冬蟲夏草水萃液對攝護腺癌所引起之小鼠蝕骨細胞分化的影響

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研究生：	袁倫浩
論文名稱：	冬蟲夏草水萃液對攝護腺癌所引起之小鼠蝕骨細胞分化的影響 Effects of Cordyceps sinensis on the prostate cancer cell-induced mice osteoclast cell differentiation
指導教授：	江啟勳
口試委員:
學位類別：	碩士 Master
系所名稱：	原子科學院 - 生醫工程與環境科學系 Department of Biomedical Engineering and Environmental Sciences
論文出版年：	2010
畢業學年度：	98
語文別：	中文
論文頁數：	60
中文關鍵詞：	冬蟲夏草、攝護腺癌、蝕骨細胞
相關次數：	點閱：3 下載：0
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冬蟲夏草被中國人視為保健聖品，為一良好的補益藥材，經研究證實在臨床上可應用在對抗腫瘤、肝臟細胞修復、提昇免疫功能等療效，在最近的研究中也指出冬蟲夏草水萃液有效的抑制由腫瘤細胞所誘發的蝕骨細胞分化，所以，本實驗旨在建立蝕骨細胞的體外培養模式，並且更進一步地探討冬蟲夏水萃液抑制蝕骨細胞分化的機轉。由本論文的實驗結果得知，冬蟲夏草水萃液可以抑制單核球細胞/巨噬細胞的共同前驅細胞分化成單核的前驅蝕骨細胞，並且也顯著地抑制單核的前驅蝕骨細胞進行融合作用，形成成熟的蝕骨細胞。另外，我們觀察到冬蟲夏草能有效地抑制由攝護腺癌所誘發的蝕骨細胞分化，且主要是抑制在前驅蝕骨細胞融合的階段；而我們也發現攝護腺癌細胞會分泌細胞激素CCL2促進前驅蝕骨細胞進行融合作用。總結來說，冬蟲夏草的確可以有效地抑制由腫瘤細胞間接或直接地引起的蝕骨細胞分化，在日後臨床上的應用將有重大的貢獻。

摘要 ---------------------------------------------------- IV
Abstract ------------------------------------------------ IV
誌謝 ---------------------------------------------------- IV
目錄 ---------------------------------------------------- IV
第一章 序論 ---------------------------------------------- 1
1冬蟲夏草----------------------------------------------- 1
1.1簡介 ------------------------------------------------ 1
1.2冬蟲夏草的藥理療效 ---------------------------------- 2
2蝕骨細胞 ---------------------------------------------- 2
3研究動機與目的 ---------------------------------------- 5
第二章 材料與方法 ---------------------------------------- 8
1材料來源----------------------------------------------- 8
1.1冬蟲夏草水萃液來源 ---------------------------------- 8
1.2冬蟲夏草的萃取 -------------------------------------- 8
1.3動物來源 -------------------------------------------- 8
1.4抗體來源 -------------------------------------------- 9
2 細胞培養 --------------------------------------------- 9
2.1配製DMEM細胞培養液 ---------------------------------- 9
2.2配製α-Medium細胞培養液 ----------------------------- 9
2.3細胞培養液的配方 ------------------------------------ 9
2.3.1小鼠攝護腺癌細胞株 (TRAMP-C1) --------------------- 9
2.3.2小鼠巨噬細胞株 (RAW264.7) ------------------------ 10
2.4細胞繼代 ------------------------------------------- 10
2.5小鼠攝護腺癌細胞株條件培養液 ----------------------- 10
2.6蝕骨細胞培養 --------------------------------------- 11
2.6.1小鼠巨噬細胞株 (RAW264.7) ------------------------ 11
2.6.2小鼠骨髓細胞 ------------------------------------- 12
2.7抗酒石酸酸性磷酸酵素染色法(Tartrate-resistant Acid Phosphatase Stain ; TRAP stain) ------------------------- 14
2.8抗酒石酸酸性磷酸酵素活性分析(Tartrate-resistant Acid Phosphatase activity assay ; TRAP activity assay) ------- 15
2.9蛋白質濃度偵測 ------------------------------------- 16
3質體建構 --------------------------------------------- 17
3.1 pIRES-mTRAP-EGFP和pIRES-mTRAP-Rluc質體建構 -------- 17
3.1.1限制酶酵素反應 ----------------------------------- 17
3.1.2限制酶酵素反應 ----------------------------------- 18
3.1.3洋菜膠電泳 --------------------------------------- 19
3.1.4 DNA的接合反應 ----------------------------------- 19
3.2所有質體製備方式 ----------------------------------- 20
3.2.1細菌熱休克轉殖法 --------------------------------- 20
3.2.2微量純化製備質體 --------------------------------- 20
3.2.3中量純化製備質體 --------------------------------- 21
4質體在轉染細胞中表現情況 ----------------------------- 22
4.1細胞存活試驗 --------------------------------------- 22
4.2細胞轉染 ------------------------------------------- 23
5螢光顯微鏡觀察 --------------------------------------- 23
第三章實驗結果 ------------------------------------------ 25
1質體建構 --------------------------------------------- 25
1.1 pIRES-mTRAP-EGFP質體建構 -------------------------- 25
1.2 pIRES-mTRAP-Rluc質體建構 -------------------------- 27
2質體在轉染細胞中的表現 ------------------------------- 29
2.1巨噬細胞株(RAW264.7)對Neomycin (G418)耐受性測試 ---- 29
2.2 把質體DNA送入細胞中 ------------------------------- 30
2.3利用細胞激素誘導轉染細胞的表現 --------------------- 31
3利用巨噬細胞株(RAW264.7)建立蝕骨細胞體外培養模式 ----- 31
4利用小鼠骨髓幹細胞建立蝕骨細胞體外培養模式 ----------- 32
5冬蟲夏草在體外培養模式中對蝕骨細胞分化的影響 --------- 32
6攝護腺癌在體外培養模式中對蝕骨細胞分化的影響 --------- 33
7冬蟲夏草對攝護腺癌在體外培養模式中所引起蝕骨細胞分化的
影響 ---------------------------------------------------- 35
第四章討論 ---------------------------------------------- 37
圖表及說明 ---------------------------------------------- 42
附錄 ---------------------------------------------------- 58
參考文獻 ------------------------------------------------ 59

