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系統識別號 U0007-1301200913331400
論文名稱(中文) 辣椒素capsaicin抑制人類大腸癌細胞株Colo 205增生並誘發細胞凋亡之機轉
論文名稱(英文) Capsaicin inhibits proliferation of human colon cancer Colo 205 cells via inducing cell cycle arrest and apoptosis
校院名稱 臺北醫學大學
系所名稱(中) 醫學檢驗生物技術學研究所
系所名稱(英) Graduate Institute of Biomedical Technology
學年度 97
學期 1
出版年 98
研究生(中文) 劉嘉又
研究生(英文) Jia-you Liu
學號 G160095001
學位類別 碩士
語文別 中文
口試日期 2008-12-10
論文頁數 61頁
口試委員 指導教授-楊沂淵
共同指導教授-呂旭峰
委員-何元順
中文關鍵字 辣椒素 
英文關鍵字 Capsaicin 
學科別分類
中文摘要 近年來在台灣癌症十大死因中,結腸直腸癌一直高居第三位。辣椒素(Capsaicin ),為辣椒(Hot Pepper)中主要產生辛辣的成分,許多相關研究指出capsaicin具有抗致癌、抗突變及化學防治(chemoprotective)的活性,能引發多種癌症細胞,如:胃癌細胞、前列腺癌細胞及乳癌細胞的凋亡,並具有抑制血管新生的能力。目前一些研究已證明Capsaicin可使癌細胞內產生大量ROS、並使Ca2+由內質網釋放出來,引發癌細胞內凋亡機制啟動。但以Capsaicin用於人類大腸癌研究卻很少。所以在此次實驗中,我們利用capsaicin 與人類大腸癌細胞(Colo 205 cell)作用,藉以探討capsaicin對於人類大腸癌細胞Colo 205所導致的細胞凋亡(Apoptosis)、細胞週期停滯(Cell cycle arrest)之機制、細胞內蛋白表現、Ca2+的變化),用以評估capsaicin對於大腸癌的抗癌成效。
將Colo 205細胞以Capsaicin作用後,利用流式細胞儀分析細胞的存活率及細胞內粒線體膜電位(Δ )、Ca2+濃度與ROS變化。Real-Time PCR技術偵測引發細胞凋亡的基因表現。最後以Western Blot方法觀察能引發或抑制細胞凋亡的蛋白質之相關性及表現程度。結果發現,作用後Colo 205細胞內p53大量表現,並誘使p21進一步抑制G1時期的蛋白激酶複合物活性,造成細胞停滯於G0/G1時期(G0/G1 arrest)。同時也觀察到,在150 μM Capsaicin作用24小時能誘發細胞凋亡之內、外在路徑,造成細胞凋亡。其中細胞內Caspase-8、-9及-3的mRNA表現上升,而Caspase-8、-9及-3蛋白質也被活化。
在外在路徑方面,細胞中Fas、FADD蛋白表現增加,造成Caspase-8及-3被活化。內在路徑方面發現,細胞內的ROS、Ca2+產生增加,進而使粒線體上抗凋亡Bcl-2家族蛋白表現下降,無法繼續抑制促凋亡Bcl-2 家族蛋白之活性,最後造成粒線體膜電位改變,Cytochrome c釋放出粒線體,活化Caspase-9及-3,造成細胞凋亡。此外,實驗中觀察到AIF蛋白表現增加,可推測除了Caspase-dependent凋亡路徑活化外,caspase-independent凋亡路徑亦被活化。
目前知道有一群統稱為IAPs (inhibitor of apoptosis proteins) 的蛋白,可以藉由和caspase 結合抑制caspase 活性,進而阻止細胞凋亡。但在實驗中,我們觀察到細胞內xIAPs蛋白表現下降,造成caspase 活性無法被抑制,進而使細胞走向細胞凋亡。
綜合以上結果,Capsaicin可使Colo 205細胞生長停滯於G0/G1時期,並同時啟動多條細胞凋亡路徑,造成細胞凋亡。證明其具有抗腫瘤功效,是一種具開發潛力的抗癌藥物。
英文摘要 In Taiwan, Colorectal cancer is the third most common cancer in men and women. The spicy ingredient in hot pepper was isolated by Thresh , and was named it capsaicin .According to some studies, Capsaicin is an effective component of nature Chinese herb medicine and with a powerful anti-tumor, against mutation and chemoprotective activity. Capsaicin is capable of inducing apoptosis of cancer cells, including gastric cancer, prostate cancer and breast cancer etc. In addition, Capsaicin also inhibits human angiogenesis.Several studies have already shown that Capsaicin could produce a large number of intracellular reactive oxygen species (ROS) and promote that calcium release from endoplasmic reticulum (ER) to damage cancer cells and then cause the activation of apoptosis mechanism in cancer cells. However, there are few studies of the effect of Capsaicin on Colorectal cancer. Because Capsaicin could induce a large of intracellular reactive oxygen species in cancer cells, we will detect the drug-induced DNA damage and the influence on cell cycle process which promotes the proliferation effect on cancer cells. Finally, we will examine the other apoptosis mechanism- endoplasmic reticulum stress (ER stress) associated with the release of intracellular calcium, and then evaluate the inhibition effect of Capsaicin on human Colo 205 cells.
