臺灣博碩士論文加值系統

骨質疏鬆症、癌症骨轉移、類風濕性關節炎等疾病都會導致骨質的流失。當蝕骨細胞的活性大於造骨細胞的活性時，整體骨質就會減少。抑制蝕骨細胞的分化和成熟是改善骨質流失的策略之一。本研究使用RAW264.7巨噬細胞株為細胞模式，發現丁香精油具有抑制蝕骨細胞分化作用，並找到其有效成分為eugenol。因此，本實驗目的為探討eugenol對蝕骨細胞分化的影響和其分子機制。實驗結果顯示eugenol呈現濃度相關性地抑制RANKL所誘導的蝕骨細胞分化，且無明顯的細胞毒性，而其一半有效抑制劑量(IC50)為54.7 μM。此外，eugenol會抑制c-Fos和NFATc1的mRNA和蛋白表現量，而real time-PCR實驗結果顯示NFATc1的下游基因OSCAR、CTR、DC-STAMP、TRAP及MMP-9的mRNA表現量也都受到eugenol處理而降低。除此之外，eugenol會抑制phospho-p38和phospho-ERK的表現，也會抑制IκB被降解以及細胞核內NF-κB的表現量。最後pit formation assay結果證實eugenol會抑制骨質蝕損的作用。綜合以上結果，eugenol可能具有治療骨質流失的潛力。

Several major bone diseases are directly attributable to bone loss, including osteoporosis, bone metastasis, and rheumatoid arthritis. Osteoporosis reflected the enhancement of osteoclast activity. Therefore, blocking the differentiation and maturation of osteoclast is a possible strategy to address this problem. In this study, RAW264.7 macrophage cell line was used as cell model to find anti-osteoclastogenesis effect of essential oil. We found clove oils showed the inhibition effect of osteoclast differentiation, and identified the effective component is eugenol. The aim of this study was to investigate the inhibition effect of eugenol on osteolcastogenesis and the molecular mechanism. The results showed eugenol had the ability to suppress RANKL inducing osteoclast differentiation in a dose dependent manner, and the half maximal inhibitory concentration (IC50) of eugenol is 54.7 μM. The mRNA and protein expression of c-Fos and NFATcl were decreased under treatment of eugenol. The gene expression of OSCAR, CTR, DC-STAMP, TRAP, and MMP-9 induced by RANKL was also suppressed by eugenol. In addition, eugenol inhibited phospho-p38, phospho-ERK and NF-κB expression, and decreased the degradation of IκB. Finally, pit formation assay demonstrated eugenol can block bone resorption. In conclusion, we suggested eugenol shows potential as a treatment for osteoporosis.