                                

1. Xiao YQ. [Studies on chemical constituents of Cordyceps sinensis I]. Zhong Yao Tong Bao 1983; 8: 32‐3.
2. Ng TB, Wang HX. Pharmacological actions of Cordyceps, a prized folk medicine. J Pharm Pharmacol 2005; 57: 1509‐19.
3. Zhu JS, Halpern GM, Jones K. The scientific rediscovery of a precious ancient Chinese herbal regimen: Cordyceps sinensis: part II. J Altern Complement Med 1998; 4: 429‐57.
4. Nakamura K, Yamaguchi Y, Kagota S, Kwon YM, Shinozuka K, Kunitomo M. Inhibitory effect of Cordyceps sinensis on spontaneous liver metastasis of Lewis lung carcinoma and B16 melanoma cells in syngeneic mice. Jpn J Pharmacol 1999; 79: 335‐41.
5. Seeman E, Delmas PD. Bone quality‐‐the material and structural basis of bone strength and fragility. N Engl J Med 2006; 354: 2250‐61.
6. Hattner R, Epker BN, Frost HM. Suggested sequential mode of control of changes in cell behaviour in adult bone remodelling. Nature 1965; 206: 489‐90.
7. Martin TJ, Sims NA. Osteoclast‐derived activity in the coupling of bone formation to resorption. Trends Mol Med 2005; 11: 76‐81.
8. Segovia‐Silvestre T, Neutzsky‐Wulff AV, Sorensen MG, et al. Advances in osteoclast biology resulting from the study of osteopetrotic mutations. Hum Genet 2009; 124: 561‐77.
9. Udagawa N, Takahashi N, Akatsu T, et al. Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow‐derived stromal cells. Proc Natl Acad Sci U S A 1990; 87: 7260‐4.
10. Halleen JM, Raisanen SR, Alatalo SL, Vaananen HK. Potential function for the ROS‐generating activity of TRACP. J Bone Miner Res 2003; 18: 1908‐11.
11. Halleen JM, Raisanen S, Salo JJ, et al. Intracellular fragmentation of bone resorption products by reactive oxygen species generated by osteoclastic tartrate‐resistant acid phosphatase. J Biol Chem 1999; 274: 22907‐10.
12. Janckila AJ, Takahashi K, Sun SZ, Yam LT. Tartrate‐resistant acid phosphatase isoform 5b as serum marker for osteoclastic activity. Clin Chem 2001; 47: 74‐80.
13. Roodman GD. Biology of osteoclast activation in cancer. J Clin Oncol 2001; 19: 3562‐71.
14. Piper PK, Jr., Gruntmanis U. Management of osteoporosis in the aging male: focus on zoledronic acid. Clin Interv Aging 2009; 4: 289‐303.
15. Mundy GR. Metastasis to bone: causes, consequences and therapeutic
opportunities. Nat Rev Cancer 2002; 2: 584‐93.
16. Lu Y, Cai Z, Xiao G, et al. Monocyte chemotactic protein‐1 mediates prostate cancer‐induced bone resorption. Cancer Res 2007; 67: 3646‐53.
17. Thomas RJ, Guise TA, Yin JJ, et al. Breast cancer cells interact with osteoblasts to support osteoclast formation. Endocrinology 1999; 140: 4451‐8.
18. Guo Y, Tiedemann K, Khalil JA, Russo C, Siegel PM, Komarova SV. Osteoclast precursors acquire sensitivity to breast cancer derived factors early in differentiation. Bone 2008; 43: 386‐93.
19. Kim MS, Magno CL, Day CJ, Morrison NA. Induction of chemokines and chemokine receptors CCR2b and CCR4 in authentic human osteoclasts differentiated with RANKL and osteoclast like cells differentiated by MCP‐1 and RANTES. J Cell Biochem 2006; 97: 512‐8.
20. Mizutani K, Sud S, Pienta KJ. Prostate cancer promotes CD11b positive cells to differentiate into osteoclasts. J Cell Biochem 2009; 106: 563‐9.
21. Morgan H, Tumber A, Hill PA. Breast cancer cells induce osteoclast formation by stimulating host IL‐11 production and downregulating granulocyte/macrophage colony‐stimulating factor. Int J Cancer 2004; 109: 653‐60.
22. Mizuha Y, Yamamoto H, Sato T, et al. Water extract of Cordyceps sinensis (WECS) inhibits the RANKL‐induced osteoclast differentiation. Biofactors 2007; 30: 105‐16.
23. Jordan JL, Sullivan AM, Lee TD. Immune activation by a sterile aqueous extract of Cordyceps sinensis: mechanism of action. Immunopharmacol Immunotoxicol 2008; 30: 53‐70.

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