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研究生:鍾綺雯
研究生(外文):Yee-Mun Choong
論文名稱:五味子萃取物對人類乳癌細胞株輻射增敏作用之機轉- 增強輻射誘導細胞凋亡經由調控凋亡相關蛋白與caspase非依賴性凋亡誘導因子(AIF)路徑
論文名稱(外文):Extract of Schisandra chinensis Enhances Radiation-Induced Apoptosis via Regulations of Apoptosis-Related Proteins and Caspase-Independent AIF Pathway in Human Breast Cancer Cells
指導教授:姚俊旭姚俊旭引用關係
指導教授(外文):Chun-Hsu Yao
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:中醫學系碩士班
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:77
中文關鍵詞:乳癌細胞株五味子輻射增敏
外文關鍵詞:Breast cancer cell linesSchisandra chinensisRadiosensitivity
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近年來,乳癌的發生率及死亡率逐年攀升,高居女性十大癌症發生率第一位,死亡率則排名第四位。放射線治療是乳癌綜合治療中不可或缺之治療方式;但是,腫瘤細胞對放射線產生之抗性的確為乳癌治療失敗最常見之原因。傳統的輻射增敏劑因有較大的副作用,故限制其臨床應用。因此,本實驗希望能從中草藥中尋找高效低毒的輻射增敏劑,以期提高乳癌放射線治療的效果。
本實驗之目的係探討放射線配合中藥五味子萃取物對MCF-7乳癌細胞株之輻射增敏的效果及作用機轉。實驗材料與方法以MTT評估五味子萃取物之毒性,以細胞群落生成探討MCF-7細胞存活曲線,利用流式細胞儀之PI染色法、Annexin V-FITC/PI雙染法、粒線體膜電位變化等進行細胞凋亡分析,以及Hoechst 33258細胞核染色檢視細胞凋亡後期之DNA斷裂,再以西方墨點法檢測AIF及凋亡相關蛋白之表現。
本實驗結果發現五味子萃取物對MCF-7之抑制率與濃度及作用時間呈正相關性。放射線併用五味子萃取物對MCF-7細胞群落生成比例均有減少的趨勢,表示五味子萃取物具輻射增敏性而達到抑制腫瘤生長之效果。此外,放射線併用五味子萃取物誘導MCF-7停滯在G2/M期。JC-1染色發現放射線併用五味子萃取物降低粒線體膜電位(ΔΨm),經由Annexin V-FITC/PI 雙染法得知放射線併用五味子萃取物之apoptotic cells亦明顯增加,且以Hoechst螢光染色偵測DNA斷裂,而五味子萃取物會增加放射線所造成的凋亡蛋白Bcl-2與Cytosolic AIF的表現,並調降抗凋亡蛋白Bcl-2及Mitochondrial AIF的表現。此均證實MCF-7細胞走向凋亡。
本實驗結論,中藥五味子萃取物對MCF-7細胞株具增強「輻射誘導細胞凋亡路徑(Radiation-induced apoptotic pathway)」之作用,具有進一步研究與乳癌輻射增敏劑開發臨床應用之潛力。

Purpose:
We investigate the radiosensitive mechanism of Schisandra chinensis induced apoptosis in MCF-7 cell line, hormone sensitive breast cancer cells with caspase 3-deficiency.

Materials & Methods:
In our study, the crude sample was extracted from Schisandra chinensis (Trucz.) Baill seed in a serial process. The cell viability of human breast cancer MCF-7 cells was determined by MTT colorimetric assay. We used clonogenic assay to count cell survival fraction of MCF-7 cells after treating S. chinensis with radiation (RT) at different condition. Moreover, we analyzed MCF-7 cell-cycle distribution, mitochondrial membrane protential (ΔΨm), and proportion of apoptosis by using flow cytometry. DNA damage of MCF-7 cells was detected by Hoechst 33258 nucleus staining. Finally, we measured expression of apoptosis-related proteins, such as Bax, Bcl-2, and AIF, by using western blotting assay.

Results:
The MCF-7 survival curve decent was proportional to doses of extract of S. chinensis, and IC50 were 196.33 μg/ml for 24 h, 118.63 μg/ml for 48 h and 57.13 μg/ml for 72 h, respectively. The clonogenic assay of MCF-7 cells treated with RT combining S. chinensis showed that survival curve was more radiosensitive than RT alone. The cell-cycle arrest in G2/M phase in RT 15Gy and S. chinensis 40 μg/ml was 48.36% ± 3.11 vs. 42.24 ± 4.43% in RT 15Gy alone (p < 0.05). Loss of ΔΨm were 23.23 ± 3.90% and 27.15 ± 8.96% in RT combining S. chinensis 20 μg/ml and 40 μg/ml, respectively, compared with 15.08 ± 3.71% in RT 15Gy alone. The percentage of apoptotic cells evaluated by the AnnexinV/PI assay were 27.10 ± 0.53% and 25.30 ± 1.60% in RT combining S. chinensis 20 μg/ml and 40 μg/ml, respectively, vs. 19.83 ± 0.8% in RT 15 Gy alone (p < 0.01). Loss of ΔΨm were 23.23 ± 3.90% and 27.15 ± 8.96% in RT combining S. chinensis 20 μg/ml and 40 μg/ml, respectively, compared with 15.08 ± 3.71% in RT 15Gy alone. The indices of DNA staining detected by Hoechst 33258 nuclear stain calculated 28/250 in IR alone vs. 53/250 in RT and S. chinensis (p < 0.01). The expression of apoptosis-related proteins showed that Bax and cytosolic AIF up-regulated, while Bcl-2 and mitochondrial AIF down-regulated in RT combining S. chinensis.

