臺灣博碩士論文加值系統

成骨細胞數目是造成骨質疏鬆症的重要因素之一。黑升麻活性成分之ㄧ，cimicifugin為三萜類衍生物，本研究發現cimicifugin對於成骨細胞具有分化作用。使用cimicifugin處理MG 63細胞，cimicifugin無法促進成骨細胞的增生作用，但會促進成骨細胞轉錄因子Runx2的轉錄作用。
Cimicifugin可促進成骨細胞成熟的早期分化指標(鹼性磷酸酶的活性ALP activity)與晚期分化指標(骨鈣素的含量osteocalcin levels以及礦化作用Minerlization )。Cimicifugin誘導成骨細胞的分化作用伴隨著JNK的磷酸化並具有增加雌激素受體-?Estrogen receptor-? 磷酸化的現象。接著，藉由Estrogen receptor-ㄖ磻蹌?ICI 182,780 可有效降低成骨細胞的早期與晚期分化指標(ALP activity與osteocalcin levels)，因此Estrogen receptor-ㄕbcimicifugin誘導成骨細胞分化作用中扮演著重要的角色。
此外，cimicifugin影響腫瘤壞死因子-?(TNF-? 誘導ㄧ氧化氮 (NO) 與IL-6(介白素-6)的產生，並抑制內生性前列腺素(PGE2)的含量，當cimicifugin處理MG 63細胞後，可減少TNF-˙冗冇O與IL-6的產生以及內生性PGE2。因此得知，cimicifugin可降低MG 63細胞中，由TNF-ㄘ瓟梇悸熊o炎性損傷(inflammatory damage)。在本篇研究中，我們證實了cimicifugin透過JNK及Estrogen receptor-ㄧ纁|誘導成骨細胞進行分化作用，或野戎i以成為治療骨質疏鬆症的新藥物。

The survival of osteoblast cells is one of the determinants of the development of osteoporosis in patients. This study is the first to investigate the osteoblast differentiation of cimicifugin, a triterpene derivative in a human osteoblast-like cell lines, MG 63. Cimicifugin did not exhibit significant effects on cell growth in MG 63 cells. In contrast, cimicifugin increased the marker of osteoblastic maturation (ALP activity) and differentiation (osteocalcin production). Cimicifugin also enhanced the transcript level of osteoblast-specific transcription factor Runx2. The effect of cimicifugin on the TNF-?induced production of nitric oxide (NO) in osteoblasts was examined. Treatment with cimicifugin decreased the TNF-?induced production of NO in osteoblasts. Induction of differentiation by cimicifugin was associated with increased JNK phosophorylation, and upregulation and phosphorylation of estrogen receptor. Blocking estrogen by antagonist (ICI 182,780) significantly decreased osteoblastic differentiation, suggesting estrogen receptor plays an important role in cimicifugin -induced cell differentiation. In addition, cimicifugin also decreased TNF-?mediated inflammatory in human osteoblast-like cell line. In this study, we demonstrate that cimicifugin induced osteoblastic differentiation through JNK and estrogen pathway, and it is a promising agent for treating osteoporosis.

中文摘要………………………………………………………………Ⅰ
英文摘要………………………………………………………………Ⅲ
本文目錄………………………………………………………………Ⅴ
圖表目錄………………………………………………………………Ⅷ
縮寫表…………………………………………………………………Ⅸ

第一章 緒論……………………………………………………………1
1-1骨質疏鬆症………………………………………………… 1
1-2 MAPKs與骨質疏鬆症……………………………………… 5
1-3 雌激素受體與骨質疏鬆症…………………………………8
1-4 發炎因子與骨質疏鬆症……………………………………10
1-5 黑升麻之cimicifugin的介紹…………………………… 11
1-6 研究動機……………………………………………………14
第二章 材料與方法……………………………………………………16
2-1 化學藥品和試劑（Chemicals and reagents）…………16
2-2 儀器…………………………………………………………17
2-3 藥品製備（Chemicals and reagents）…………………18
2-4 細胞培養（Cell cultures）…………………………… 18
2-5 細胞增生分析（Cell proliferation assay）…………18
2-6 成骨細胞分化指標分析……………………………………19
2-6-1 鹼性磷酸酶分析Alkaline phosphatase activity
(ALP activity)………………………………………19
2-6-2 骨鈣素分析 (Osteocaclin)…………………………20
2-6-3 礦化分析 (Minerlization)…………………………20
2-7 成骨細胞分化路徑…………………………………………21
2-7-1 利用西方墨點法偵測MAPKs、Estrogen receptor-ㄙ
熙J白質表現…………………………………………21
2-7-2 利用JNK 抑制劑和ER-ㄖ磻蹌砥A分析鹼性磷酸鹽
alkaline phosphatase (ALP activity) 及西方墨點
法，探討細胞內訊號路徑……………………………23
2-7-3 利用RT-PCR分析Runx2 mRNA的表現量………………23
2-7-4 利用kinase活性分析JNK活性……………………… 25
2-8 成骨細胞之細胞發炎反應路徑……………………………26
. 2-8-1 探討cimicifugin對TNF-ㄘ珨冗阞截L-6與NO活性抑制
分析……………………………………………………26
2-8-2 探討cimicifugin對PGE2活性抑制分析…………… 27
2-9 統計分析……………………………………………………27
第三章 結果……………………………………………………………28
3-1 Cimicifugin對MG 63細胞株增生影響…………………… 28
3-2 Cimicifugin對MG 63細胞株的成熟與分化指標分析…… 28
3-3Cimicifugin增加Runx2 mRNA與蛋白質的表現…………… 29
3-4 Cimicifugin調控MG 63細胞株之MAPKs的活性……………30
3-5 Cimicifugin調控MG 63細胞株之ER-ㄙ漪〝吽K.…………31
3-6 Cimicifugin調控MG 63細胞株之MAPKs與ER-ㄙ疑鰜Y……31
3-7 Cimicifugin對TNF-ㄘ珨冗阞摸O及IL-6產物的抑制能力探
討…………………………………………………………… 32
3-8 Cimicifugin對抑制內生性PGE2作用影響…………………32
第四章 討論……………………………………………………………34
第五章 結論……………………………………………………………37
參考文獻……………………………………………………………… 38
圖表…………………………………………………………………… 50

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