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系統識別號 U0026-0812200911343524
論文名稱(中文) Genistein 對於血管內皮新生因子生成之作用機制
論文名稱(英文) The Action Mechanism of Genistein on VEGF production
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 93
學期 2
出版年 94
研究生(中文) 陳志遠
研究生(英文) Chih-Yuan Chen
電子信箱 s3691108@ccmail.ncku.edu.tw
學號 s3691108
學位類別 碩士
語文別 英文
論文頁數 71頁
口試委員 指導教授-蔡美玲
口試委員-陳淑慧
口試委員-李碧雪
中文關鍵字 血管內皮新生因子  雌激素受體 
英文關鍵字 estrogen receptor  VEGF 
學科別分類
中文摘要   雌激素受體-貝他 (Estrogen receptor )是於西元一九九六年所發現的第二種雌激素受體。在人類子宮內膜中,位於月經週期內分泌期晚期之蛻膜化基質細胞 (decidualized stroma cell)、平滑肌細胞、內皮細胞,以及位於動情週期中的母鼠子宮裡,其基質細胞、平滑肌細胞、與內皮細胞,皆可偵測至雌激素受體-貝他之表現。在肺的上皮細胞與肺癌細胞內,雌激素受體-貝他是主要顯明的雌激素受體。

  血管內皮生長因子 (Vascular endothelial growth factor, VEGF)為一種多效的血管新生因子,其能於老鼠蛻膜化基質細胞、人類子宮內膜基質細胞所表現。雌激素 (17-estradiol) 能在由人類子宮內膜所分離而出的基質細胞中增加血管新生因子表現。血管新生因子亦能由肺上皮細胞所製造,在乳癌細胞,MCF7 cells,雌激素在高量的雌激素-阿法 (此為首先鑑定而出的雌激素受體) 存在下,增加血管新生因子之信息核醣核酸的表現。

  對於刺激血管新生因子之信息核醣核酸需要雌激素受體之活化,在人類子宮內膜細胞中,已有完整研究,而位在人類血管內皮新生因子基因中的啟動子 (Promoter) 區域中也已鑑定出含有雌激素反應元素序列 (Estrogen response element, ERE) 的特定序列存在。再者,在乳癌細胞,MCF7 cells,雌激素受體-阿法單獨存在下,能增加雌激素所誘導之血管內皮新生因子之表現,然而,當與雌激素-貝他共同表現下,則反轉了這項誘導作用。雌激素受體-阿法也同樣被指出,在乳癌移植系統與成年老鼠子宮中,能抑制血管內皮新生因子的表現。然而,對於雌激素受體-貝他是否對於調控血管內皮新生因子之表現扮演重要角色,至今仍不甚清楚。近來,不同的雌激素反應元素序列被認為其作為可能的調節者 (Modulator) 而涉入依賴雌激素受體之基因轉錄作用。我們假設雌激素受體-貝他可透過雌激素反應元素序列而達至增加血管內皮新聲音子表現。

  雌激素-阿法與雌激素-貝他兩者皆能增加內皮細胞中內皮一氧化氮合成酶 (endothelial nitric oxide synthase, eNOS) 之活性。在培養之老鼠肝臟內皮細胞中,長期曝露雌激素能增加一氧化氮合成酶之表現與一氧化氮之生成。短期曝露雌激素,則能增加在內皮細胞內一氧化氮之產生。一氧化氮活化鳥糞嘌呤環化酶 (soluble guanyl cyclase, sGC) 進而活化環化鳥糞嘌呤核甘酸依賴性蛋白激酶 (cGMP-dependent kinase, PKG) 導致目標蛋白或轉錄因子之磷酸化。因此,我們進一步欲了解會否一氧化氮/ 環化鳥糞嘌呤/環化鳥糞嘌呤核甘酸依賴性蛋白激酶這個路徑會藉由影響雌激素受體-貝他與雌激素反應元素序列之建結而影響血管內皮新生因子之生成。

  懷孕老鼠子宮與人類肺臟上皮細胞為我們的實驗系統。四項實驗目的,分別探討,首先,雌激素對於血管新生因子的影響,再者,genistein 對於血管新生因子在肺臟上皮細胞表現的影響,第三,genistein 對於人類血管新生因子啟動子中可能的雌激素反應元素序列,其與雌激素受體-貝他鍵結作用之影響,最後,一氧化氮/ 鳥糞嘌呤/環化鳥糞嘌呤核甘酸依賴性蛋白激酶此一路徑對於雌激素受體-貝他與雌激素反應元素序列之鍵結作用之影響。

