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系統識別號 U0026-2608201416532200
論文名稱(中文) 細胞凋亡在窄化所誘導的腹主動脈瘤豬模式中扮演的角色
論文名稱(英文) The Role of Apoptosis in the Coarctation-Induced Abdominal Aortic Aneurysm in a Porcine Model
校院名稱 成功大學
系所名稱(中) 細胞生物與解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 102
學期 2
出版年 103
研究生(中文) 尹志銘
研究生(英文) Chih-Ming Yin
學號 T96011038
學位類別 碩士
語文別 英文
論文頁數 55頁
口試委員 指導教授-江美治
口試委員-莫凡毅
口試委員-陳玉怜
中文關鍵字 腹主動脈瘤  細胞凋亡  原位末端轉移酶標記  自噬作用 
英文關鍵字 abdominal aortic aneurysm  apoptosis  TUNEL  autophagy 
學科別分類
中文摘要 動脈瘤是血管壁不正常的膨脹而造成的疾病,六十五歲以上的人口中有4~8%的人患有此疾病。在所有的動脈瘤中,以腹主動脈瘤(AAA)最為常見,而腹主動脈瘤的好發位置在腎動脈以及髂總動脈分枝處之間。當腹主動脈瘤破裂時,其致死率高達90%,是臨床上棘手的難題。腹主動脈瘤的病理特色主要有持續的慢性發炎、細胞外基質的降解以及血管平滑肌細胞(VSMCs)的損失。過去的文獻指出,在病人的腹主動脈瘤組織檢體中,血管平滑肌細胞的數量較正常的主動脈減少,而凋亡的細胞(Apoptosis)數則增加。這些觀察顯示,血管平滑肌細胞的凋亡在動脈瘤形成的過程中可能扮演重要的角色。在本實驗室先前的研究,透過在蘭嶼迷你豬的腹主動脈進行窄縮,成功的建立退化性腹主動脈瘤形成的模型。在此動物模型中,腹主動脈瘤於窄縮後12周在窄縮的遠心端形成。因此我們提出一個假設:在腹主動脈進行長時間的窄縮會誘發血管壁細胞的凋亡,進而促使腹主動脈瘤的生成。在本實驗中,我們將腎動脈下端的腹主動脈以人工血管進行4周、8周以及12周的窄化。首先,以蘇木素-伊紅(H&E)染色的結果顯示,血管在窄縮後12周,在遠心端處,血管的內徑較腎動脈上方的腹主動脈有顯著的增加。在彈性纖維與膠原蛋白的染色結果顯示,在遠心端的腹主動脈隨著窄縮的時間增加,血管中層的彈性纖維呈現明顯的降解和片斷化,而膠原蛋白則是有重塑的現象產生。利用原位末端轉移酶標記(TUNEL)技術來偵測細胞凋亡的結果顯示,在遠心端處,血管中層內的細胞凋亡在窄縮後4周有增加的趨勢,之後逐步地減少在窄縮後12周回到基值。此外,在血管內層及外層也觀察到不少的細胞在進行細胞凋亡作用。我進一步利用螢光雙重染色偵測在腹主動脈瘤形成的過程中,進行凋亡的細胞類型。結果顯示,血管內層中的內皮細胞(endothelial cells)的細胞凋亡在對照組及實驗組均偏高,但在窄縮後12周則明顯的減少;而在血管外層中的滋養小血管(vasa vasorum)之內皮細胞的細胞凋亡則是在窄縮後4周有明顯的增加。血管中層的平滑肌細胞主要是在窄縮後4周有明顯而局部的細胞凋亡現象。此外,在血管內皮下內層利用S100A4所偵測到的修飾型血管平滑肌細胞在窄縮後4周及8周均有明顯的細胞凋亡。相對的,分佈在血管外層的巨噬細胞(macrophages)及纖維母細胞(fibroblasts)則無明顯的細胞凋亡現象。在細胞凋亡與自噬作用(autophagy)相關的指標蛋白部分,我們偵測了半胱氨酸天冬氨酸蛋白酶-3(caspase-3)與微管相關蛋白輕鏈-3(LC-3),並沒有檢測到顯著的變化。綜合上述結果,在腹主動脈進行長期的窄縮,可誘發遠心端處的腹主動脈血管內層與中層的平滑肌細胞及血管內層與外層的內皮細胞進行細胞凋亡,這些變化可能進一步促進窄化所誘導的腹主動脈瘤的生成。
英文摘要 Aneurysms, an abnormal expansion of the vascular wall, occur in 4-8% of the population over 65 years old. Abdominal aortic aneurysm (AAA) is the most common aneurysm that usually occurs between the renal arteries and the aortic bifurcation. The mortality rate of AAA reaches 90% when ruptured and presents a significant clinical problem. AAA is characterized by chronic inflammation, extracellular matrix degradation, and loss of vascular smooth muscle cells (VSMCs). In human AAA tissues, VSMC density decreases while apoptotic cells increase compared with normal aorta. Therefore, apoptosis of VSMCs may play an important role in aneurysm formation. Our laboratory developed a coarctation-induced degenerative AAA model by surgically narrowing an infrarenal abdominal aorta (AA) segment in Lanyu mini pigs. In this model, AAA was formed in the aortic segment distal to coarctation at 12 weeks post-coarctation. We hypothesized that prolonged coarctation of abdominal aorta induces apoptosis of vascular cells that participate in AAA formation. An infrarenal AA segment was wrapped with an ePTFE Teflon strip for 4 weeks (4w), 8 weeks (8w) or 12 weeks (12w). Histological analysis showed that aortic lumen perimeter markedly increased in the distal AA compared to the suprarenal AA at 12w post-coarctation. In the distal AA, fragmentation and degradation of elastic lamellae in the media increased with time and collagen remodeling was detected. Cellular apoptosis examined with terminal transferase dUTP nick end labeling (TUNEL) assay showed that TUNEL-positive cells in the media increased at 4w and gradually decreased to basal levels at 12w post-coarctation. Interestingly, TUNEL-positive cells were abundant in the intima and adventitia. Double immunofluorescence staining was used to identify cell types which underwent apoptosis during AAA formation. Apoptotic rate of endothelial cells in the intima appeared high in all groups except for the 12w post-coarctation whereas endothelial apoptosis in the vasa vasorum of the adventitia appeared to increase at 4w post-coarctation. Apoptosis of VSMC in the media appeared focal and was mainly detected at 4w post-coarctation. In addition, in the sub-endothelial intima, apoptosis was detected in S100A4-positive cells which may represent phenotypically modified VSMCs at 4w and 8w post-coarctation. In contrast, apoptotic rate of macrophages and fibroblasts, both were distributed in the adventitia, was low. Using immunoblotting analysis, no change in active caspase-3 and LC3 was detected in the distal AA segment. In summary, prolonged coarctation of the abdominal aorta induces apoptosis of endothelial cells in the intima and vasa vasorum and VSMCs in the intima and media of the distal AA segment, which may contribute to coarctation-induced AAA formation.
論文目次 Chinese abstract 一
English abstract 三
Acknowledgements 五
Introduction 1
Objective of this study 6
Materials and Methods 7
Results 24
Discussion 29
References 33
Figures 38
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