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系統識別號 U0026-0812200910392123
論文名稱(中文) 血管生長素-1對胎盤血管內皮之鈣離子依賴性黏著分子表現調控的可能機轉
論文名稱(英文) The possible mechanism of angiopoietin-1 on the expression of placental vascular endothelial cadherin
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 91
學期 2
出版年 92
研究生(中文) 羅士傑
研究生(英文) Shih-Chieh Lo
學號 s3690104
學位類別 碩士
語文別 英文
論文頁數 86頁
口試委員 口試委員-李文森
召集委員-陳淑慧
指導教授-蔡美玲
口試委員-呂增宏
中文關鍵字 血管生長素-1  鈣離子依賴性黏著分子 
英文關鍵字 angiopoietin-1  vascular endothelial cadherin 
學科別分類
中文摘要 血管生長素-1 (Angiopoietin-1)是一種血管新生因子 (angiogenic factor)可存在於血管的內皮細胞及胎盤組織的細胞滋養層細胞 (cytotrophoblasts)和融合滋養層細胞 (syncytiotrophoblasts)中。對內皮細胞來說,Angiopoietin-1被視為是一種anti-permeability factor,同時也被認為具有促進 endothelial survival and vascular maturation的功能。但是到目前為止,Angiopoietin-1在胎盤中對於胎盤的形成究竟扮演什麼樣的角色仍然不清楚。血管內皮的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,且ve-cadherin亦可以在胎盤組織中的間質絨毛 (intermediate villi)和絨毛(terminal villi)中被偵測到。當細胞與細胞間的接合處間的ve-cadherin的表現量增加時會降低微血管的滲透性進而增加血管的完整性,同時ve-cadherin也被認為與促進endothelial survival and vascular maturation的功能有關。綜合以上所述,由於 Angiopoietin-1與ve-cadherin對於血管的內皮細胞具有相同的功能,且Angiopoietin-1的接受器 Tie-2與 ve-cadherin在血管內皮細胞與胎盤細胞滋養層細胞的細胞膜上皆有表現,因此本論文之目的在利用體外培養 (in vitro)的懷孕大白鼠胎盤為研究模式來探討在胎盤組織中存在的Angiopoietin-1是否可以調控ve-cadherin的表現以及可能的作用機轉。本實驗首先探討懷孕時 Angiopoietin-1和Tie-2蛋白質的表現量,並且更進一步去探討 Angiopoietin-1是否會隨著其給予劑量的增加而使得 ve-cadherin的表現量增加。根據我們的實驗結果指出:(1) Angiopoietin-1的接受器Tie-2蛋白質表現量在懷孕第十八天的胎盤中表現量最多,顯示在懷孕後期Angiopoietin-1對於胎盤的發展扮演很重要的角色且Angiopoietin-1的接受器Tie-2的表現量多寡亦可能決定胎盤血管新生的發展。(2) Angiopoietin-1在低濃度(1-10 ng/ml)時會增加 ve-cadherin的表現量,但是在高濃度則不會出現上述的情況,顯示Angiopoietin-1對於它的接受器Tie-2來說在相對低濃度的Angiopoietin-1可能具有促進ve-cadherin的表現的功能進而降低微血管的滲透性並增加血管的完整性, 而Angiopoietin-1在對於它的接受器Tie-2相對高濃度的時候則可能具有抑制ve-cadherin表現而使得微血管的滲透性增加而有促進更多物質交換功能以供應日異增大的胎兒營養所需。在過去的研究中已經報告過Angiopoietin-1可以藉由活化PI3K/AKT signaling pathway使得一氧化氮產量 (NO production)增加。而對於平滑肌來說,NO 的產生具有活化soluble guanylate cyclase, sGC的能力。當sGC被活化時則可以增加cGMP在細胞內的濃度。就另一方面來說,當細胞內的cGMP濃度增加時,這些增加的cGMP又可能藉由Type 1α PKG的磷酸化並透過細胞內訊息調節傳導路徑如ERK/MAP kinase pathway去控制基因的表現。除此之外NO-cGMP-PKG系統在目前亦被認為可能藉由控制細胞與細胞間的接合間隙來調整微血管的通透性。因此接下來,我們更進一步想要去研究Angiopoietin-1是如何影響ve-cadherin的蛋白表現以及Angiopoietin-1是否能透過NO-cGMP依賴性訊息路徑來調控ve-cadherin的蛋白表現。首先為了證明Angiopoietin-1是否可以透過NO去影響ve-cadherin的蛋白表現,因此本實驗先研究Angiopoietin-1是否會去影響NO production以及之後的ve-cadherin蛋白表現,並且利用一氧化氮合成酉每的抑制劑 (L-NAME)來確認NO在Angiopoietin-1所調控的ve-cadherin蛋白表現中所扮演的角色。根據實驗結果顯示:(1) Angiopoietin-1在低濃度 (1.0 ng/ml)時會增加一氧化氮的產量同時也使得ve-cadherin的蛋白表現增加。(2) Angiopoietin-1所增加的NO production及ve-cadherin皆會被一氧化氮合成酉每抑制劑 (L-NAME)所抑制。根據上述結果可以說明Angiopoietin-1的確可以藉由改變NO production來調控ve-cadherin蛋白的表現量。 同時更進一步想證明Angiopoietin-1是否能透過NO-cGMP依賴性訊息路徑來調控ve-cadherin的蛋白表現。本實驗利用PKG phosphorylation來說明cGMP是否參與在Angiopoietin-1所調控ve-cadherin蛋白表現的訊息傳遞路徑中。為了測量PKG phosphorylation,本實驗利用免疫沈澱法 (immunoprecipitation)去濃縮磷酸化的PKG,並且利用西方點墨轉漬分析法 (Western blot analysis)來做確認。另一方面,利用固定化親和層析系統 (immobilized affinity chromatography, IAC)來測量PKG的磷酸化。同樣的在實驗結果可以看到Angiopoietin-1在低濃度 (1.0 ng/ml)時會增加磷酸化PKG的表現量,但是在高濃度 (50-100 ng/ml)時則不會出現上述的情況。而Western blot analysis的實驗結果同樣的可以證實IAC所得的結果。因此顯示Angiopoietin-1可以透過cGMP來調控ve-cadherin的蛋白表現。此外本實驗亦利用質譜分析法試圖找出其它有關Angiopoietin-1透過NO-cGMP依賴性來調控ve-cadherin蛋白表現的訊息傳遞路徑。跟據我們的實驗結果總結來說,Angiopoietin-1可能透過NO-cGMP依賴性訊息路徑來調控ve-cadherin的蛋白表現量,同時藉由調控ve-cadherin的蛋白表現量來影響胎盤形成的過程中血管及絨毛的發展。
英文摘要 Angiopoietin-1 (Ang-1), an angiogenic factor, existed in the placental tissues including cytotrophoblasts and syncytiotrophoblasts. In endothelium, angiopoietin-1, an anti-permeability factor, stabilizes the vessels and facilitate endothelial survival and vascular maturation. Therefore, Ang-1 may facilitate placental development by promote trophoblast growth and support fetoplacental vascular development and stabilization in placental tissue, but the functional roles of Ang-1 in placental formation are still unclear. Vascular endothelial-cadherin (ve-cad), a 130 kD Ca2+ -depend adhesion molecules, exclusively and constitutively expressed at interendothelial junctions. Ve-cad also can be detected in