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研究生:張同佑
研究生(外文):Tung Yu Chang
論文名稱:血管內皮生長因子透過一氧化氮依賴訊息路徑對胎盤血管內皮鈣離子依賴性黏著分子蛋白之調控機制
論文名稱(外文):The regulation mechanism of VEGF on the expression of placental VE-cadherin through a NO-dependent signaling pathway
指導教授:蔡美玲蔡美玲引用關係
指導教授(外文):Mei Ling Tsai
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:90
語文別:中文
論文頁數:43
中文關鍵詞:血管內皮生長因子一氧化氮血管內皮型鈣離子依賴黏著分子
外文關鍵詞:vascular endothelial growth factor (VEGF)nitric oxidevascular endothelial-cadherin (VE-cadherin)
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血管內皮型鈣離子依賴性黏著分子(vascular endothelial cadherin, VE-cadherin)是一個鈣離子依賴黏著分子,與滲透性(permeability)、細胞增殖(cell proliferation)、微血管形成(capillary formation)有關。在胎盤生長期間,分化的滋養層(trophoblasts)會形成類似血管之絨毛構造。而且在中期與末期的絨毛(villi)上也發現有血管內皮型鈣離子依賴性黏著分子表現。
血管內皮生長因子(vascular endothelial growth factor, VEGF)是一種有效力的血管新生因子(angiogenic factor),藉由它兩個接受器 (血管內皮生長因子接受器一型與二型) 誘導血管滲透性(vascular permeability)、內皮細胞增殖(endothelial proliferation)、微血管形成(capillary formation)。胎盤生長因子(placenta growth factor, PlGF)是血管內皮生長因子超級家族(VEGF superfamily)的成員且只能與血管內皮生長因子接受器一型結合。
目前研究已經知道血管內皮型鈣離子依賴性黏著分子和血管內皮生長因子與內皮細胞存活(endothelial survival)有關。在胎盤組織中,可以偵測到血管內皮型鈣離子依賴性黏著分子、血管內皮生長因子、胎盤生長因子與血管內皮生長因子接受器。但特別的是在胎盤組織裡血管內皮生長因子接受器一型與胎盤生長因子的蛋白質含量比血管內皮生長因子接受器二與血管內皮生長因子多。因此,本研究目的在探討血管內皮生長因子是否能透過血管內皮生長因子接受器一型活化其訊息路徑調控血管內皮型鈣離子依賴性黏著分子蛋白表現。
本實驗使用懷孕第十四、十八、二十一天大白鼠胎盤,此外將第十八天大白鼠胎盤剪成移植體(explants),然後加入試劑體外培養(in vitro)在含百分之五的二氧化碳、攝氏三十七度西培養箱中培養二十四小時。之後利用西方點墨轉漬分析法(Western blot analysis)測量細胞內血管內皮型鈣離子依賴性黏著分子、內皮型一氧化氮合成酉每(eNOS)、血管內皮生長因子接受器一型與二型的蛋白質含量,而以一氧化氮分析儀(NO analyzer, NOA 280 model)測量培養液中一氧化氮的量。 最後以免疫沉澱分析法來確定西方點墨法的結果。
根據實驗目的,有三個實驗目標要達成︰1)在胎盤組織中,懷孕對血管內皮型鈣離子依賴性黏著分子蛋白、內皮型一氧化氮合成酉每、血管內皮生長因子接受器一型與二型的影響,2)一氧化氮參與血管內皮生長因子誘導血管內皮型鈣離子依賴性黏著分子蛋白的表現,3)胎盤生長因子(placenta growth factor, PlGF)透過一氧化氮訊息路徑誘導血管內皮型鈣離子依賴性黏著分子蛋白的表現。
由結果可以說明:(1)隨著懷孕天數增加,血管內皮型鈣離子依賴性黏著分子蛋白表現趨勢與內皮型一氧化氮合成酉每表現相似。(2)懷孕十八天的胎盤,血管內皮生長因子接受器一型表現量最多而血管內皮生長因子接受器二表現量最少。(3)透過L型精氨酸(L-arginine)可以加強血管內皮生長因子誘導血管內皮型鈣離子依賴性黏著分子蛋白表現。 (4)胎盤生長因子增加血管內皮型鈣離子依賴性黏著分子蛋白與一氧化氮含量可以被一氧化氮合成酉每抑制劑(L-NAME)抑制。總結來說,血管內皮生長因子結合血管內皮生長因子接受器一可能透過一氧化氮依賴訊息路徑而增加血管內皮型鈣離子依賴性黏著分子蛋白質含量。

Vascular endothelial-cadherin (VE-cadheirn), a calcium-dependent homotypic adhesion molecule, is related to permeability, cell proliferation, and capillary formation. During placental development, the differentiation of trophoblasts forms vascular-like structures. VE-cadherin expresses in the intermediate and terminal villi.
Vascular endothelial growth factor (VEGF), a potent vascular permeability factor or angiogenic factor, induces vascular permeability, endothelial proliferation and capillary formation by activating its two receptors (VEGFR-1 and VEGFR-2). Placenta growth factor (PlGF), a member of VEGF superfamily only binds to VEGFR-1.
It is known that VE-cadherin and VEGF are related to endothelial survival. In placental tissues, VE-cadherin, VEGF, PlGF, and VEGF receptors can be detected. In particular, the protein abundance of VEGFR-1 and PlGF is greater than that of VEGFR-2 and VEGF in the placenta. Therefore, the purpose of this study was to examine whether VEGF can regulate the expression of VE-cadherin through a VEGFR-1-dependent pathway.
The placenta from rats on gestation day 14 (G14), 18 (G18) and 21 (G21) were used. Placental explants from G18 rats were pretreated with various compounds in vitro for 24 hours in a 5% CO2 incubator at 37 °C. Western blot analysis measured the protein abundance of eNOS, VE-cadherin, VEGFR-1 and VEGFR-2. A NO analyzer (NOA 280 model) measured the concentration of NO in culture media. Immunoprecipitation assay was used to confirm the results of Western Blot analysis.
According to the purpose, our aims were to examine: 1) the effect of pregnancy on the protein abundance of VE-cadherin, eNOS, VEGFR-1, and VEGFR-2 in placental tissues, 2) the involvement of NO in the induction of placental VE-cadherin by VEGF, and 3) the induction of VE-cadherin by PlGF through the NO signaling pathway.
As our data indicate, 1) the protein profile of VE-cadherin is related to that of eNOS over gestation, 2) G18 placenta expressed the greatest amount of VEGFR-1 but the lowest amount of VEGFR-2, 3) the induction of VE-cadherin protein by VEGF can be enhanced by L-arginine, and 4) the increase of VE-cadherin protein and NO production by PlGF can be inhibited by L-NAME. In conclusion, the binding of VEGF to VEGFR-1 may increase the protein abundance through a NO-dependent pathway.

Content…………………………………………………………………I
誌謝……………………………………………………………………II
中文摘要………………………………………………………………01
Abstract………………………………………………………………03
Introduction…………………………………………………………05
Purpose and Aims……………………………………………………10
Materials and Methods……………………………………………12
Result I………………………………………………………………18
Result II………………………………………………………………21
Result III……………………………………………………………25
Discussion……………………………………………………………28
References……………………………………………………………37
Figures
Appendix
Curriculum vitae

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