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  • 學位論文

蘿蔔硫素誘導人類原發性大腸結腸癌細胞株細胞週期G2/M期停止以及細胞凋亡之研究

The study of human primary colorectal cancer cell lines G2/M arrest and apoptosis induction by sulforaphane

指導教授 : 徐怡強
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摘要


蘿蔔硫素(sulforaphane, SFN) 是一種天然的異硫氰酸酯,主要存在於我們常吃十字花科的蔬菜中,其富含的代表為西蘭花。我們的研究中發現蘿蔔硫素可誘發人類大腸結腸癌細胞株(CPs)產生G2/M phase的停滯進而誘導其細胞凋亡(apoptosis)之反應。以MTT assay作細胞劑量依賴性的測試,發現SFN處理人類大腸結腸癌細胞株24小時,可觀察到明顯的劑量依賴性。再以流式細胞儀配合Propidium Iodide 染色以及Annexin V 加上 Propidium Iodide 雙染色測試是否SFN造成的抑制生長是以細胞凋亡的方式進行,實驗結果發現以SFN處理CPs 24小時後,人類大腸結腸癌細胞株的細胞週期之G2/M phase停滯以及sub G0/G1 phase DNA之含量增加約5.2倍到26.6倍,細胞膜內之phosphotidylserine轉至膜外造成late apoptosis約4.5倍到14.3倍的增加,這些結果顯示SFN是透過細胞凋亡的過程抑制人類大腸結腸癌細胞株的生長。以JC-1測試以不同濃度之SFN處理後之人類大腸結腸癌細胞株,發現會造成粒線體膜電位的下降約為3.12倍到4.35倍。另外再以共軛焦顯微鏡配合Rhodamine123加上Cytochrome C 分析,發現在粒線體膜電位的下降的情況下Cytochrome C位移現象並不顯著。再以西方墨點法探討SFN誘導細胞凋亡之機制,發現caspase-3的活性有所增加,而caspase-9的活化並不顯著。最後再以共軛焦顯微鏡配合DAPI以及NF-κB染色可以很明顯的看見NF-κB的活化。總觀以上實驗結果推測,SFN在處理人類大腸結腸癌細胞株(CPs)後,會抑制其生長、導致G2/M phase停滯、誘導細胞凋亡、phosphotidylserine外翻、caspase-3活性的增加、粒線體膜電位的下降,NF-κB的活化最終造成其細胞凋亡的結果,但不會因粒線體膜電位的下降而造成Cytochrome C的轉移使得caspase-9進行活化。本研究結果針對SFN做分子機制之探討,希望在未來有助於作為大腸結腸癌病患治療的新藥作為一個有效的參考。

並列摘要


Sulforaphane is a natural isothiocyanates, found primarily in cruciferous vegetables, the representatives of its rich in broccoli. Sulforaphane can induce human colorectal cancer cell lines (CPs) cell cycle G2 / M phase arrest and induce apoptosis. SFN treated CPs for 24 hours were observed in a dose-dependent by the MTT assay. Then Flow cytometer with Propidium Iodide staining and Annexin V with Propidium Iodide double staining assay whether the growth inhibition caused by SFN is a way of apoptosis. The cell cycle G2 / M phase arrest and its sub G0/G1 phase DNA content increased approximately 5.2 fold to 26.6 fold. The late apoptosis were observed the cell membrane eversive (phosphotidylserine) were increased 4.5 fold to 14.3 fold by SFN treatment. Our results show that SFN is the process of apoptosis through the inhibition of CPs proliferation and apoptosis induction. The mechanism of SFN induced apoptosis was analyzed by Western blot. We found SFN can induce the activity of caspase-3. With JC-1 assay with different concentrations of SFN treatment of and another to verify the confocal microscope with Rhodamine123 and Cytochrome C – FITC of CPs, found to cause mitochondrial membrane potential decline of about 3.12 fold to 4.35 fold, and Cytochrome C translocation. Overview of the results suggest that, SFN in the treatment of human primary colorectal cancer cell lines (CPs) will inhibit growth, resulting in G2 / M phase arrest, induction of apoptosis, phosphotidylserine valgus, caspase-3 activity increased, mitochondrial membrane potential decline, NF-κB activation eventually leading to the result of apoptosis, but not mitochondrial membrane potential due to the decrease caused by the transfer of Cytochrome C allows for activation of caspase-9. The results of this study may provide a new field and the turning point for the colorectal cancer prevention and therapy.

參考文獻


行政院衛生署 國民健康局http://www.bhp.doh.gov.tw/BHPnet/Portal/
行政院衛生署 衛生統計資訊網
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