻回顧 3
2-1 肌肉組織 3
2-1-1 骨骼肌構造與功能 3
2-1-2 骨骼肌收縮 4
2-2 骨骼組織結構與功能 6
2-2-1 成骨細胞 7
2-2-2 破骨細胞 7
2-3 肌肉激素(Myokine)與骨骼間關係 8
2-3-1 肌肉生長抑制素(Myostatin)和生長因子(Growth Factors) 8
2-3-2 細胞因子(Cytokine) 12
2-3-3 其它肌肉激素(Other myokines) 13
2-3-4 肌肉激素對成骨細胞及破骨細胞影響 14
2-4 其它與肌肉生長有關之肌肉激素 15
2-5 生物刺激裝置 16
2-5-1 機械刺激裝置 16
2-5-2 電刺激裝置 20
2-5-3 肌肉激素在體外培養裝置的表現及對骨組織的影響 25
第三章 實驗方法與儀器 27
3-1 實驗藥品 27
3-1-1 材料製備藥品 27
3-1-2 細胞培養、骨分化藥品 28
3-1-3 細胞定性、定量試劑 33
3-2 實驗儀器 37
3-3 實驗方法 39
3-3-1 設計、組裝生物刺激裝置 39
3-3-2 生物反應器組裝以進行循環拉伸刺激及電刺激 45
3-3-3 微接觸印刷 48
3-3-4 拉伸量測試 50
3-3-5脈衝訊號的量測 51
3-3-6 細胞培養、繼代、冷凍及解凍 52
3-3-7 分化培養液配製及分化過程 56
3-3-8 即時聚合酶反應儀 (Real-time PCR) 59
3-3-9 ELISA蛋白檢測 67
3-3-10 茜素紅染色定性分析 (Alizarin Red Staining) 69
3-3-11 Calcium-O-Cresophtalein Complexone定量分析 70
3-3-12 抗酒石酸酸性磷酸酶染色(Tartrate-Resistant Acid Phosphatase Staining) 72
3-4 實驗架構設計 74
3-4-1 循環拉伸及電刺激對肌管收縮的影響 74
3-4-2 肌管被動收縮及主動收縮分泌之肌肉激素對成骨細胞及破骨細胞的影響 75
第四章 結果與討論 77
4-1 拉伸量測試 77
4-2 循環拉伸及電刺激對肌管分泌肌肉激素的影響 80
4-2-1 電刺激對肌肉激素的表現效果 80
4-2-2 不同刺激下C2C12基因表現 83
4-3 條件培養液對成骨細胞的影響 90
4-3-1 不同條件CM下hDPSC基因表現 90
4-3-2茜素紅染色與鈣離子沉積分析骨質形成效果 96
4-4 條件培養液對破骨細胞的影響 98
4-4-1 不同條件CM下osteoclast基因表現 98
4-4-2 抗酒石酸酸性磷酸酶染色(Tartrate-Resistant Acid Phosphatase Staining) 102
第五章 結論 104
第六章 參考文獻 105
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指導教授 胡威文(Wei-Wen Wu) 審核日期 2022-9-27
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