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研究生:官沛瑩
研究生(外文):Pei-Ying Guan
論文名稱:天然物SLN選擇性殺死乳癌細胞誘導細胞G2/M期停滯及造成細胞凋亡
論文名稱(外文):Natural product SLN selectively kills breast cancer cells involving G2/M arrest and apoptosis
指導教授:黃弘文黃弘文引用關係
指導教授(外文):Hung-Wen Huang
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:41
中文關鍵詞:乳癌氧化壓力凋亡G2/M停滯軟珊瑚
外文關鍵詞:SLNsoft coraloxidative stressbreast cancerapoptosisG2/M arrest
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根據一些研究指出,從軟珊瑚純化的天然產物SLN,能夠抑制某些癌症的增殖,但對於其選擇性殺傷潛力的研究卻很少。本篇論文的目的是,評估乳癌細胞的選擇性殺傷潛力並探討其中可能的機制。選擇性殺傷潛力由MTS測定來確定,氧化壓力、粒線體膜電位、細胞週期、細胞凋亡和氧化性DNA損傷等分析則使用流式細胞儀來檢測,所有實驗皆處理SLN 24 h。在MTS測定中,SLN降低了兩種乳癌細(SKBR3和MDA-MB-231)的細胞存活率,但對乳腺正常細胞(M10)幾乎沒有損傷。在7-aminoactinomycin D (7AAD)染色下,SLN劑量效應地誘導SKBR3細胞的G2/M期停滯,經由annexinV/7AAD雙染檢測,SLN在SKBR3細胞有誘導細胞凋亡結果。SLN劑量效應地誘導氧化壓力(reactive oxygen species; ROS)產生、粒線體去極化和粒線體超氧化物的產生。總之,SLN對於乳癌細胞具有選擇性殺傷,能夠誘導ROS增加,G2/M停滯,細胞凋亡和氧化性DNA損傷。
SLN, the natural product purified from soft corals, was reported to inhibit proliferation against some cancers but its selective killing potential was seldom investigated. The purpose of this study is to evaluate the selective killing potential of breast cancer cells and to explore its possible mechanisms. The selective killing potential is determined by MTS assay. The oxidative stress (reactive oxygen species (ROS) generation, mitochondrial membrane potential (MitoMP), and mitochondrial superoxide), cell cycle analysis, apoptosis analyses (annexin V/7-aminoactinomycin D (7AAD) and pancaspase), and oxidative DNA damage (8-oxo-2''-deoxyguanosine; 8-oxodG)) were performed using flow cytometry. All cell experiments were treated with SLN for 24 h. In MTS assay, SLN dose-responsively decreased cell viability of two breast cancer cells (SKBR3 and MDA-MB-231) but only little damage to breast normal cells (M10). Under 7AAD staining flow cytometry, SLN dose-responsively induced G2/M phase arrested of SKBR3 cells. Under flow cytometry analyses, SLN dose-responsively induced apoptosis on SKBR3 cells in terms of annexin V/7AAD staining and pancaspase activity). Under flow cytometry analyses, SLN dose-responsively induced oxidative stress in terms of ROS generation, mitochondrial depolarization, and mitochondrial superoxide generation. Taken together, SLN induces selective killing against breast cancer cells involving oxidative stress, G2/M arrest, apoptosis, and oxidative DNA damage. Therefore, SLN is a potential marine drug for breast cancer therapy.
論文審定書....................................................................i
論文公開授權書............................................................ii
誌謝(Acknowledgement) .............................................iii
中文摘要......................................................................iv
英文摘要(Abstract) ......................................................v
III 前言(Introduction) ...................................................1
IV 材料與方法(Materials and methods) ......................3
1. 細胞培養(Cell cultures)........................................3
2. 細胞存活率測試(Cell viability) .............................3
2.1.1 試劑資訊 .....................................................3
2.1.2 染劑配置 .....................................................4
2.1.3 實驗步驟 .....................................................4
3. 細胞週期分析(Cell cycle distribution analysis) ....5
3.1.1 試劑資訊......................................................5
3.1.2 染劑配置 .....................................................5
3.1.3 實驗步驟 .....................................................5
4. 細胞凋亡測定(Annexin V/7AAD staining assay) ..6
4.1.1 試劑資訊 ......................................................6
4.1.2 染劑配置 ......................................................6
4.1.3 實驗步驟 ......................................................7
5. 細胞凋亡蛋白檢測(Pan-caspases assay) ............7
5.1.1 試劑資訊 ......................................................8
5.1.2 染劑配置.......................................................8
5.1.3 實驗步驟.......................................................8
6. 活性氧物質表現量測定(ROS assay) ....................8
6.1.1 試劑資訊 ......................................................9
6.1.2 染劑配置.......................................................9
6.1.3 實驗步驟 ......................................................9
7. 粒線體膜電位檢測(MitoMP staining).....................9
7.1.1 試劑資訊 .....................................................10
7.1.2 染劑配置......................................................10
7.1.3 實驗步驟......................................................10
8. 粒線體活性氧測定(MitoSox determination) ........10
8.1.1 試劑資訊......................................................11
8.1.2 染劑配置......................................................11
8.1.3 實驗步驟......................................................11
9. DNA氧化損傷分析(8-oxodG staining)..................11
9.1.1 試劑資訊......................................................12
9.1.2 染劑配置......................................................12
9.1.3 實驗步驟......................................................12
10. Statistical analysis...............................................13

V 實驗結果(Results)
1. 細胞存活率測試(Cell viability) .............................13
1.1 不同劑量下,SLN處理乳癌細胞(SKBR3和MDA-MB-231)及正長乳腺細胞(M10)的細胞存活率測試..............................................................................13
2. 流式細胞儀(Flow cytometry) 13
2.1不同劑量下,SLN處理乳癌細胞(SKBR3)的細胞週期影響 13
2.2 SLN處理SKBR3在annexin V/7AAD-based-apoptosis的影響 14
2.3 SLN處理SKBR3在pancaspase-based-apoptosis的影響 14
2.4 SLN處理SKBR3的ROS表現量變化 14
2.5 SLN處理SKBR3粒線體膜電位的變化 14
2.6 SLN處理SKBR3的mitoSOX表現量變化 14
2.7 SLN處理SKBR3細胞DNA氧化損傷的影響 15
VI 討論(Discussion) 16
VII 圖表與說明(Figures and figure legends) 18
VIII 參考文獻(Reference) 26
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