石斛對人類乳癌細胞株(MDA-MB-231)的細胞週期及細胞凋亡之影響; Effects of Shihu on cell cycle and apoptosis in human breast cancer cell line (MDA-MB-231)

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题名:	石斛對人類乳癌細胞株(MDA-MB-231)的細胞週期及細胞凋亡之影響;Effects of Shihu on cell cycle and apoptosis in human breast cancer cell line (MDA-MB-231)
作者:	郭詩晴;Shih-Ching Kuo
贡献者:	中國醫藥大學營養研究所
日期:	1993
上传时间:	2009-12-24 10:50:00 (UTC+8)
摘要:	石斛最早收載於神農本經上品，主要功效為清熱生津，滋陰養胃，清肝明目等。本篇研究探討石斛水萃取物在人類乳癌細胞（MDA-MB-231 cells）之細胞增生、細胞週期和細胞凋亡調節的作用。稱取經冷凍乾燥之石斛水提取物0.045 g，溶於1.5 mL DMSO，配製成石斛水提取物之原始溶液（stock solution）。從原始溶液（30 mg/mL）分別取1.25、2.5、5、10及20 mL（其最終濃度為18.75 、37.5、75、150及300 mg/mL）至人類乳癌細胞，經過不同的時段（12、24、36、48小時）培養後分別進行如下的試驗。在細胞增生的試驗中，結果發現於150及300 mg/mL的石斛水萃取物可以抑制經過24及48小時培養的人類乳癌細胞（MDA-MB-231 cells）之細胞增生。在流式細胞儀分析DNA含量的試驗中，投予300 mg/mL的石斛水萃取物培養24小時後，結果發現細胞週期的分佈中G0/G1期為31﹪（控制組為60﹪），與控制組比較降低了29﹪。G2/M期為44﹪（控制組為19﹪），與控制組比較增加了25﹪。在西方點墨法的試驗中，經不同濃度之石斛水萃物處理24小時後分析cyclin B1、Cdk1、p21蛋白質的表現量，結果在300 mg/mL濃度的石斛水萃取物與控制組比較，cyclin B1、Cdk1、p21蛋白質的表現量皆明顯上升，分別為2.82倍、1.42倍和2.53。p21蛋白質表現量的增加可能是石斛水萃取物引起細胞週期停滯於G2/M期的其中一個主要的因子。在流式細胞儀的分析和DNA片段膠體電泳分析試驗中，皆確認石斛水萃取物可誘發MDA-MB-231 cells之細胞凋亡。在西方點墨法的試驗中，經不同濃度之石斛水萃取物處理36小時後分析Bax、Caspase-3、Caspase-9蛋白質的表現量，結果在300 mg/mL濃度的石斛水萃取物與控制組比較，其Bax蛋白質的表現量明顯上升（1.80倍）。將300 mg/mL濃度的石斛水萃取物與75 mg/mL的石斛水萃取物進行比較，Caspase-3和Caspase-9蛋白質的表現量皆上升，分別為1.11倍和1.80倍。由本研究中的試驗結果證明，石斛會抑制人類乳癌細胞的細胞增生，引起細胞週期停滯於G2/M期和誘發細胞凋亡。因此，石斛為具有潛力的細胞週期阻斷劑。對於石斛是否可當作抗癌藥物需要再作更深入的評估其抗癌的功效。; Shihu is a Chinese medicine used as a Yin tonic to nourish the stomach, to promote the production of body fluid, and to reduce fever. In this study, water extract of Shihu was analyzed for its effects on the regulation of proliferation, cell cycle progression and apoptosis in human breast cancer cell line, MDA-MB-231. Thirty milligram per milliliter of water extract of Shihu (30 mg/mL) was prepared as the stock solution. Cultured cancer cells were treated with different concentrations (0, 18.75 , 37.5, 75, 150 and 300 μg/mL) of Shihu water extract for different times (12, 24, 36 and 48 hr). Treatment of MDA-MB-231 cells with water extract of Shihu（150 and 300 μg/mL）for 24 or 48 hr inhibited the proliferation of human breast cancer cells. Flow cytometric analysis for the DNA content in Shihu-treated cells indicated that following Shihu (300 μg/mL) treatment for 24 hr , the cell cycle distribution in the G0/G1 phase decreased; 60.02﹪vs. 31.30﹪concomitant with an increase in cells in the G2/M; 18.45﹪vs. 44﹪, for control vs. Shihu, respectively. Immunoblot analysis of the levels of cyclin B1, cyclin-dependent kinases (Cdk1), and p21 in the MDA-MB-231 cells treated with Shihu water extract (300 μg/mL) for 24 hr showed that expressions of cyclin B1, cyclin-dependent kinases (Cdk1), and p21 increased 2.82 fold, 1.42 fold, and 2.53 fold, respectively, comparing to the control group. The increase of the level of p21 may be one of the major factors for Shihu water extract to cause G2/M arrest in the examined cells. Flow cytometric assays and DNA fragmentation gel electrophoresis both confirmed that Shihu water extract induced apoptosis in MDA-MB-231 cells. In cell treated with Shihu（300 μg/mL）for 36 hr, the level of Bax increased 1.80 fold, comparing to the control group. The levels of Caspase-3 and Caspase-9 in cells treated with 300 μg/mL of Shihu for 36 hr were increased by 1.11 fold and 1.80 fold, respectively, comparing to the cells treated with 75 μg/mL of Shihu for 36 hr. Results in this study demonstrate that Shihu inhibited the proliferation of cancer cells, caused G2/M arrest, and induced apoptosis. Thus, Shihu is promising as an important cell cycle bloker and is important for further evaluation for its application as a cancer chemopreventive agent.
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