the placental tissues including intermediate and terminal villi. The increased expression of ve-cad on intercellular junctions decreases capillary permeability by increasing vessel integrity. In addition to ve-cad, Ang-1 and its receptor Tie-2 are also detected in human and primate placental tissues including syncytiotrophoblasts, cytotrophoblast, and perivascular cells. Even though ve-cad, Ang-1 and its receptor Tie-2 exist in cytotroblasts of human term placenta, no current studies further examine whether Ang-1 can modulate the expression of ve-cad. Therefore, we examined whether Ang-1 could increase the expression of ve-cad. First of all, we characterized the expression of Ang-1 and it receptor (Tie-2) throughout gestation and examined the dose-effect of Ang-1 on ve-cad expression. As our data indicated, its receptor was highly expressed in the G18 placenta. Ang-1 at lower concentrations (1-10 ng/ml) increased the expression of ve-cad but that at higher concentrations did not. Accordind our data indicate that the ve-cad expression may regulate by Ang-1 in placental tissues. Secondly, we further investigate how Ang-1 increased ve-cad. It has been reported that Ang-1 can increase NO production by activation of PI3K/AKT pathway. In smooth muscle, the most important target for NO is soluble guanylyl cyclase (sGC). Activation of sGC by NO increases the production of cGMP. Binding of cGMP to cGMP-dependent protein kinase (PKG) type Ⅰα causes PKG autophosphorylation and triggers PKG-mediated cellular responses such as sooth muscle relaxation. The induction of cGMP-dependent protein kinase (Type 1α PKG) phosphorylation by cGMP also could modulate gene expression through cellular signal transduction pathway, foe example, the ERK/MAP kinase pathway. In recent studies, the NO-cGMP-PKG system also considered that may regulate capillary permeability by modulating intercellular junctions. Therefore, to examine whether the increased expression of ve-cad by Ang-1 was through a NO-cGMP-dependent pathway, we first examined the influences of Ang-1 on NO production and then the expression of ve-cad. As our data indicated that Ang-1 at 1.0 ng/ml increases NO production and facilitate ve-cad expression. The increased NO and ve-cad by Ang-1 were reversed by L-NAME. These data suggest that Ang-1 can modulate the expression of ve-cad by change the NO production. To comfirm cGMP is involved in Ang-1 induced ve-cad expression signal transduction pathway, we measure the PKG phosphorylation. The phosphorylated Type 1 PKG was concentrated by immunoprecipitation and confirmed by Western blot analysis. PKG phosphorylation was measured by immobilized affinity chromatography (IAC). Ang-1 at 1.0 ng/ml increased the abundance of phospho-PKG but at higher concentrations (>1.0 ng/ml) did not. IAC data was supported our findings from western blotting analysis. We also use MS/MS assay to find out other signal pathway which are involved in Ang-1 induced ve-cad expression through a NO-cGMP dependent pathway. In conclusion, Ang-1 increased the expression of ve-cad may through a NO-cGMP dependent signal pathway. And then Ang-1 may play an important role in placental formation by regulate the expression of ve-cad.
論文目次 Contents............................................1
誌謝................................................2
中文摘要............................................3
Abstract............................................6
Introduction........................................9
Hypothesis and Aims................................13
Materials and methods..............................15
Results............................................29
Discussion.........................................41
References.........................................48
Figures............................................59
Appendix...........................................77
Curriculum vitae...................................84
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