The assays methods are using : 1) flow cytometry for examining the cell cycle arrest and apoptosis; inclusive of cell viability, the levels of ROS, Ca2+, and mitochondrial membrane potential in human Colo 205 cells. 2) Real-Time PCR and flow cytometry to detect mRNA expression. 3)Western blotting methods for examining the apoptosis associated proteins to find out the mechanism of apoptosis. The approach taken in this experiment is to demonstrate that p53 level elevates obviously and causes p21 to inhibit the activity of protein kinase complex , that decreased the percentage of the Colo 205 cells in the S- and G2/M- phases, and increased the percentage in G0/G1- phase (G0/G1 arrest). Based on the result of this study, the extrinsic and Intrinsic pathways of apoptosis were induced. The caspase-3, -8 and -9 mRNA levels increased after treatment with 150μM Capsaicin. The protein of caspase caspase-3, -8 and -9 also be activated after treatment with 150μM Capsaicin .
The extrinsic apoptosis pathway is triggered by Fas ligand (FasL) on account of the amount of Fas and FADD protein elevation. These proteins results in the formation of caspase-8 and caspase-3, which trigger the execution of apoptosis. The percentage of ROS and Ca2+ levels are significantly different between Capsaicin treated group and control. We also found the expression of anti-apoptosis Bcl-2 family lower down. Due to anti-apoptosis Bcl-2 not able to inhibit apoptosis in these examined cells, mitochondria membrane release cytochrome c to cause the activations of Caspase-9、-3 which result in apoptosis.
On account of the elevation of AIF protein, we suggest that both Caspase-Dependent and independent pathways of apoptosis were activated.
So far as we know, a group of inhibitor of apoptosis protein (IAPs) can bind to caspase and block apoptosis. In this study, the decrease of xIAPs lead to apoptosis due to no inhibition of caspase activity.
From the above cited, Capsaicin induced cell cycle arrest of Colo 205 cells in G0/G1- phase and caused apoptosis via extrinsic and Intrinsic pathways. Capsaicin is a strong potential agent for the treatment of Colorectal cancer since it induced apoptosis through the activation of caspase activity in Colo 205 cells.
論文目次 目錄
目錄-----------------------------------------------Ⅰ
表圖目錄-------------------------------------------Ⅲ
中文摘要-------------------------------------------Ⅳ
英文摘要- -----------------------------------------Ⅵ
第壹章 緒論
第一節 台灣癌症流行病學統計 ---------------------- 1
第二節 台灣結腸直腸癌流行病學統計 ---------------- 1
第三節 辣椒素(Capsaicin)簡介 -------------------- 2
第四節 細胞週期的調控 ---------------------------- 3
一、細胞週期(cell cycle) ------------------------ 3
二、Cyclins 家族 (Cyclins family) ---------------- 4
三、CDKs 家族 (Cyclin-dependent kinases family) --- 5
四、CDKIs 家族 (Cyclin-dependent kinase inhibitors family) ------------------------------------------- 5
五、細胞週期檢查哨 (Cell-cycle checkpoints) ------- 6
六、G0/G1 時期的調控 (Regulation of G0/G1 phase) - 7
第五節 細胞凋亡 (Apoptosis) ---------------------- 8
一、細胞凋亡之外在路徑 ---------------------------- 9
二、細胞凋亡之內在路徑 ---------------------------- 9
第六節 粒腺體膜電位(Mitochondrial membrane potential)、鈣離子(Ca2+)與細胞凋亡之關係 -------------------------- 11
第七節 活性氧化物(Reactive oxygen species) -------- 11
第貳章 實驗目的 ----------------------------------- 13
第參章 研究材料與方
第一節 實驗材料 ----------------------------------- 14
第二節 實驗方法 ----------------------------------- 18
一、辣椒素配製 ------------------------------------ 18
二、細胞冷凍保存方法------------------------------- 18
三、冷凍細胞解凍之方法 ---------------------------- 18
四、繼代培養 -------------------------------------- 19
五、細胞分盤 -------------------------------------- 19
六、藥物處理 -------------------------------------- 19
七、酵素免疫分析儀測定(MTT分析法) ----------------- 20
八、流式細胞儀測定 -------------------------------- 20
1.細胞週期測 -------------------------------------- 20
2.細胞內粒線體膜電位測定--------------------------- 21
3.細胞內Ca2+濃度測定------------------------------- 22
4.細胞內活性氧(ROS)測定--------------------------- 22
九、西方墨點分析法 -------------------------------- 23
十、Real-Time PCR技術 ----------------------------- 27
十一、統計方法 ------------------------------------ 29
第肆章 實驗結果與分析
一、Capsaicin導致細胞存活率下降 ------------------- 30
二、Capsaicin對細胞細胞週期的影響並引發細胞凋亡 --- 30
三、Capsaicin作用後細胞內粒線體膜電位(Δ )下降------ 30
四、Capsaicin作用後細胞內Ca2+濃度增加-------------- 31
五、Capsaicin作用下細胞內ROS增加------------------- 31
六、Capsaicin引發細胞內Caspase-8、-9及-3的mRNA表現增加 --------------------------------------------------------31
七、Capsaicin作用後引發細胞凋亡之外在路徑 --------- 32
八、Capsaicin作用活化caspase-dependent凋亡路徑 ---- 32
九、Capsaicin作用活化caspase-independent凋亡路徑 -- 33
十、Capsaicin引發Bcl-2家族調控粒線體內外膜通透度--- 33
十一、Capsaicin引發p53蛋白及p21蛋白大量表現 ------- 34
十二、Capsaicin抑制xIAPs蛋白表現 ------------------ 35
第伍章、討論 -------------------------------------- 36
表圖 ---------------------------------------------- 39
附錄 ---------------------------------------------- 50
參考文獻------------------------------------------- 53
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