  我們的實驗結果包括,首先,雌激素受體-阿法在懷孕第七天Mesometrium大量表現,此次,雌激素受體-貝他於懷孕過程的Mesometrium與Antimetrium並沒有顯著差異的表現,第三,第七天懷孕子宮所表現出的血管新生因子相較於懷孕第十四天的老鼠子宮,具有顯著差異,再者,給予雌激素刺激,對於懷孕第七天之Antimesometrium中,並不具有刺激血管新生因子表現的作用,而genistein雖能些微增加血管新生因子表現,然並不具有顯著差異。在肺臟上皮細胞,H23 cells,genistein能藉由增加雌激素受體-貝他與雌激素反應元素序列鍵結而進而增加血管新生因子的表現,接著在肺臟上皮細胞與乳癌細胞中,不同的雌激素受體組成,對於特定的人類血管新生因子中5’端內的雌激素反應元素序列具有不同的鍵結作用。進一步,genistein能些微增加肺臟上皮細胞中一氧化氮的產生,給予一氧化氮給予者 (NO donor),則發現一氧化氮產生量與雌激素受體與雌激素反應元素序列鍵結作用呈現負相關,而在含溴環化鳥糞嘌呤核甘酸 (8-bromo-cGMP) 於 1 M條件下則能增加雌激素受體-貝他與雌激素反應元素序列之鍵結。

  結論而言,genistein能透過雌激素受體-貝他活化進而增加血管新生因子表現,而一氧化氮/ 環化鳥糞嘌呤這條路徑亦可能涉入此一過程。







英文摘要  Estrogen receptor beta (ER) is the second estrogen receptor (ER) identified in 1996. ER is detected in the decidualized stroma cells, smooth muscle cells, and endothelial cells of human endometrium at the late-secretory phase as well as the stroma cells, smooth muscle cells, and endothelial cells of the rat uteri in the estrous cycle. ERis the predominant estrogen receptor in the lung epithelium and lung cancer cells. . .

 Vascular endothelial growth factor (VEGF), a potent angiogenic factor, is detected in the mouse decidualized stroma cells, human endometrial stroma cells, and 17-estradiol can increase the production of VEGF in isolated stroma cells from the human endometrium. VEGF can also be produced by lung epithelial cells. In breast cancer cells, MCF7 cells, 17-estradiol increases VEGF mRNA expression through ER, the first identified estrogen receptor.
The enhancement of VEGF mRNA required for ER activation in human endometrial cells is documented, and human VEGF gene promoter region containing estrogen response element (ERE) is characterized. Furthermore, ER alone can increase E2-induced VEGF expression, but cotransfection with ER reverses the effect in MCF7 cells. ER was also reported to inhibit VEGF expression in breast tumor xenograft and adult murine uteri in vivo. However, it is still unclear whether the presence of ER played a crucial role on the regulation of VEGF expression. Currently, different EREs were discussed as a possible modulator in the involvement of ER-dependent gene transcription. We hypothesized that ER functions by enhancing VEGF production through an ERE-dependent pathway.

 ER and ER both increase the activity of endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. Chronic exposure to estrogens increases the protein abundance of nitric oxide synthase and the production of NO in cultured rat hepatic sinusoidal endothelial cells. Acute exposure to estrogens increases the production of NO in endothelial cells. Nitric oxide activates soluble guanylatre cyclase (sGC) to activate cGMP-dependent protein kinases (PKG) causing phosphorylation of target proteins or transcription factors. Therefore, we further questioned whether NO/cGMP/PKG influenced the binding of ER to ERE to regulate VEGF expression in lung epithelium.

 Pregnant uteri and lung epithelium were used as our experimental models. Four objectives were 1) to characterize the influence of 17-estradiol on on the expression of VEGF, 2) to characterize the effect of genistein on VEGF expression in H23 cells, 3) to characterize the effect of genistein on the binding of nuclear extraction (NE) of lung cancer cells to consensus ERE (cERE) and cERE-like sequences in the promoter region and 5’-untranslated region (5’-UTR) of human VEGF gene, and 4) to characterize the influence of NO/cGMP/PKG pathway on the binding of ER to ERE in lung cancer cells.

 Our data have shown that 1) ER was greatly expressed in G7 mesometrium, 2) No significant difference on ER expression was observed in mesometrium and antimesometrium, 3) the expression of VEGF on G7 was significantly higher than that on G14 in mesometrium and antimesometrium, 4) the treatment with 17-estradiol on VEGF expression in G7 antimesometrium was ineffective, 5) the treatment with genistein slightly increased VEGF production but without significant difference in G7 antimesometrium, 6) genistein increased VEGF expression in H23 cells by increasing the binding of ER to ERE, 7) ERand ER exhibited the different binding affinity with specific ERE-like sequences in 5’UTR of human VEGF gene, 8) genistein slightly increased NO production in H23 cells, 9) NO production was negatively correlated to the binding of ER to ERE, and 10) 8-bromo-cGMP at 1 M significantly increased the binding of ER to ERE.

 In conclusion, these results suggested genistein can increase VEGF production through the activation of ERand the NO/cGMP pathway may involve in this process.






論文目次 Content……………………………………………………………………………1

中文摘要……………………………………………………………………………2

Abstract……………………………………………………………………………5

Introduction………………………………………………………………………8

Materials and Methods…………………………………………………………13

Objectives and Results…………………………………………………………21

Discussion…………………………………………………………………………32

References…………………………………………………………………………37

Figures

Curriculum vitae
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