臺灣博碩士論文加值系統

在先前的研究發現, 大豆異黃酮可以抑制膀胱癌，然而其分子機轉並不清楚。為了去探討其中的抑癌機制，我們在抑制膀胱癌細胞生長的情況下，利用subtractive suppression hybridization (SSH)的方法尋找表現量
不同的基因。在75 個表現量不同的基因，其中epithelial membrane
protein 2 (EMP2) 被我們挑出是在大豆異黃酮抑制癌症中扮演一個重要的角色。在實驗室先前的研究裏，從foci formation 和soft agar 的實驗也發現，過度表現EMP2 可以抑制癌細胞的轉型(孫瑩，2004)。然而，血
管新生、癌細胞和正常細胞之間的訊息傳遞、以及癌細胞和基質之間的作用，在癌症發生的過程中也都扮演重要的角色。在這篇研究，我們利用可以持續表現EMP2 的stable cell lines 來探討EMP2 對於細胞凋亡、細胞週期、血管新生、細胞和基質之間的影響。我們也利用microarray 來找尋EMP2 在抗血管新生的作用上，可能參與的訊息傳遞路徑。利用流式細胞儀分析annexin V 和propidium iodide 的雙重染色，我們發現當細胞表現EMP2 會造成細胞走向細胞凋亡的前期，然而卻不會造成細胞死亡。更進一步發現EMP2 的表現會造成細胞週期停留在G2/M phase，這在先前我們實驗室發現大豆異黃酮可以造成膀胱癌細胞的細胞週期停留G2/M phase 是一致的。而在血管新生的研究中，我們發現當EMP2 大量表現會造成血管新生的分子表現降低，例如VEGF、TF、HDGF、PDGF-A，而且造成抗血管新生的分子表現增加，例如enodstatin。在癌症發生的過程中，除了血管新生，癌細胞和正常細胞之間的訊息傳遞、癌細胞和基
質間的作用在癌症發生的過程中都會有一定的影響。在先前與EMP2 相關的研究發現，EMP2 可以去調控細胞膜上integrin 的表現，而我們同樣也發現EMP2 可以造成β1 integrin 的表現上升並且改變細胞和基質間的作用，特別是collagen IV。然而有關EMP2 詳細的機制到目前並不清楚，因此我們試著利用cDNA microarray 來探討EMP2 的分子機制，從microarray 的資料，我們發現當EMP2 高度表現時，會有104 個基因增加表現和270 個基因表現被抑制。同時利用BIOCARTA, GENMAPP 和KEGG 的資料庫，去統整EMP2 可能調控的訊息傳導路徑。繼而利用RT-PCR 和western blot 去確認microarray 的資料，我們發現EMP2 會去調控Tuberous sclerosis 2 (TSC2)的表現並且去抑制mTOR-VEGF 訊息傳導路徑，進而抑制血管新生。

Soy isoflavones have been demonstrated to exhibit tumor suppressor effect on human bladder cancer cells. To explore the underlying mechanisms, subtractive suppression hybridization was undertaken to identify the
differentially expressed genes related to growth arrest of tumor cells. Among 75 genes identified, epithelial membrane protein 2 (EMP2) was proved to involve in anticancer effect of isoflavones. In our prior study, EMP2 can inhibit the cell transformation analyzed by foci formation and soft agar experiments (Sun Ying, 2004). However, the potential importance of
angiogenesis, cell-cell communication and cell-matrix interaction in tumorigenesis in EMP2-mediated biological effects is currently unknown. In this study, EMP2 stable cell line was used to examine its effects on cell apoptosis, tumor angiogenesis, cell cycle control, cell-matrix interaction and novel signaling events associated with tumor angiogenesis of bladder cancer cells. The flow cytometry of propidium iodide and annexin V double staining supports that the effect of EMP2 in the early stage of apoptosis. Furthermore,
EMP2 expression seems to induce G2/M cell cycle arrest, consistent with that of suppressor effect of isoflavones. As for angiogenesis, expression of angiogenic factors-VEGF, TF, PDGF-A, and HDGF was all suppressed, while angiogenesis inhibitor-endostatin was stimulated by EMP2. Besides, RT-PCT experiment demonstrated that EMP2 could up-regulate the expression of β 1 integrin and the associated cell-matrix interaction, especially collagen IV. To investigate the novel molecular mechanisms,
profiling of EMP2 overexpression stable cell line was carried out by cDNA microarrays. The profiling revealed a total of 104 up-regulated and 270 down-regulated genes associated with overexpression of EMP2. Bioinformatic analysis of the databases (BIOCARTA, GENMAPP and KEGG)
has revealed several potential signaling pathways or protein-protein interactions related to EMP2. The microarray data were confirmed by western blot and RT-PCR. The results indicate that EMP2 may regulate Tuberous sclerosis 2 (TSC2), and then inhibit the mTOR-VEGF pathway. By using this strategy, anti-angiogenesis is one of the molecular mechanisms underlying anticancer effect of EMP2 for human cancer cells.

Contents
Abstract in Chinese ……………………………. i
Abstract in English ……………………………… iii
Acknowledgements ……………………………. v
Contents .....….…………………………………… vi
Index of Tables …………..……………………… vii
Index of Figures …………………………..…….. viii
Abbreviations …………...………………….….. ix
Introduction ……………....………………..…… 1
Materials and Methods ………………………… 7
Results …….………………………………...……. 12
Discussion ….…………………………………….. 18
References ...………..…………………..……… 22
Appendix of Tables …………………..……….. 26
Appendix of Figures ..………………..…………. 43

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