臺灣博碩士論文加值系統

台灣因惡性腫瘤而死亡的案例中，位居十大死因之首，而惡性腫瘤所造成的死亡種類中，大腸癌更是常年位居第三名。大腸癌也是常見的癌症之一，在臨床上一般的治療方式是以外科手術的方式切除癌細胞再以化療藥物或者放射線來搭配治療，但是癌細胞會慢慢對於舊的抗癌藥物產生抗性，化學治療所帶來的副作用也會降低患者的生活品質，尋找出新的治療方法對於治療大腸癌細胞是非常重要的，因此天然植物萃取物就是一個很好的來源。
2',4',6',4-Tetrahydroxy-3'-C-geranylchalcone (TG)是從麵包樹果實中萃取出來的類黃酮成分， TG中的主要骨架結構chalcone是許多天然化合物的基本骨架，在蔬菜，水果，茶和其他植物中皆能找到，具有多種生物活性如抗發炎、抗菌、抗微生物、抗病毒，這種成分在1999年時已經有人從啤酒花中分離出來，對於人類卵巢癌細胞ovarian cancer cell line (A-2780) 有抑制生長的作用，但是其抗大腸癌的作用至今尚未清楚。本篇的目的即是觀察TG對於大腸癌細胞DLD-1 的效用，並探討其機制和路徑。結果發現大腸癌細胞DLD-1經由TG處理後，
藥物處理後對於細胞凋亡相關蛋白BAX、Fas、FasL、caspase 3、p-JNK、caspase-9有加強的作用而使PARP-1蛋白裂解最終導致DLD-1凋亡，對於抗凋亡蛋白Bcl-2則有抑制蛋白表現量的影響。TG也會經由粒線體膜電位的電子傳遞鏈complex Ⅱ和Ⅲ來誘導細胞內的ROS濃度上升進而耗空細胞體內的GSH含量，最終導致細胞內的粒線體功能受損而影響細胞的生長。這些結果與發現表示TG可能具有發展成為新抗癌藥物的潛力，也有可能成為臨床治療上的新藥物選擇。

Human colorectal cancer is a common cancer around world. The general treatment is removed surgically the cancer cells in the clinic and then treated with chemotherapy or radiation, however, many cancer cells will develop resistance to many chemotherapeutic drugs. The chemotherapy comes with the side effect that will decrease the life quality to patient. Therefore, it is very important to find a new lead compound for the treatment of colorectal cancer cells.
2',4',6',4-Tetrahydroxy-3'-C-geranylchalcone (TG) is a flavonoid extracted from the fruit of breadfruit. Its anti-colorectal cancer has not known yet. Chalcone is a natural compound. The basic skeleton, found in vegetables, fruits, tea and other plants, has a variety of biological activities such as anti-inflammatory, anti-bacterial, anti-microbial, and anti-viral.
The aim of my study is to find out the mechanism of TG on anti-colon cancer. My experiment results showed that TG (extract from fruit of breadfruit) can inhibit DLD-1 cells viability via increase the expression of apoptosis protein, ex : Bax、Fas、FasL、caspase 3、p-JNK、caspase-9 and decrease the expression of anti-apoptotic protein, ex : Bcl-2. In the end, PARP-1 protein associated with cell alive will cleavage, then DLD-1 will apoptosis. TG also can enhanced intracellular reactive oxygen species (ROS) of DLD-1 via affected mitochondrial membrane potential complex Ⅱ and complex Ⅲ of mitochondrial respiration chain. In conclusion, TG can induce apoptosis. Therefore, in this study suggested that TG treatment may had the potential to be a strategy of colon cancer therapy.

