花生四烯酸對於乳癌細胞株增生與細胞週期的影響; Effects of Arachidonic Acid on the Proliferation and Cell Cycle of Human Cancer Cell Lines

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题名:	花生四烯酸對於乳癌細胞株增生與細胞週期的影響;Effects of Arachidonic Acid on the Proliferation and Cell Cycle of Human Cancer Cell Lines
作者:	葉仲宜;Chung-Yi Yeh
贡献者:	中國醫藥大學醫學研究所
关键词:	肝細胞癌腫瘤;DNA甲基化
日期:	2004
上传时间:	2010-01-20 16:53:22 (UTC+8)
摘要:	據本實驗室先前的研究得知，在乳癌患者之乳房癌化組織過氧化?體增生接受器? (PPARα)蛋白質表現量明顯高於周邊非癌化組織，且分析脂肪酸組成時也發現癌化組織的arachidonic acid (AA, 20:4, n-6)含量明顯高於周邊非癌化組織，而AA是人體的必需脂肪酸，也是一種天然性活化PPARα的配體。根據這些結果，本實驗想要探討PPARα經arachidonic acid活化後，對於乳癌細胞生長的影響。本實驗將以三種不同的人類乳癌細胞株作探討，分別為MDA-MB-231 [ERα(－)/HER2/neu(－)]、MCF-7[ERα(＋)/HER2/neu(＋)]、BT-474[ERα(＋)/HER2/neu(＋＋＋)]。觀察以AA處理後，各種乳癌細胞的生長情形、細胞週期變化、PPARα與其他細胞週期調節相關蛋白質的表現。實驗結果發現到在三種細胞株以AA處理24、48及72小時後，細胞數目皆會隨著添加AA劑量的增加而增加。其中AA處理48小時，10μM的AA可以顯著性的促進MDA-MB-231及MCF-7細胞的生長，而且達到最大的促進效果。同樣的，10μM的AA於72小時後也顯著性促進BT-474細胞的生長。分析細胞週期的變化得知，經10μM AA處理後的三種細胞，處於S期與G2/M期的細胞群族比控制組多，顯示AA可以加速此三種細胞的細胞週期運行，快速進入S 期。為了進一步探討AA促進細胞生長的機制，因此以西方墨點法分析PPARα與其他細胞調節相關蛋白質的表現，實驗結果得知，MDA-MB-231與MCF-7經AA處理後48及72小時，PPARα表現量有上升的情形，BT-474細胞在AA處理後72小時，PPARα表現量才有上升的情形。在細胞週期調節蛋白的表現上發現，三株細胞經AA處理過後4小時，Cyclin E的表現量皆明顯增加，Cyclin D1、CDK 2、CDK 4的表現與控制組並無明顯差異。在MAPK蛋白質表現方面，ERK 2與p38的表現與控制組比較並無明顯差異。在調控細胞凋亡的蛋白質表現上，Bax / Bcl-2的比例與控制組比較並無明顯差異。由這些結果顯示，AA會促進乳癌細胞株的生長，並會誘導Cyclin E的表現量增加，加速細胞週期運行而使得細胞產生增生現象。; Our previous study showed that peroxisome proliferator-activated receptor α(PPARα) protein was highly expressed in breast cancer tissue when compared with surrounding cancer-free tissues in breast cancer patients. In addition, the proportion of arachidonic acid (AA, 20:4, n-6) was significantly higher in cancer tissues. AA is an essential fatty acid and an natural ligand of PPARα. In this study we investigated the effects of PPARα which was activated by AA on breast cancer cell lines. Three breast cancer cell lines included MDA-MB-231 [ERα(－)/HER2/neu(－)]、MCF-7[ERα(＋)/HER2/neu(＋)]、BT-474[ERα(＋)/HER2/neu(＋＋＋)]were used in this study. We examined the cell growth rate, cell cycle distribution, protein expressions of PPARα, and regulatory proteins of cell cycle in these breast cancer cell lines after 10 μM AA treatment. We found that the proliferation of these three cell lines increased time-dependently at various AA doses. AA caused a maximal stimulatory effect at 10 μM in MDA-MB-231 and MCF-7 cells. The proliferative effect of 10 μM AA was also observed in BT-474 cells after 72-hrs treatment. Cell cycle analysis showed that 10 μM AA accelerated the cell cycle progression in MDA-MB-231, MCF-7, and BT-474 cells. The proportions of cells at S phase were predominant as compared with control after treatment with AA in these three cancer cells. In addition, the proliferative effect of AA was accompanied by an increase in the proportion of cells at G2/M phase. To explore the proliferative effect of AA, the expression of PPARα protein and regulatory proteins of cell cycle were further confirmed by Western blot. In MDA-MB-231 and MCF-7 cells, PPARα protein expression increased significantly after 48 and 72 hours of treatment with 10 μM AA. In BT-474 cells, PPARα protein level was significantly higher after 72 hours. The expressions of cyclin E increased significantly after 4 hours in these three kinds of cancer cells. However, the expressions of cyclin D1, CDK2, and CDK4 did not significant differences as compared with control group. Regulatory proteins of mitogen-activated protein kinase (MAPK) cascades, such as ERK2 and p38, did not changed significantly in these three cancer cell lines, also. Moreover, ratio of Bax/Bcl-2 had no significant difference as compared with control group in MDA-MB-231 and MCF-7 cells. These results suggested that AA may exert an effect on the breast cancer cell growth and this effect possibly involves the induction of cyclin E expression and leading to cell cycle progression.
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