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研究生:黃柏勝
研究生(外文):Bo-Sheng Huang
論文名稱:嶺南野菊水萃物藉由誘導G2/M期停滯抑制骨肉留細胞生長與遷移能力
論文名稱(外文):The growth inhibition with migrationof human osteosarcoma cells through induced G2/M arrest by Vernonia patula (Dryand.) Merr water extraction
指導教授:施科念施科念引用關係黃文濤黃文濤引用關係
指導教授(外文):Ko-Nien ShihWen-Tao Huang
學位類別:碩士
校院名稱:元培科技大學
系所名稱:放射技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
畢業學年度:102
語文別:中文
論文頁數:70
中文關鍵詞:嶺南野菊骨肉瘤細胞存活細胞遷移
外文關鍵詞:Vernonia patulaosteosarcomaviabilitymigration
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本研究以水蒸氣蒸餾法所萃取之嶺南野菊精油利用MTT assay法來探討,以細胞模式來探討嶺南野菊精油(Crol)對於人類骨肉瘤細胞株的抗癌活性。結果發現嶺南野菊精油可以抑制143B與MG-63細胞生長,實驗結果顯示半致死劑量各為103.87±7.8、57.32±5.0 (μg/mL),由細胞學觀察可知Crol誘導143B細胞凋亡和DNA受損,DNA condensation和細胞週期G2/M arrest。
最後將嶺南野菊Crol利用管柱分析,依極性不同分離出7個(A~G)分離物,以 MTT assay抑制骨肉瘤細胞活性分析,結果顯示A、B、G對143B與MG-63細胞皆有抑制效果,IC50分別為9.01±5.66μg/mL、54.28±3.32μg/mL、60.97±2.09μg/mL。
癌症的轉移是多重因素造成的,且也常使治療更複雜。本研究也探討嶺南野菊精油對人類骨肉瘤癌細胞的抑制細胞遷移與貼附能力測試。我們發現Crol可經由抑制MMP-9的活性,來抑制人類骨肉瘤癌細胞腫瘤遷移能力。 以上結果顯示嶺南野菊確實對人類骨肉瘤癌細胞有毒殺作用、抑制細胞生長、抑制癌細胞轉移。將來擬進一步研究考量可做為治療人類骨肉瘤癌的新選擇方向。
關鍵字:嶺南野菊、骨肉瘤、細胞存活、細胞遷移
The objectives of this study were to investigate the effects of various concentrations of Vernoni patula oil on 143Band MG-63 cell viability, was investigated by MTT assay. Our results demonstrated the Vernonia patula oil Crol inhibition in 143B and MG-63 cell growth.Results showed that half maximal inhibitory concentration value(IC50)103.87±7.8、57.32±5.0 (μ g/mL), induced apoptosis in 143B cell DNA condensation and G2/M arrest. Through silica gel chromatography and successively eluted, varioue fractions were investigatol the bioactivity by MTT assay investigated by MTT assay. Results showed that A, B, and G inhibition in 143B and MG-63 cell growth, the IC50 was 9.01±5.66μg/mL、54.28±3.32μg/mL、60.97±2.09μg/mL. Cancer metastasis is a multifactorial disease which complicates the treatments .In the present study inhibition of migration and attachment of Vernonia patula oil.We foundthatVernoniapatula oil Crolsignificant inhibiting MMP-9 activities expression by inhibition human steosarcoma 143B and MG-63 of migration. So we believe that the Vernonia patula oil perhaps containing the capability of anti-cancer effects,and could be considered as a new strategy for anti-cancer treatment on human osteosarcoma cells
Keywoeds: Vernonia patula,osteosarcoma, viability, migration
致謝 I
摘要 II
英文摘要 III
目錄 IV
第一章緒論 1
1.1背景介紹 1
1.1.2嶺南野菊功效及相關研究 2
1.2天然物與中草藥使用 3
1.3中草藥蒸餾萃取 3
1.3.1精油萃取不同方式 3
1.4植物精油成分及運用 5
1.4.1精油的化學結構 5
1.5骨肉瘤癌細胞介紹 8
1.5.1骨肉瘤癌 8
1.5.2細胞週期控制異常 9
1.5.2骨肉瘤癌細胞生長 9
1.5.3細胞凋亡(apoptosis)與細胞壞死(necrosis) 9
1.6細胞遷移(Cell migration)與貼附(Cell attachment) 10
1.7基質金屬蛋白酶(Matrix metalloproteinase, MMPs) 11
1.8抗癌藥物的發展 12
1.9研究動機與目的 13
第二章實驗材料 14
2.1實驗試劑與藥品 14
2.1.1化學試劑與藥品 14
2.1.2實驗儀器 14
2.1.3實驗材料 15
第三章實驗方法與步驟 16
3.1實驗流程與架構 16
3.1.1萃取方法與極性分離方法 17
3.1.2管柱層析 17
3.2萃取方法 18
3.3極性分離方法 19
3.3.1管柱的填充 19
3.3.2精油極性分離 19
3.4細胞培養與實驗室藥品配製 21
3.4.1細胞來源 22
3.4.2細胞株解凍方法 22
3.4.3細胞冷凍保存與解凍 23
3.4.4細胞培養方法 23
3.4.5細胞培養液配製 24
3.4.6細胞計數 24
3.4.7細胞存活率測試(MTT assay) 25
3.4.8流式細胞儀(Flow cytometry) 26
3.4.9細胞遷移實驗(Wound healing assay) 30
3.4.10細胞貼附實驗 31
3.4.11基質金屬蛋白酶活性測試- gelatin zymography assay 32
3.4.12. DAPI細胞核螢光染色 28
3.5總類黃酮測試 33
3.6統計方法 34
第四章結果與討論 35
4.1嶺南野菊精油之萃取 35
4.1.1水蒸餾萃取 35
4.2嶺南野菊Crol極性分離結果 35
4.3嶺南野菊Crol對骨肉瘤細胞生長之影響-細胞活性測試 36
4.4嶺南野菊Crol對143B細胞週期之變化 39
4.5骨肉瘤細胞經嶺南野菊Crol處理後的細胞核DNA螢光染色 41
4.5.1嶺南野菊Crol抑制143B細胞生長影響-細胞生長型態觀察 42
4.6嶺南野菊Crol萃取物抑制骨肉瘤細胞遷移能力 43
4.7嶺南野菊Crol萃取物抑制骨肉瘤細胞貼附實驗 47
4.8基質金屬蛋白酶活性測試- Gelatin gel zymographic assay 49
4.9嶺南野菊Crol總類黃酮當量測試 51
4.10 嶺南野菊Crol極性分離後的分離物對骨肉瘤細胞之細胞活性分析 52
第五章結論 55
第六章參考文獻 59

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