中文摘要 Ⅰ
英文摘要 Ⅲ
致謝 Ⅳ
縮寫對照表 1
第一章 緒論 2
第一節 前言 2
第二節 程序性細胞凋亡(Programmed cell Death, PCD) 2
一、 細胞壞死(Necrosis) 3
二、 細胞自嗜(Autophagy) 3
三、 細胞凋亡(Apoptosis) 3
第三節 ROS與細胞凋亡之關聯 8
第四節 MAPK路徑與細胞凋亡之關聯 9
第五節 2',4',6',4-Tetrahydroxy-3'-C-geranylchalcone (TG) 10
第二章 研究目的 12
第三章 材料與方法 13
第一節 材料 13
一、 細胞株 13
二、 藥品 13
三、 西方墨點法-使用抗體 15
四、 實驗儀器 15
五、 分析軟體 16
第二節 實驗配方 17
一、 細胞培養液配方 17
二、 蛋白質定量 17
三、 蛋白質電泳配方 17
第三節 研究方法 19
一、 細胞培養(cell culture)-貼壁細胞 19
二、 細胞生存率測試(MTT assay) 19
三、 總蛋白萃取(Total Protein Extraction) 20
四、 蛋白質定量分析(Quantitative of protein concentration) 21
五、 西方墨點法(Western blot) 21
六、 流式細胞儀(Fluorescence-activated cell sorter) 23
七、 統計分析 25
第四章 結果 26
第一節 觀察TG對人類大腸癌細胞DLD-1細胞存活率的影響 26
第二節 TG對人類大腸癌細胞DLD-1細胞凋亡作用的影響 26
一、 TG有誘導細胞凋亡的能力 26
二、 TG有影響細胞週期與誘導細胞凋亡的能力 26
三、 TG誘導DLD-1細胞早期與晚期細胞凋亡 27
四、 TG對凋亡蛋白PARP-1表現量的影響 28
五、 TG對促凋亡蛋白Bax表現量的影響 28
六、 TG對抗凋亡蛋白Bcl-2表現量的影響 29
七、 TG對促凋亡蛋白Fas、FasL、caspase-3表現量的影響 29
八、 TG對MAPK路徑蛋白p-P38、p-JNK、p-ERK表現量的影響 29
九、 TG對促凋亡蛋白caspase-9表現量的影響與Bax和Bcl-2的關聯 30
第三節 TG對人類大腸癌細胞DLD-1內ROS值的影響 30
一、 TG對人類大腸癌細胞DLD-1內ROS值的影響 30
二、 TG對人類大腸癌細胞DLD-1內GSH值的影響 31
三、 TG誘導的ROS值增加源自粒線體電子傳遞鍊complex Ⅱ和complex Ⅲ 32
四、 TG對人類大腸癌細胞DLD-1誘導ROS上升對粒線體膜電位的影響 32
第五章 討論 34
第一節 2',4',6',4-tetrahydroxy-3'-C-geranylchalcone (TG)與ROS的關聯性 34
第二節 2',4',6',4-tetrahydroxy-3'-C-geranylchalcone (TG)與Bax和Bcl-2的關聯性 35
第三節 2',4',6',4-tetrahydroxy-3'-C-geranylchalcone (TG)與MAPK蛋白的關聯
性 36
第四節 2',4',6',4-tetrahydroxy-3'-C-geranylchalcone (TG)與△Ψm的關聯性 37
第六章 結論 38
參考文獻 39
附表 46
圖目錄 47
Fig. 1. 2',4',6',4-tetrahydroxy-3'-C-geranylchalcone (TG)化學結構圖 47
Fig. 2. 大腸癌細胞DLD-1經由TG處理後的細胞生存百分率變化 48
Fig. 3. 大腸癌細胞DLD-1經由TG處理後細胞週期與凋亡程度的變化 49
Fig. 4. TG能誘導大腸癌細胞DLD-1前期與後期的細胞凋亡 50
Fig. 5. TG對大腸癌細胞DLD-1的凋亡指標性蛋白PARP-1、促凋亡蛋白Bax
與抗凋亡蛋白Bcl-2的影響 51
Fig. 6. TG對大腸癌細胞DLD-1外在路徑上的促凋亡Fas、FasL和caspase-3蛋白的影響
52
Fig. 7. TG對大腸癌細胞DLD-1 MAPK路徑蛋白的影響 53
Fig. 8. TG對大腸癌細胞DLD-1的促凋亡蛋白Bax、caspase-9與抗凋亡蛋白Bcl-2的影響 54
Fig. 9. 大腸癌細胞DLD-1經由TG處理後的ROS變化量 55
Fig. 10.大腸癌細胞DLD-1經由TG處理後對胞內GSH耗空的影響 56
Fig. 11. TG誘導的ROS值上升是透過complex Ⅱ與complex Ⅲ 57
Fig. 12. TG對大腸癌細胞DLD-1在3、6與24小時的粒線體膜電位變化 59
Fig. 13. TG對大腸癌細胞DLD-1的作用機轉圖 60
附錄 61

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