高壓氧處理之人類臍靜脈內皮細胞所誘導之血管內皮細胞生長因子VEGF表現的分子機制探討

Mechanisms of The Induction of Vascular Endothelial Growth Factor Expression by Hyperbaric Oxygen in Human Umbilical Vein Endothelial Cells

陳淑貞(Shu-Chen Chen)

指導教授 ： 李宏謨 博士 ； 徐國基 博士

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2004年)

摘要

高壓氧治療可促進血管新生，進而加速和缺氧相關的傷口癒合，而血管內皮細胞生長因子 (VEGF) 及其接受器系統無論在生理或病理性的血管新生都是最主要的調控者。然而高壓氧調控血管內皮細胞活化的機制尚未明瞭。本實驗即是在探討高壓氧活化血管內皮細胞生長所扮演的角色及其可能的調控機制。我們由Real-time PCR及北方墨點分析數據證實，高壓氧可誘發血管內皮細胞生長因子基因的表現，並於4小時達到最高峰，且高壓氧所誘發VEGF mRNA的表現可被MEK的抑制劑PD98059及JNK的抑制劑SP600125所抑制。另一方面，西方墨點分析法顯示，VEGF蛋白最大表現量在8小時，ERK1/2的磷酸化則在15分鐘最強。此外，VEGF 的promoter上有hypoxia inducible factor-1 (HIF-1) 和Activator protein-1 (AP-1) 轉錄因子的結合位，由electrophoretic mobility shift assay (EMSA) 結果顯示，高壓氧處理1小時可明顯活化此二轉錄因子，且其DNA-protein的結合能力皆會被PD98059及SP600125所抑制。因此推測高壓氧可能是透過JNK-mitogenic-activated protein kinase及ERK-mitogenic-activated protein kinase 的訊息傳遞路徑，活化HIF-1和AP-1轉錄因子而啟動VEGF基因的表現，進而促進血管的新生。

關鍵字

血管內皮細胞生長因子 ； 高壓氧 ； Mitogen Activated Protein Kinases ； ERK1/2 ； JNK ； 活化蛋白-1 ； 缺氧誘發因子-1α

並列摘要

One of the biological effects of hyperbaric oxygen (HBO) therapy in enhancing ischemia-related wound healing is the induction of angiogenesis. The vascular endothelial growth factor VEGF/VEGF receptor system plays a central regulatory role in physiological and pathological angiogenesis. During embryogenesis, the VEGF/VEGF receptor system is critically involved in the formation of the vascular system by regulating both the growth and the survival of blood vessels. The mechanism by which hyperbaric oxygen regulates angiogenesis is presently unclear. The aim of the present study was to investigate the mechanism of the activation of VEGF gene activation in human umbilical vein endothelial cells (HUVECs) exposed to HBO. Preliminary Real-time quantitative PCR studies revealed that the expression of VEGF mRNA was induced after 4 hours of exposure. The induction of VEGF mRNA was further confirmed by Northern blot analysis. Western blot analysis showed that HBO enhanced the VEGF protein level and increased the ERK1/2 activity, with a maximal level after 8 hours and 15 min, respectively. Whereas, JNK inhibitor SP600125 and MEK inhibitor PD98059 blocked the HBO-induced VEGF mRNA expression. Since VEGF promoter region contains AP-1 and HIF-1 binding sites, stimulation of HUVECs with HBO enhanced AP-1 and HIF-1 binding to their DNA binding sites as demonstrated by electrophoretic mobility shift assay(EMSA). This effect was also inhibited by SP600125 and PD98059. These data suggested that HBO-induced VEGF expression might be through AP-1 and HIF-1 stimulation, and required the involvement of JNK and ERK-mitogenic-activated protein kinase signaling pathways, which may play an important role in HBO-induced angiogenesis.

並列關鍵字

Vascular Endothelial Growth Factor ； hyperbaric oxygen ； Mitogen Activated Protein Kinases ； ERK1/2 ； JNK ； Activator Protein-1 ； HIF-1α

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