Conclusion:
The radiosensitization of extract of Schisandra chinensis may relate with cell apoptosis through cell cycle arrest at G2/M phase, depolarization of ΔΨm, and regulation of Bax, Bcl-2 and caspase-independent AIF pathway in MCF-7 human breast cancer cells.


目錄

第一章 前言..............................................1
第二章 文獻回顧..........................................3
第一節 台灣地區常見之癌症................................3
第二節 女性首號殺手-乳癌................................5
第三節 乳癌的中醫治療....................................8
第四節 五味子的介紹.....................................12
第五節 細胞凋亡(Apoptosis)............................15
第六節 腫瘤與細胞凋亡...................................19
第七節 輻射生物效應(Radiobiological effects)..........19
第八節 輻射增敏劑(Radiosensitizer)....................21
第三章 材料與方法.......................................24
第一節 材料.............................................24
第二節 方法.............................................26
第四章 結果.............................................38
壹、五味子的萃取與高效液相層析(HPLC)..................38
貳、不同濃度五味子萃取物對MCF-7細胞之影響...............38
參、放射線合併五味子萃取物對MCF-7細胞群落生長之影響.....39
肆、放射線合併五味子萃取物對MCF-7細胞週期之影響.........39
伍、放射線合併五味子萃取物誘發MCF-7細胞粒線體膜電位△Ψm的改變......................................................40
陸、放射線合併五味子萃取物誘導MCF-7細胞凋亡之分析.......40
(一)流式細胞儀偵測凋亡的細胞..........................40
(二)Hoechst 33258偵測凋亡細胞之片段化DNA..............41
(三)西方墨點法偵測凋亡蛋白之表現量....................41
(四)西方墨點法偵測AIF蛋白之表現量.....................42
第五章 討論.............................................63
第六章 結論.............................................67
參考文獻................................................68
英文摘要................................................76

圖目錄

圖2.1. 細胞凋亡路徑圖...................................18
圖4.1. 五味子萃取物之HPLC分析圖.........................43
圖4.2. 五味子萃取物(S. chinensis)對MCF-7細胞之藥物毒性分析(Drug cytotoxicity)...................................45
圖4.3. MCF-7細胞群落生長抑制情形........................46
圖4.4. 五味子萃取物對MCF-7之細胞群落生成分析結果........47
圖4.5. 放射線合併五味子萃取物對MCF-7細胞週期之PI染色分析......................................................48
圖4.6. 放射線合併五味子萃取物對MCF-7細胞粒線體膜電位△Ψm之流式細胞儀分析..........................................50
圖4.7. 放射線合併五味子萃取物誘發MCF-7細胞粒線體膜電位△Ψm的改變..................................................51
圖4.8. 放射線合併五味子萃取物對MCF-7細胞之Annexin V-FITC細胞凋亡分析................................................52
圖4.9. 放射線合併五味子萃取物對MCF-7細胞之流式細胞儀分析......................................................53
圖4.10. MCF-7經Hoechst 33258染色偵測片段化DNA...........54
圖4.11. 放射線合併五味子萃取物誘發MCF-7細胞出現片段化DNA.....................................................56
圖4.12. 以Western blot偵測凋亡相關蛋白AIF、Bcl-2以及Bax在MCF-7人類乳癌細胞株之表現量.............................57
圖4.13. 放射線合併五味子萃取物對抗凋亡蛋白Bcl-2之影響...58
圖4.14. 放射線合併五味子萃取物對凋亡蛋白Bax之影響.......59
圖4.15. 放射線合併五味子萃取物對Mitochondrial AIF之影響......................................................60
圖4.16. 放射線合併五味子萃取物對Cytosolic AIF之影響.....61
圖4.17. β-actin之表現量,作為internal control...........62
圖6.1. 五味子萃取物(Schisandra chinensis)增強放射線誘導之細胞凋亡作用機制........................................67

表目錄

表2.1. 2011年台灣地區10大癌症死亡統計....................4
表3.1. 五味子萃取物之HPLC梯度條件.......................27
表4.1. 放射線合併五味子萃取物對MCF-7細胞週期之影響......49

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