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系統識別號 U0026-0812200910411132
論文名稱(中文) 人類血管內皮細胞的細胞刺激對白血球黏著及氧活性物質產生之研究
論文名稱(英文) Investigation on Cellular Stimulations That Regulate Leukocyte Adhesion and ROS Production in Human Vascular Endothelial cells
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 91
學期 2
出版年 92
研究生(中文) 黃盈婷
研究生(英文) Ying-Ting Huang
電子信箱 tita65@ms33.hinet.net
學號 S4690103
學位類別 碩士
語文別 中文
論文頁數 73頁
口試委員 指導教授-謝奇璋
口試委員-莊季瑛
口試委員-戴任恭
中文關鍵字 白血球黏著  氧活性物質  血管內皮細胞 
英文關鍵字 ROS  Leukocyte Adhesion  Vascular Endothelial cells 
學科別分類
中文摘要 血管內皮細胞(vascular endothelial cells)乃位於血管最內層、與血液直接接觸的地方,對於調控血管功能扮演重要角色。其主要功能在調控血管收縮、細胞激素(cytokines)的產生及與血液中的免疫細胞作用等。但當內皮細胞機能失調時(endothelial dysfunction),便無法維持各項生理平衡,不但改變內皮細胞抗凝固(anti-coagulatory)及抗發炎(anti-inflammatory)的特性,也降低內皮細胞的生長。在正常情況下,白血球在血管中流動,並不會與血管內皮細胞黏著。但當體內有外來物入侵或發炎反應產生時,因組織受損,導致血管內皮細胞受到細胞刺激 (如tumor necrosis factor-α(TNF-α)、C-reactive protein (CRP)、lipopolysaccharide (LPS) 、IL-1、IL-6)作用之下,影響血管內微環境的發炎反應。而發炎反應的發生乃藉由血管內皮細胞表現黏著分子(如ICAM-1及VCAM-1),促使循環白血球黏著到血管壁上,進一步移行到發炎組織部位,執行免疫反應。

內皮細胞功能缺損(endothelial dysfunction)出現在心血管疾病或發炎性疾病(如高血壓、動脈粥狀硬化、糖尿病、過敏性疾病)的病人當中。在此類病人體內,可偵測到大量的ROS產生,形成氧化壓力(oxidant stress),除了造成血管內NO的活性下降,同時也造成血管內皮細胞的滲透性增加及白血球黏著增加。細胞內ROS也扮演著訊息傳遞的角色,促進細胞生長及細胞激素的產生。

內皮細胞在發炎反應中,是透過怎樣機制來調控細胞黏著及ROS的產生,目前仍不清楚。為了進一步瞭解內皮細胞在受到細胞刺激時如何調控其生理功能,我們利用不同的細胞刺激(TNF-a, CRP, LPS, IFN-g)刺激人類臍靜脈內皮細胞 (Human Umbilical Vein Endothelial Cells;HUVECs),模擬體內受到發炎反應時,內皮細胞在白血球黏著及ROS產生方面所造成之影響。結果顯示,在上述的刺激之下,會造成細胞表面不同程度ICAM-1及VCAM-1的表現。我們從cellular ELISA method及flow cytometric analysis的結果發現,TNF-aand LPS造成ICAM-1及VCAM-1的高度表現,而CRP與IFN-g則只造成黏著分子較小的表現。而當合併不同細胞刺激時,我們發現而CRP有增強TNF-a所引發HUVECs表現ICAM-1及VCAM-1的能力。因此,CRP在此可能扮演了一個增強其它細胞刺激功能的角色,但其牽涉的機制仍不清楚。此外,不同的細胞刺激,也影響到內皮細胞對於白血球的黏著能力的增加,且是透過白血球與內皮細胞間LFA-1-ICAM-1 and VLA-4- VCAM-1的連結所達成。

