臺灣博碩士論文加值系統

將新合成的α-咔吧啉合成衍生物進行人類大腸癌細胞株COLO 205及人類正常胚胎皮膚細胞Detroit 551的細胞毒性評估。結果發現化合物TLS-11對於人類大腸癌細胞株COLO 205具有優越之毒殺活性，IC50 值為0.49 μM，細胞存活率分析顯示TLS-11抑制細胞生長呈現劑量及時間依存性；其對人類正常胚胎皮膚細胞Detroit 551則具低毒性。故本論文進一步探討TLS-11之抗癌作用機轉。。
細胞週期分析結果，發現人類大腸癌細胞株COLO 205經過TLS-11處理後造成細胞週期停滯在G2/M期並且造成G2/M期相關蛋白質：CDK1、cyclin B1、p53及p21蛋白質增加。
為探討人類大腸癌細胞株COLO 205經TLS-11處理後造成細胞死亡的機制，進行型態觀察、Hoechst 33258染色法染色、Annexin V / PI 雙染法染色、粒線體膜電位、DNA膠體電泳及西方墨點法分析，結果顯示人類大腸癌細胞株COLO 205經過TLS-11處理後造成細胞的死亡與細胞凋亡有關。
在細胞凋亡內在途徑方面，TLS-11造成人類大腸癌細胞株COLO 205的 Bax/ Bcl-2蛋白質比例增加以及由粒線體釋出cytochrome c ，cytochrome c與Apaf-1蛋白質結合後進而活化caspase 9及caspase 3蛋白質；粒線體也會釋放出AIF及Endo G蛋白質使細胞產生凋亡。在細胞凋亡外在途徑方面，TLS-11對於death-receptor蛋白：FAS/CD95、TNFR1及TRAILR1 (DR4)增加有明顯的影響；但相對而言，TRAILR2 (DR5)卻沒有顯著的增加。在有絲分裂活化蛋白質激酶路徑方面，TLS-11對於JNK及ERK訊息傳導路徑有明顯的影響；對於p38較無明顯之影響。
綜合以上實驗數據顯示，TLS-11誘導人類大腸癌細胞株COLO 205產生細胞凋亡與死亡受體路徑、粒線體凋亡路徑及JNK-MAPKs訊息傳導路徑有關，希望可以成為一個對正常細胞具低毒性之治療大腸癌之抗癌候選藥物。

The newly synthesized α-carboline derivatives were evaluated for cytotoxicity against human colon cancer cell line COLO 205 and normal human embryonic skin cell Detroit 551. The results showed that the compound TLS- 11 exhibited potent cytotoxicity against human colon cancer line COLO 205 with IC50 value 0.49 μM. TLS-11 induced dose- and time-dependent decrease in viability in TLS-11 treated COLO 205 cells. Beside, it exerts low cytotoxicity on Detroit 551 normal human embryonic skin cell. Further, the purpose of this study was to investigate the anti-cancer effect and the molecular action mechanisms of TLS-11 on COLO 205 cells.
Cell cycle analysis showed that TLS-11induced significant G2 / M phase arrest. Western blotting showed that TLS-11caused increase in CDK1,cyclin B1, p53 and p21 proteins .
After the TLS-11 treatment COLO 205 cells caused cell death by carry out cell morphological observation , Hoechst 33258 staining, Annexin V / PI double staining, Mitochondrial membrane potential, DNA electrophoresis and Western blot analysis. The results showed that TLS-11 treatment caused cell death and apoptosis.
In apoptosis intrinsic pathway, TLS-11 treatment caused Bax / Bcl-2 protein ratio increase and cytochrome c released from mitochondria. And then cytochrome c will binding with Apaf-1 and thus activation of caspase 9 and caspase 3 protein. Mitochondria also released AIF and Endo G protein to cause apoptosis.In apoptosis extrinsic pathway, TLS-11 treatment caused increase in FAS/CD95, TNFR1 and TRAILR1 (DR4). The TRAILR2 (DR5) is not. In mitogen activated protein kinase pathway, TLS-11 treatment caused JNK and ERK increase. The p38 is not.
Taken together, we conclude that TLS-11 induced COLO 205 cells death by G2/M phase arrest and death-receptor associated and mitochondrial - apoptotic pathway and JNK-MAPKs signaling pathways. It may be a low cytotoxicity drug candidate for the treatment of human colon cancer in the future.

目錄
圖目錄 IV
表目錄 VI
略字表 VII
中文摘要 IX
Abstract XI
第一章 緒論 1
第一節 癌症流行病學 1
第二節α-咔吧啉 (α-Carboline)類化合物研究概況 4
第三節TLS-11之合成方法 5
第四節 細胞週期 (Cell cycle)之調控機制 6
一、細胞週期 (cell cycle) 6
二、細胞週期 (cell cycle)之調控因子 8
三、p53蛋白質與細胞週期關係 10
第五節 細胞凋亡 (apoptosis) 11
一、死亡接受器細胞凋亡路徑 13
二、粒腺體細胞凋亡路徑 13
第六節 有絲分裂活化蛋白質激酶之簡介 16
第七節 大腸癌(Colorectal cancer)之簡介 17
一、大腸功能介紹 17
二、大腸癌病因 17
三、大腸癌臨床徵狀 18
四、大腸癌治療 19
第二章 研究動機與目的 22
第三章 結果與討論 23
第一節 結果 23
一、TLS-11化合物對COLO 205細胞的細胞毒性 23
二、TLS-11誘導COLO 205細胞之型態變化 23
三、TLS-11誘導COLO 205細胞產生細胞凋亡 24
四、TLS誘導COLO 205細胞產生DNA 斷裂情形 24
五、TLS誘導COLO 205細胞產生粒線體膜電位改變 25
六、TLS-11對COLO 205細胞的細胞週期影響 26
七、TLS-11對COLO 205細胞的細胞週期調控蛋白之影響 26
八、TLS-11對COLO 205細胞之Bcl-2家族蛋白質調控 27
九、TLS-11誘導COLO 205細胞凋亡時會造成cytochrom c的累積及caspase 9、caspase 3的活化 27
十、TLS-11誘導COLO 205細胞凋亡時對AIF及Endo G的影響 28
十一、TLS-11誘導COLO 205細胞凋亡時對death-receptor的影響 29
十二、TLS-11誘導COLO 205細胞凋亡時對MAPKs影響 29
第二節 討論 31
第四章 結論 34
第五章 實驗部分 35
第一節 實驗材料 35
一、細胞株 35
二、細胞培養材料 35
三、儀器 36
四、試劑 37
五、溶液配製 40
第二節 實驗方法 44
一、藥物配製 44
二、細胞培養 44
三、細胞計數及存活測試 44
四、細胞增殖分析 45
五、細胞型態分析 46
六、Hoechst 33258染色法 46
七、Annexin V / PI 雙染法 47
八、DNA萃取及電泳分析 47
九、粒線體膜電位染色 48
十、細胞週期分析 48
十一、蛋白質萃取 49
十二、蛋白質定量分析 49
十二、西方墨點法 50
十三、統計分析 51
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