中文摘要 I
Abstract II
英文縮寫表對照表 III
第一章、緒論 1
一、骨質疏鬆症： 1
(一) 骨質疏鬆症的定義 1
(二) 骨質疏鬆症的流行病學 1
(三) 骨質疏鬆症的分類 2
(四) 骨質疏鬆症的診斷 3
二、骨骼重塑 (bone remodeling) 3
三、蝕骨細胞的分化 5
四、RANKL訊息傳遞路徑 6
五、抗酒石酸酸性磷酸酶 (TRAP) 8
六、蝕損作用 8
七、臨床上骨質疏鬆症的治療 9
八、草藥、芳香療法與精油 11
九、丁香精油 13
十、研究動機與目的 14
十一、實驗設計 15
(一) 精油篩選平台的建立 15
(二) 具抑制蝕骨細胞分化的精油有效成分鑑定 15
(三) 有效成分對於蝕骨細胞分化的影響與分子機制研究 16
(四) 有效成分抑制骨質蝕損的情形 16
第二章、材料與方法 17
一、實驗材料 17
二、儀器設備 19
三、實驗方法 19
第三章、結果 27
一、精油之抑制蝕骨細胞分化活性篩選 27
二、丁香精油的主要成分分析 27
三、丁香精油和其主要成分eugenol對於RAW264.7的細胞毒性 27
四、丁香精油和eugenol對於蝕骨細胞分化的影響 28
五、以TRAP活性來確認eugenol對蝕骨細胞的抑制影響 29
六、Eugenol對於不同分化時期的蝕骨細胞影響 29
七、Eugenol對轉錄因子NFATc1和c-Fos mRNA表現的影響 30
八、Eugenol對NFATc1下游基因表現的影響 30
九、Eugenol對NFATc1和c-Fos 蛋白在核內表現的影響 31
十、Eugenol對NF-κB和IκB蛋白表現量的影響 32
十一、Eugenol對p38、ERK磷酸化蛋白表現的影響 32
十二、Eugenol結構相似物對蝕骨細胞分化的影響 33
十三、Eugenol對於RANKL誘導蝕骨細胞蝕損作用的影響 33
第四章、討論 35
一、丁香精油和eugenol的安全性探討 35
二、篩選系統的探討 35
三、Eugenol對於蝕骨細胞不同分化時期的影響探討 36
四、Eugenol對於蝕骨細胞訊號傳遞影響的探討 37
(一) MAPK訊號傳遞 37
(二) NF-κB訊號傳遞 37
(三) PI3K訊號傳遞 38
(四) Ca2+訊號傳遞 38
五、Eugenol對於NFATc1和其下游基因影響的探討 39
六、抑制骨質流失的策略探討 40
(一) 阻止RANKL與它的ligand結合 40
(二) 抑制RANKL訊號傳遞過程 41
(三) 抑制蝕骨細胞的融合和成熟 41
(四) 促進蝕骨細胞凋亡 42
七、Eugenol相似物對抑制蝕骨細胞影響的探討 42
八、總結 43
第五章、表格與附圖 44
表一、Real time-PCR引子序列 44
表二、抑制蝕骨細胞分化有效精油篩選表 45
圖一、骨骼重塑過程圖 46
圖二、造血幹細胞的分化流程圖 47
圖三、蝕骨細胞的新生和骨質蝕損 48
圖四、蝕骨細胞的新生流程圖 49
圖五、RANKL的訊號傳遞 50
圖六、NFATc1調控蝕骨細胞分化流程圖 51
圖七、進行骨質蝕損之蝕骨細胞 52
圖八、NFATc1的自我放大效應 53
圖九、Eugenol對於蝕骨細胞不同分化時期的影響之實驗設計 54
圖十、丁香精油的GC-MS圖 55
圖十一、細胞存活率分析 56
圖十二、丁香精油和eugenol對蝕骨細胞分化的影響-TRAP染色圖 57
圖十三、丁香精油和eugenol對蝕骨細胞分化的影響-蝕骨細胞計數 58
圖十四、Eugenol對蝕骨細胞分化的影響-TRAP活性分析 59
圖十五、Eugenol對蝕骨細胞不同分化時期的影響 60
圖十六、Eugenol對RANKL所誘導c-Fos和NFATc1 mRNA表現的影響 61
圖十七、Eugenol對RANKL所誘導OSCAR和CTR mRNA表現的影響 62
圖十八、Eugenol對於RANKL所誘導TRAP、DC-STAMP和MMP-9 mRNA表現的影響 63
圖十九、Eugenol對於RANKL誘導NFATc1和c-Fos蛋白在核內表現的影響 64
圖二十、Eugenol對於RANKL誘導IκB分解和NF-κB細胞核中表現量的影響 65
圖二十一、Eugenol對於RANKL誘導p38、ERK磷酸化的影響 66
圖二十二、Eugenol相似物對蝕骨細胞分化的影響 67
圖二十三、Eugenol相似物結構圖 68
圖二十四、細胞存活率-isoeugenol 69
圖二十五、Eugenol對於RANKL誘導蝕骨細胞蝕損作用的影響 70
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