臺灣博碩士論文加值系統

人類骨骼的形態與構成，涉及成骨細胞的骨基質合成和蝕骨細胞的骨骼再吸收這兩者之間微妙的調控。蝕骨細胞是一個巨大並具有多核的細胞，主要由巨噬細胞和單核球的前驅細胞分化而來。當人體蝕骨細胞活性大於成骨細胞時就會造成骨質疏鬆症。 RANK信息藉由細胞質因子調控幾種不同的蛋白激酶訊息路徑，影響蝕骨細胞的生成和活化；這些蛋白路徑分別是 NF-κB kinase、 JNK、 p38、 ERK。
異甜菊醇（isosteviol）是甜葉菊葉內成份之一甜菊糖（stevioside）經酸水解而成的衍生化合物；異甜菊醇衍生化合物在先前文獻中即具有良好的生物活性，包括可以抑制發炎反應、細菌活性。本研究主角 NC-8 (ent-16-oxobeyeran-19-N-methylureido)是以異甜菊醇為基礎後進行生物催化或化學修飾而成的新型化合物。
在最近文獻中， NC-8透過干擾 NF-κB路徑有效抑制 B型肝炎病毒（HBV）表面抗原分泌與基因表現。蝕骨細胞分化的活性與 NC-8抑制 HBV，兩者都與 NF-κB羠訊息路徑有關聯。因此，在本研究中我們將探討 NC-8是否透過 NF-κB訊息路徑影響 RANKL誘導 RAW 264.7細胞形成蝕骨細胞的功能。
我們利用 MTT Assay觀察 NC-8對 RAW 264.7細胞毒性並使用抗酒石酸酸性磷酸酶(TRAP)染色，做為鑑定蝕骨細胞專一性染色法。實驗中發現 NC-8對 RAW 264.7細胞具毒性濃度 IC50在 40 μg/ml；在抗酒石酸酸性磷酸酶(TRAP)染色結果顯示 RAW 264.7 細胞受到 RANKL誘導分化成為蝕骨細胞情形會被 NC-8所抑制。我們的實驗中發現， RANKL所誘發的 ERK， p38和 JNK磷酸化會因加入 NC-8而被抑制下來。因此在本研究中， NC-8不單單可以成為新型治療病毒性肝炎之潛力藥物，還可以進一步抑制單核球或者巨噬前驅細胞分化成為蝕骨細胞以間接避免骨質疏鬆的產生。

Morphogenesis and remodeling of bone involve the synthesis of bone matrix by osteoblasts and the coordinate resorption of bone by osteoclasts. Osteoclasts are multinucleated giant cells differentiated from the precursors of monocyte/macrophage cell lineage. When osteoclasts more activated than osteoblasts cells can cause osteoporosis in human body. RANK signaling pathways is mediated by cytoplasm factor several different protein kinases signaling; these proteins are NF-κB kinase, JNK, p38, ERK, influence osteoclast formation and activation.
Stevioside is a natural sweetener that exists in leaves of Stevia rebaudiana Bertoni. Stevioside synthesis of new compounds derived from acid hydrolysis modified. Isosteviol derived compounds in the previous paper, having good biological activity, including anti-inflammation, antibacterial activity. In this study, the NC-8 (ent-16-oxobeyeran-19-N-methylureido) that is modification by biocatalytic or chemical of isosteviol .
Recently paper show, NC-8 inhibited HBV viral secretion of surface antigen and gene expression by interfere NF-κB signaling pathway. Both osteoblasts formation activation and NC-8 inhibits HBV by NF-κB signaling pathway. Thus, in this study we explore whether NC-8 will influence the formation of osteoclasts cells from RANKL induced RAW 264.7 cells by NF-κB signaling pathway.
We use MTT Assay observed NC-8 will result in RAW 264.7 cell toxicity and use tartrate- resistant acid phosphatase(TRAP) staining as the identification of osteoclast- specific staining. The experimental, we found that cytotoxic of NC-8 for RAW 264.7, the IC50 value is 40 μg/ml. In TRAP staining that NC- 8 is a novel effect which inhibits osteoclasts-like cell formation. Our experiments found that, RANKL induced ERK, p38 and JNK phosphorylation was inhibited by NC-8.
The novel function of NC- 8 demonstrated in this study indicates a new role for NC- 8 in negative regulation osteoclastogenesis and will be valuable in developing better strategies for treating osteopenic disorders in the future.

目 錄

誌 謝 - 1 -
中文摘要 - 3 -
ABSTRACT - 5 -
目 錄 - 7 -
圖 目 錄 - 9 -
第 一 章 前 言 - 10 -
第一節 研究背景 - 11 -
1. Isosteviol - 11 -
2. NC-8 - 11 -
3. 骨的生理學 - 12 -
4. 成骨細胞(osteoblast) - 13 -
5. 蝕骨細胞(Osteoclast) - 14 -
6. 蝕骨細胞分化信息路徑(Osteoclast differentiation signaling pathway) - 15 -
第二節 研究目標 - 19 -
第 二 章 材料與方法 - 20 -
第一節 研究設計 - 21 -
1. NC-8的製備 - 22 -
2. NC-8的細胞毒性實驗 - 22 -
3. TRAP染色觀察蝕骨前驅細胞分化情形 - 22 -
4. 蝕骨細胞 MAPKs與 NF-κB分子蛋白表現 - 23 -
5. 蝕骨細胞溶蝕能力 - 24 -
第二節 研究材料 - 25 -
1. 細胞株 - 25 -
2. 抗體 - 25 -
3. 一般材料 - 26 -
4. 實驗儀器 - 26 -
5. 化學藥品與試劑 - 27 -
6. 緩衝液製備 - 28 -
第三節 實驗方法 - 29 -
1. 細胞培養 - 29 -
2. 細胞毒性試驗 - 29 -
3. 蝕骨細胞分化實驗 - 30 -
4. 抗酒石酸酸性磷酸染色(TRAP Staining) - 30 -
5. 西方墨點法(Western blotting) - 31 -
6. 骨質再吸收實驗 - 32 -
7. 分析統計 - 33 -
第 三 章 實驗結果 - 34 -
1. RAW 264.7細胞處理 NC-8之細胞毒性 - 35 -
2. RANKL誘導蝕骨細胞處理不同濃度 NC-8之影響 - 35 -
3. RANKL誘導蝕骨細胞處理10 μg/ml NC-8不同時間之影響 - 36 -
4. NC-8影響 RANKL誘導蝕骨細胞MAP kinase路徑與 NF-κB蛋白活化的情形 - 36 -
5. NC-8影響 RANKL誘導蝕骨細胞的溶蝕骨能力 - 38 -
第 四 章 討 論 - 39 -
第 五 章 結 論 - 43 -
參 考 文 獻 - 45 -
圖 表 說 明 - 51 -

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