探討植物凝集素E誘導人類肺癌細胞凋亡之機制

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题名:	探討植物凝集素E誘導人類肺癌細胞凋亡之機制 Induction of the mitochondria apoptosis pathway by phytohemagglutinin erythroagglutinating in human lung cancer cells
作者:	郭韋廷;Wei-Ting Kuo
贡献者:	醫學院臨床醫學研究所碩士班
关键词:	肺癌;凋亡;粒線體;植物凝集素E;表皮生長因子受體;lung cancer;apoptosis;mitochondria;phytohemagglutinin erythroagglutinating;epidermal growth factor receptor
日期:	2010
上传时间:	2010-09-29 12:17:06 (UTC+8)
摘要:	凋亡是維持人體生理功能的重要機制，當其失去調控時會導致疾病的發生，而癌症就是其中之一。因此，發展具有啟動凋亡機制之抗癌藥物在臨床治療上有其必要性。根據過去的研究報告指出，從大紅豆中所萃取的植物凝集素具有抗癌的功效，然而“E型”的植物凝集素在抑制肺癌上並未被探討。本研究將以植物凝集素E來誘導肺癌細胞A-549凋亡，並探討相關的訊息傳遞路徑之作用。實驗中，植物凝集素E所使用的劑量分別為20, 40, 80, 160 μg/ml，而由MTT分析法及G6PD釋放分析法之結果得知，在經過植物凝集素E作用兩天後，會毒殺肺癌細胞A-549且抑制其生長。接著，由Annexin V/PI雙染法、TUNEL/PI雙染法和DAPI/TUNEL螢光染色的結果證實，植物凝集素E會誘導肺癌細胞A-549凋亡。最後，從西方點墨法和反轉錄聚合酶連鎖反應的結果發現，植物凝集素E會促使cytochrome c的釋放、增加活化態caspase-9及caspase-3之蛋白表現、活化Bcl-2家族蛋白中促凋亡蛋白與抑制抗凋亡蛋白的表現、抑制EGFR磷酸化態和蛋白表現與阻斷其下游PI3K/Akt及MEK/ERK訊息傳遞路徑。總結上述結果，植物凝集素E是藉由活化粒線體凋亡路徑來誘導肺癌細胞A-549凋亡並且抑制其生長，植物凝集素E具有發展抗肺癌藥物之潛力。 Apoptosis is a physiological mechanism required for maintaining cell numbers and removing unnecessary cells. De-regulation of apoptosis will influence the balance of cell proliferation and cell death, resulting in various fatal diseases that can include cancer. Lung cancer is the leading cause of cancer-related death all over the world. Therefore, it is necessary to develop a potential drug that can be used to re-develop the apoptosis of cells for cancer treatment. In prior research reports related to cancer therapy, phytohemagglutinin (PHA), the lectin extracted from red kidney beans (phaseolus vulgarus), demonstrated the ability to inhibit the growth of human cancer cells. However, one of its isoforms, PHA-E (erythroagglutinating) has yet to be evaluated on its anti-cancer effects against lung cancer cells A-549. In this study, PHA-E was used to induce apoptosis of A-549 lung cancer cells in order to determine the possible signal transduction pathway, as measured by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, G6PD (glucose-6-phosphate dehydrogenase) release assay, flow cytometry assays, fluorescent stains, western blot analysis and RT-PCR. The results showed that PHA-E treatment (20-160 μg/ml; 48 hr) caused a dose-dependent increase of cell growth inhibition and cytotoxicity on A-549 cells. In Annexin V/PI and TUNEL/PI assay, we found that the rate of apoptotic cells was raised as the concentration of PHA-E increased. In addition, cell morphological changes, chromatin condensation and fragmentation, were observed by DAPI/TUNEL stain after treatment with PHA-E. Treatment of A-549 cells with PHA-E resulted in enhancing the release of cytochrome c, which thus activated an increase in protein levels of caspase-9 and caspase-3, up-regulation of Bax and Bad, down-regulation of Bcl-2 and phosphorylated Bad, and finally the inhibition of epidermal growth factor receptor (EGFR) and its downstream signal pathway PI3K/Akt and MEK/ERK. In summary, our study has demonstrated that PHA-E can induce growth inhibition and cytotoxicity of lung cancer cells, which is mediated through activation of the mitochondria apoptosis pathway. These results suggest that PHA-E can be developed into a new therapeutic treatment that can be applied as an effective anti-lung cancer drug in the near future.
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