臺灣博碩士論文加值系統

乳癌為婦女最常見之惡性腫瘤，每年造成460,000婦女死亡。其主要死因為癌症擴散至其他部位，其中有30%為乳癌之骨轉移，而乳癌之骨轉移主並非透過乳癌細胞直接影響，而是藉由乳癌細胞所分泌之細胞激素影響骨骼重塑之過程。牛樟芝及猴頭菇富含多種生理活性物質，本研究主要之兩種樣品分別為由牛樟芝所萃取出類似antroquninonol之純化合物NB-AB，另為由猴頭菇所萃取出之erinacine類純化合物NB-EA，兩者均具抗癌活性。結果顯示NB-EA和NB-AB在正常分化組別及乳癌細胞培養基共培養組別皆能顯著抑制RAW 264.7分化成蝕骨細胞，此抑制作用可能透過MAPK傳訊路徑。此外，NB-EA及NB-AB皆抑制MDA-MB-231乳癌細胞中Erk及JNK之磷酸化活化，進而抑制MDA-MB-231之移行能力及基質金屬蛋白酶9(MMP 9)之活性。本研究於細胞模式中證明NB-EA和NB-AB對抗乳癌細胞之轉移潛力及基礎機制。

Breast cancer is the most prevalent female tumor which causes 460,000 annual deaths. Most of the fatality does not come from breast cancer in its primary site, but rather from the metastasis malignancy. Bone is the major metastasis site of breast cancer,and bone metastasis, which arises mainly by osteoclasts,accounts for over 30% among all secondary tumor cases of breast cancer. Antrodiacinnamomea and Hericiumerinaceusare rich sources of several classes of bioactive compounds. NB-AB, an antroquinonol derivative, was purified from Antrodiacinnamomea,andNB-EA, an erinacine purified from Hericiumerinaceus,both possess anticancer activities and were employed in this study. NB-EA and NB-AB significantly inhibited the differentiation of Raw 264.7 to osteoclast, possibly through MAPK pathway, either in normal differentiation media or in medium supplemented with conditioned media from the culture media of breast cancer cells. Furthermore,NB-EA and NB-ABalso suppressed matrix metalloproteinase 9 (MMP 9) activity and the migration of MDA-MB-231 cells, which might be related to the inactivation of Erk and JNK.In this study, we have demonstrated the anti-metastatic potential and underlying mechanisms of NB-EA and NB-AB in an in vitro cell model.

CONTENTS
摘要 1
Abstract 2
Chapter 1 Literature review 3
1-1 Antrodia cinnamomea 3
1-2 Antrodia cinnamomea bioactive components 3
1-2-1 4-Acetylantroquinonol B 4
1-3 Hericium erinaceus 4
1-3-1 Erinacine A 5
2-4 Breast cancer and bone metastasis 5
1-5 Bone remodeling 7
1-6 Matrix metalloproteinases 8
1-7 Mitogen-activated protein kinases(MAPKs) 8
1-7-1 Extracellular signal-regulated kinases (Erk) 9
1-7-2c-Jun NH2-terminal protein kinase (JNK) 9
Chapter 2 Experiment design 10
Chapter 3 Materials and methods 11
3-1 Experimental materials 11
3-2 Experimental reagents 11
3-2-1 Cell culture reagents 11
3-2-2 TRAP activity assay 11
3-2-3 Protein quantification assay 12
3-2-4 ELISA 12
3-2-5 Western blotting 12
3-2-6 Gelatin zymography 13
3-2-7 Wound-healing assay 14
3-3 Experiment equipment 14
3-4 Methods 15
3-4-1 Cells and cultural conditions 15
3-4-2 Cell proliferation assay 16
3-4-3 Osteoclast differentiation and TRAP activity assay 16
3-4-4 Wound-healing assay 17
3-4-5 Gelatin zymography 17
3-4-6 Western blot analysis 18
3-4-7 ELISA 18
3-4-8 Statistical analysis 19
Chapter 4 Results 20
4-1 Proliferation 20
4-1-1 Cell proliferation with NB-EA 20
4-1-2 Cell proliferation with NB-AB 20
4-2 TRAP activity 23
4-2-1 TRAP activity treated with NB-EA 23
4-2-2 TRAP activity treated with NB-AB 23
4-3 Wound-healing 28
4-3-1 Wound-healing of MCF-7 and MDA-MB-231 treated with NB-EA 28
4-3-2 Wound-healing of MCF-7 and MDA-MB-231 treated with NB-AB 28
4-4 TGF-β concentration by ELISA assay 35
4-4-1 TGF-β concentration of osteoclast treated NB-EA 35
4-4-2 TGF-β concentration of osteoclast treated NB-AB 36
4-5 Gelatin Zymography 39
4-6 Western blot 40
4-6-1 MDA-MB-231 treated with NB-EA 40
4-6-2 MDA-MB-231 treated with NB-AB 40
4-6-3 Normal differentiation group treated with NB-EA 43
4-6-4 Normal differentiation group treated with NB-AB 43
4-6-5 Conditioned medium group treated with NB-EA 46
4-6-6 Conditioned medium group treated with NB-AB 46
Chapter 5 Discussion 49
Chapter 6 References 51

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