臺灣博碩士論文加值系統

本篇論文的主旨，在研究化學合成物質SDil-1 （5,5-Diphenyl-2-thiohydantoin） 對於人類臍帶靜脈內皮細胞 HUVEC 在體外生長的影響， 並探討其中作用的機制。 我們的初步實驗結果發現SDil-1 在不造成細胞死亡的濃度下， 能對HUVEC 產生生長抑制的作用。 3H-Thymidine incorporation 的實驗結果顯示， SDil-1 會抑制內皮細胞DNA 的合成作用。 利用流式細胞儀分析細胞週期，發現SDil-1 增加了細胞停滯在G0/G1 phase 的比例。西方墨點法（Western blot）的實驗結果， 觀察到SDil-1 處理21小時的細胞，其和細胞週期停滯有關的蛋白p21， 表現量高於對照組， 同時cdk4、 cyclin D3、cyclin A 蛋白的表現量有顯著的降低， p53、p27、cdk2、cyclin D1， 以及cyclin E 蛋白的表現則無明顯變化。 免疫沉澱（immunoprecipitation）與蛋白激酶活性測定的實驗結果， 發現以SDil-1處理後，能增加p21與cdk2和cdk4的結合量， 並降低cdk2的激酶活性。 根據實驗結果， 我們認為SDil-1 會干擾內皮細胞的細胞週期進行而減少細胞的增生， 其作用主要是透過抑制cdk活性的途徑， 因此， SDil-1或許具有潛力， 能成為抗血管新生的藥物。

The aim of this study is to examine the antiproliferation effect of SDil-1 （5,5-Diphenyl-2-thiohydantoin）in human umbilical vein endothelial cells （HUVEC）and its possible underlying mechanism. Our data demonstrated that SDil-1 caused an inhibition in HUVEC proliferation. The result of 3H-Thymidine incorporation showed that SDil-1 decreased DNA synthesis in HUVEC. Flow cytometric analysis demonstrated that treatment of HUVEC with SDil-1 arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis showed that treatment of HUVEC with SDil-1 for 21h increased the levels of p21 protein but decreased the level of cdk4, cyclin D3, and cyclin A proteins. The expression of p53、p27, cdk2, cyclinD1 and cyclin E levels did not significantly change as compared with untreated cells. Furthermore, immunoprecipitation study showed that the cdk4-cyclin-linked p21 and the cdk2-cyclin-linked p21 proteins were increased after SDil-1 treatment. Using kinase assay method to measure the kinase activity, we demonstrated that the cdk2 activity was decreased in the SDil-1 treated HUVEC. Thus, our results suggest that SDil-1 can interrupt the cell cycle progression and proliferation of human endothelial cells by inhibiting the cyclin-dependent kinase activity. The findings from the present study suggest that SDil-1 might have the potential to inhibit the occurrence of angiogenesis.

誌謝……………………………………………………………………...vi
目錄……………………………………………………………………..vii
圖次……………………………………………………………………...ix
中文摘要………………………………………………………………...xi
英文摘要………………………………………………………………..xii
緒論………………………………………………………………………..1
一、 細胞週期……………………………………………………….....1
二、 血管新生……………………………………………………….....2
三、 血管的新生與腫瘤的治療…………………………………….....3
四、 SDil-1 的相關背景……………………………………………....6
五、 實驗目的……………………………………………………….....7
實驗材料與方法…………………………………………………………..8
一、 實驗材料……………………………………………………….....8
二、 實驗方法……………………………………………………......12
實驗結果………………………………………………………………...23
一、 SDil-1 對人類臍靜脈內皮細胞的生長抑制作用………….....23
二、 SDil-1 對其它種類細胞的影響…………………………….....24
三、 SDil-1 對內皮細胞存活率的影響………………………….....24
四、 SDil-1 對於內皮細胞之細胞週期的影響………………….....25
五、 SDil-1 對內皮細胞週期調控蛋白表現的影響…………….....26
六、 p21 antisense oligonucleotide 對SDil-1
抑制DNA合成作用的影響………………………………..............27
討論……………………………………………………………………...29
一、 Cyclins與cyclin dependent kinases
調控細胞週期進行的機制……………………………….............29
二、 增加cdk抑制蛋白所帶來的影響…………………………….....30
三、 SDil-1 抑制人類臍靜脈內皮細胞生長…………………….....34
四、 SDil-1 成為抗癌藥物的潛力……………………………….....35
參考文獻………………………………………………………………...36
附圖……………………………………………………………………...44

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