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系統識別號 U0026-0309201514133200
論文名稱(中文) 在新生兒缺血缺氧性腦傷中,血管內皮細胞IRS-1透過增加神經發炎反應加重神經血管的傷害
論文名稱(英文) Endothelial IRS-1 aggravates neurovascular damage after neonatal hypoxic-ischemic brain injury through increasing neuroinflammation
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 103
學期 2
出版年 104
研究生(中文) 黃名儀
研究生(英文) Ming-Yi Huang
學號 S96021032
學位類別 碩士
語文別 英文
論文頁數 49頁
口試委員 指導教授-黃朝慶
指導教授-杜伊芳
口試委員-謝奇璋
口試委員-楊尚訓
中文關鍵字 缺氧窒息  血管內皮細胞  神經血管單元  胰島素受體底物-1  神經發炎反應  黏附因子  新生兒大腦 
英文關鍵字 hypoxic-ischemia  vascular endothelium  neurovascular unit  insulin receptor substrate-1  neuroinflammation  adhesion molecules  neonatal brain 
學科別分類
中文摘要 周產期缺氧窒息性腦傷是造成新生兒死亡和神經性後遺症的一大原因,缺氧窒息不只會造成神經血管單元中神經元的死亡,也會傷害血管內皮細胞,而維持良好的神經血管單元的完整性,對於大腦功能的發揮很重要。破壞神經血管單元的完整性,特別是大腦的微血管內皮細胞,會破壞血腦屏障,並導致血管源性的腦水腫和繼發性的神經元損傷,最終加劇了長期的神經功能障礙。因此,尋找針對於保護血管以維持神經血管單元的完整性之方法,可期為目前對於缺氧窒息性腦傷的治療新方向。
在我們之前的研究發現,攝食量節制後減重的幼鼠能改善缺氧窒息性腦傷,並提供長期的保護,而這個保護機制是經由胰島素受體底物-1 (IRS-1)-Akt訊息傳遞路徑來達到保護效果。在體外細胞實驗中發現,IRS-1過度表現可減少缺氧缺糖誘導的細胞死亡,而且主要是血管內皮細胞。因此,為了瞭解IRS-1在新生兒缺氧窒息腦傷中對於血管內皮細胞的保護機制,我們建立了一隻IRS-1過度表現於血管內皮細胞的轉殖基因大鼠。
然而,我們發現血管內皮細胞過度表現IRS-1,反而惡化幼鼠的缺氧窒息腦傷。它增加了神經血管單元的破壞,像是基質金屬蛋白酶-9(MMP 9)的產生、緊密連接蛋白(tight junction protein)的缺損和血腦屏障的傷害,而在缺氧窒息後第七天,也發現我們的轉殖基因大鼠,有較多的腦梗塞體積。因為缺氧缺血性腦傷中若讓血管內皮細胞上的黏附因子(例如ICAM-1或 VCAM-1)表達上升,會增加周邊的白血球黏附,進而穿透血管進入受傷的腦組織,且引發更大的神經發炎反應,會導致更嚴重的腦傷。因此,我們進一步測試內皮細胞過度表現IRS-1的轉殖基因大鼠,在缺氧窒息後的黏附因子表現。我們的結果發現內皮細胞過度表現IRS-1的轉殖基因大鼠之仔鼠,相較於野生型大鼠,在缺氧窒息後三小時的大腦皮質,就有較多的黏附因子VCAM-1的表現,而缺氧窒息後二十四小時則會增加另一種黏附因子ICAM-1表現,且在體外的細胞實驗發現,人類大腦微血管內皮細胞過度表現IRS-1,不論缺氧缺糖實驗前或後,VCAM-1和ICAM-1都有表現上升的現象。在缺氧窒息後二十四小時,轉殖基因大鼠,相較於野生型大鼠,有較多的小膠質細胞的活化和中性粒細胞的浸潤。當我們在轉殖基因大鼠中利用抗體抑制VCAM-1和 ICAM-1的表現後,發現抑制VCAM-1或抑制VCAM-1和 ICAM-1 都減少了轉殖基因大鼠在缺氧窒息後腦細胞的死亡、MMP 9的產生、小膠質細胞的活化和中性粒細胞的浸潤。
總結,在新生兒的缺血缺氧性腦傷中,血管內皮細胞的IRS-1過度表現,會增加VCAM-1和ICAM-1的表現而加重神經發炎反應,最終惡化腦傷。
英文摘要 Perinatal hypoxic-ischemia (HI) is a major cause of neonatal mortality and long-term neurological morbidity among survivors. HI injury not only causes neuron death but also damages endothelial cells within neurovascular unit. Maintaining the integrity of whole neurovascular unit is required for proper brain function. Disruption of neurovascular unit, especially cerebral microvascular endothelial cells, leads to BBB perturbation and causes vasogenic cerebral edema and secondary neuronal damage, which eventually exacerbates long-term disability. Thus, there is a new idea to develop means to protect the vasculature to improve HI outcome.
Our previous study found that dietary restriction (DR) reduced neurovascular damage after HI and conferred long-term protection in the neonatal brain through insulin receptor substrate-1 (IRS-1)-Akt pathway. Moreover, IRS-1 over-expression protected against oxygen-glucose deprivation (OGD) cell death mainly in the endothelial cells. Therefore, an endothelium specific IRS-1 transgenic rat was created to delineate the role of IRS-1 in vascular endothelial cells against HI in the neonatal brain.
We found that over-expressed IRS-1 in the vascular endothelial cells worsened the HI infarct volume with decreased tight junction proteins expression, and increased matrix metalloprotein 9 (MMP 9) production and BBB damage. Since upregulating adhesion molecules of endothelium increases peripheral leukocytes infiltration in to the damaged brain tissue and subsequently worsen the brain injury, we further investigated the expression of adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in our transgenic rats. We found endothelial IRS-1 transgenic pups had upregulated VCAM-1 expression as early as 3 hours and ICAM-1 expression at 24 hours after HI. It associated with significantly increased microglia activation and neutrophils infiltration at 24 hours after HI. The in vitro experiments confirmed that IRS-1 over-expression in human brain microvascular endothelial cells could increase VCAM-1 and ICAM-1 expression before and after oxygen-glucose deprivation (OGD). Blockage of VCAM-1 and ICAM-1 using anti-VCAM-1 and anti-ICAM-1 antibody in our transgenic rats decreased neuronal apoptosis, and attenuated MMP 9 production, microglia activation and neutrophils infiltration after HI.
In conclusion, IRS-1 specific over-expression in the vascular endothelial cells enhanced VCAM-1 and ICMA-1 expression, and exaggerated neuroinflammation and neurovascular damage after HI in the neonatal brain.
論文目次 中文摘要…………………………………………………...…… Ⅰ
Abstract………………………………………………………… Ⅳ
Acknowledgement………………………………………………..Ⅵ
Contents………………………………………………………… Ⅷ
Figure Contents…………………………………………………Ⅸ
Abbreviation Index………………………………………..……Ⅹ
Introduction…………………………………………...………..… 1
Materials and Methods………………………………………..… 6
Results…………………………………………………………. 13
Discussion…………………………………………………...… 17
Figures and Figure Legends… ……………………………...… 22
References…………………………………………………… 32
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