臺灣博碩士論文加值系統

血管新生 (angiogenesis)從原有的血管衍生出新生的微血管，在正常生理功能以及諸多疾病都扮演著重要的角色。而在血管新生的過程中，血管內皮細胞會進行增生、爬行、分化的作用，最終形成成熟的血管。抗血管新生藥物可以減少腫瘤內部新生的血管數量以及限制腫瘤生長的大小，因此可當作一個治療癌症的目標。在本篇研究中，我們以MTT assay篩選了一系列的化合物對於人類臍帶靜脈內皮細胞 (HUVECs) 存活率的影響，並選擇較佳活性的Than42來探討其抗血管新生作用與相關機轉。
Than42呈現劑量相關性地抑制內皮細胞的增生。經流式細胞儀分析，Than42會增加細胞週期中sub-G1相的分佈，顯示出Than42可能是藉由引發內皮細胞凋亡 (apoptosis) 來抑制其增生的現象。Than42能夠劑量相關性顯著地抑制內皮生長因子 (VEGF) 所誘發之內皮細胞移行現象。Than42也能夠抑制內皮細胞經由內皮生長因子所刺激的管狀生成。Than42的局部給予會完全抑制內皮生長因子引發大鼠主動脈血管環所爬行出來之先生血管環的形成。另一方面，在活體內(in vivo) Matrigel plug實驗中，Than42可抑制活體內的血管新生作用。另外，我們發現Than42對內皮生長因子及其接受器結合所活化之訊息傳遞分子 PI3K-Akt和ERK1/2也具有劑量相關性的抑制作用。
總結，Than42可展現其試管和動物活體之抗血管新生的藥理活性，它會引起內皮細胞凋亡來抑制細胞的增生，而且可以抑制內皮生長因子所誘發之內皮細胞移行、管狀生成及其相關之訊息傳遞等作用來抑制血管新生的現象，顯示出Than42具有良好的抗血管新生能力，可進一步研發成防治血管新生之藥物。

Angiogenesis, forming new capillaries from the pre-existing blood vessels, is essential for many physiologic processes as well as many pathological disorders. Endothelial cells play an important role in the processes of angiogenesis including cell proliferation, migration, differentiation and tube formation. Anti-angiogenic agents reduce the tumor vasculature and growth, thereby being a therapeutic strategy for cancer. In this study, we screened a series of compounds by MTT assay to investigate the action on human umbilical vein endothelial cells (HUVECs). Among these compounds, Than42 exerts a more potent effect, therefore we further explore the mechanism of Than42 on HUVECs and angiogenesis.
We found Than42 inhibited HUVEC proliferation by BrdU proliferation assay. Furthermore, we found Than42 inhibited cell proliferation by inducing cell apoptosis as evidenced by the increased sub-G1 phase using flow cytometry. In addition, Than42 inhibited VEGF-induced cell migration and tube formation in vitro in a concentration-dependent manner. Moreover, Than42 also inhibited endothelial cells sprouting from aortic ring stimulated by VEGF. We also found Than42 inhibited VEGF-induced angiogenesis in Matrigel plug assay in vivo. Besides, Than42 blocked the activation of PI3K-Akt pathway and ERK1/2 stimulated by VEGF treatment. In conclusion, Than42 exhibits anti-angiogenic activity both in vitro and in vivo by inducing endothelial cell apoptosis and inhibiting cell migration, tube formation, and signaling induced by VEGF. These results suggest that Than42 has a potential to be a candidate for drug development in field of anti-angiogenesis.

口試委員會審定書……………………………………………………….i
中文摘要………..………………………………………………………ii
英文摘要……………………………………………………….…….. iv
Abbreviations…..…….………………………….…………….... vi
第一章 Introduction………………………………………………… 1
1.1 Angiogenesis………………………………………………….....1
1.2 Tumor angiogenesis……………………………………………...2
1.3 Vascular endothelial growth factor (VEGF) and receptor
(VEGFR)………………………………………………….…….....3
1.3.1 Vascular endothelial growth factor
(VEGF)………….........................................3
1.3.2 Vascular endothelial growth factor receptor
(VEGFR)…..............................................5
1.4 VEGF receptor-2 signal transduction…………….……….…6
1.5 Aims of this thesis………………………………….………… 8
第二章
Materials and methods……………………………...…………... 18
第三章
Results……………………………………………………... 25
3.1 The exploration of an anti-angiogenic compound from
5250-derivatives……………………………………………….25
3.2 Than42 inhibited endothelial cell viability and
proliferation…..................................... 25
3.3 Than42 exhibits no cytotoxicity to
HUVEC…………………................................. 26
3.4 Than42 caused endothelial cell cycle arrest and
apoptosis……....................................... 26
3.5 Than42 inhibited VEGF-induced endothelial cell
migration..........................................…27
3.6 Than42 inhibited VEGF-induced tube formation in
vitro……............................................27
3.7 Effect of Than42 on VEGF-induced aortic ring
sprouting……........................................28
3.8 Effect of Than42 on VEGF-induced signal
transduction…….....................................29
3.9 Effect of Than42 on VEGF-induced angiogenesis in
Matrigel plug
assay………………………………………………………......30
第四章
Discussion……………………………………………………...... 49
第五章
Conclusion and future work……………………………… ......53
References………………………………………………….…….… 56

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