臺灣博碩士論文加值系統

運動會刺激骨骼肌肉膜蛋白 (Fibronectin type III domain-containing protein 5； FNDC5)大量表現， 大量表現， FNDC5裂解後以 鳶尾素 (Irisin) 型式分泌， 釋放到血液循環型式分泌， 釋放到血液循環型式分泌， 釋放到血液循環改善肥胖與穩定血糖， 對更年期慢性併發症亦有幫助。依先前研究Irisin第 75個胺基酸 Arg-75與 Irisin形成雙體結構 (dimer) 高度相關。 若將 Irisin Arg-75胺基酸點突變為 Glu，產生之 IrisinR75E突變將導致 Irisin雙體結構的形成及功能 受影響。類固醇藥物 受影響。類固醇藥物 受影響。類固醇藥物 Prednisolone是過敏或發炎反應病患緩解症狀的常用藥，然 是過敏或發炎反應病患緩解症狀的常用藥，然 該類藥物會誘發骨質疏鬆症 (Glucocorticoid-induced osteoporosis；GIOP)，是繼 發性骨質疏鬆症最普遍生的原因，率僅亞於停經相關。
本研究探討 Irisin和 IrisinR75E在體外 (in vitro) 可否刺激骨細胞增生和分化， 可否刺激骨細胞增生和分化， 可否刺激骨細胞增生和分化， 進而有效預防和治療 GIOP，降低骨折發生率。以藥物 Prednisolone對成骨細胞 hFOB1.19進行處理，並於 24小時後觀察對細胞毒殺性；另將 Predniolone合併 給予 Irisin和 IrisinR75E後，觀察細胞存活率之影響。於斑馬魚骨鬆模式中在養 殖水中添加 Prednisolone 50 μM，於 2週後觀察魚鱗，模擬 GIOP的形成。結果 顯示， 顯示， Irisin會促進成骨細胞的增生，但 會促進成骨細胞的增生，但 會促進成骨細胞的增生，但 IrisinR75E則否。動物實驗顯示， 則否。動物實驗顯示， 則否。動物實驗顯示， 則否。動物實驗顯示， Prednisolone會抑制成骨細胞生長，但外加 會抑制成骨細胞生長，但外加 會抑制成骨細胞生長，但外加 Irisin可增加成骨細胞的 ALP活性，並抑制 活性，並抑制 活性，並抑制 TRAP的活性，代表 Irisin可能促進成骨細胞增生，加密度改善質疏鬆。由本研究建立之斑馬魚骨鬆模式，發現 Irisin蛋白具有延緩類固醇誘發骨質疏鬆症的治療之潛力，並可利用此斑馬魚平台進一步探討人類骨質疏鬆及方式。

Regular exercise stimulated the expression of the Fibronectin type III domain-containing protein 5 (FNDC5) in skeletal muscle. In post-translation modification process, FNDC5 cleavage to a new hormone: Irisin. Through released into blood circulation, Irisin help to improve obesity and stable blood sugar and menopause chronic complications. Base on previous studies, the IrisinR75E mutant impair Irisin dimerization through interfere salt bridge formation, it also leading to the functional deficiency in Irisin. Prednisolone, one kind of Glucocorticoids (GCs) drugs, that widely used in inflammatory and autoimmune conditions (allergy) treatment, however, it induced osteoporosis. Glucocorticoid-induced osteoporosis (GIOP) is the most common cause of secondary osteoporosis, the incidence rate was second to menopause-related osteoporosis. In this study, we have explored whether Irisin and IrisinR75E could stimulate osteoblast proliferation and differentiation in vitro, and clarified the Irisin and IrisinR75E in GIOP prevention effect in vivo. To discuss the proliferation effect, “Prednisolone” was added to the hFOB1.19 osteoblast cells, and cell viability was measured 24 hours post-treatment; In zebrafish osteoporosis model, zebrafishes were cultured and immersed in Prednisolone (50 µM) for 2 weeks. The results indicated that Irisin stimulated osteoblast proliferation, but not IrisinR75E. Prednisolone inhibited osteoblast growth, but this phenomenon could be reversed by Irisin through upregulation of ALP activity and downregulation of TRAP activity. The conclusion represents that irisin enhances of osteoblast proliferation, bone density and improve osteoporosis. We suggest that Irisin could be used as a therapeutic agent for GIOP, besides, the established zebrafish platform able to apply for human osteoporosis studies.

第一章 緒論 1
第一節 Irisin和IrisinR75E介紹 1
第二節 類固醇引起的骨質疏鬆症(Glucocorticoid-induced osteoporosis，以下簡稱GIOP)介紹 4
第三節 斑馬魚(Zebrafish)當作研究骨質疏鬆平台介紹 9
第四節 本論文研究目的和重要性 10
第二章 實驗材料與研究方法 11
第一節、 細胞株之培養 11
第二節、實驗動物 14
第三節、細胞增生率測定 15
第四節、蛋白質定量分析 16
第五節、SDS-PAGE 電泳膠製作 16
第六節、西方墨點法分析（Western Blot Analysis） 17
第七節、斑馬魚鱗ALP(alkaline phosphatase)活性實驗 18
第八節、斑馬魚鱗TRAP(tartrate-resistant acid phosphatase)活性實驗 18
第九節、即時定量聚合酶連鎖反應(quantitative real-time polymerase chain reaction, Q-PCR) 19
第十節、細胞型態分析(Cell morphology assay) 21
第十一節、統計學分析 21
第三章 研究結果 22
第四章 討論 27
圖表說明 29
附錄 40

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