此外,我們也製備了對抗內皮細胞表面分子的單株抗體(EN1-EN6),試著去了解內皮細胞表面分子調控白血球黏著及對其ROS產生之關係。這些單株抗體除了染上HUVECs外,有些也可染上其它不同種類的細胞。同時,隨著不同細胞刺激作用在HUVECs,這些單株抗體與HUVECs的結合也隨之變化。這群anti-HUVEC mAbs也會對內皮細胞黏著或ROS產生有不同程度的影響。而我們發現其中一株單株抗體(EN2)可以在30分鐘內,增加內皮細胞與白血球的黏著,且非經由刺激ROS的產生所達成。我們也證實這株單株抗體乃辨識內皮細胞表面的ICAM-1分子。因此,這群單株抗體對於研究內皮細胞表面分子調控細胞黏著及ROS產生可能是有用的工具。藉由此研究將能對於發炎性疾病中,調控血管內皮細胞生理機制方面,能有更進一步的瞭解,而達到抑制發炎性疾病的目的。


英文摘要 Vascular endothelial cells cover the internal surface of blood vessels and play important roles in the regulation of multiple vascular functions. The functions of vascular endothelial cells include modulation of vascular tone, production of cytokines, and interaction with circulating immune cells. Endothelial dysfunction leads to several pathological conditions including altered anti-coagulatory and anti-inflammatory properties of the endothelium and reduced regulation of vascular growth. In inflammation, multiple cellular stimulations (e.g. TNF-a, CRP, LPS, IFN-g, IL-1 and IL-6) activate endothelial cells to express adhesion molecules (e.g. ICAM-1 and VCAM-1). The expression of adhesion molecules on endothelial cells promotes circulating leukocytes to adhere and then transmigrate through vascular endothelium. Finally, the leukocytes reach the inflammatory sites and induce immune response.

Endothelial dysfunction has been found in cardiovascular and inflammatory diseases including hypertension, atherosclerosis, diabetes, and allergic diseases. Excessive ROS was detected in the inflamed or damaged sites in these patients. More and more evidences suggest that oxidant stress reduces NO bioactivity, increases vascular endothelial permeability and promotes leukocyte adhesion. ROS also can serve as intracellular signaling molecules participating in cell growth and cytokine production.

The regulation mechanisms of leukocyte adhesion and ROS production in inflamed endothelium are largely unknown. In order to investigate the cellular stimulations that regulate cell function, we treated human umbilical endothelial cells (HUVECs) with LPS, TNF-a, CRP, and IFN-g to mimic the inflammatory microenvironment. Our data showed that TNF-a and LPS induced high expression of ICAM-1 and VCAM-1 on HUVECs. However, CRP and IFN-g only had minor effects on adhesion molecule expression. When we combined TNF-a and CRP treatment, we found that CRP plays a role in enhancing TNF-a induced ICAM-1 and VCAM-1 expression on HUVECs. The mechanisms through which CRP modulates adhesion molecule expression and enhances other cytokines effects are still unknown.

Furthermore, we prepared monoclonal antibodies against HUVECs to study how endothelial cell surface molecules are involved in the regulation of cell adhesion and ROS production. These anti-HUVEC mAbs (EN1-EN6) stained HUVECs, and some also stained other cell type. Their binding levels to HUVECs vary on cells treated with different stimulations. Some of them influenced leukocyte adhesion and induced ROS production on HUVECs. Interestingly, we found that an anti-HUVEC mAb (EN2) against ICAM-1 had the ability to enhance leukocyte-endothelial interaction in a ROS independent manner. Therefore, these anti-HUVEC mAbs will be useful tools to investigate endothelial functions including cellular adhesion and ROS production of HUVECs. These studies may facilitate the understanding of the regulation mechanisms on inflamed endothelial cells and lead to novel strategies in controlling inflammatory diseases.



論文目次 中文摘要 Ⅰ
英文摘要 Ⅲ
目錄 Ⅴ
圖表目錄 Ⅶ
第一章 緒論 1
第二章 材料與方法 5
1.細胞株與培養基 5
2.人類臍靜脈內皮細胞 (HUVECs)之分離與繼代培養 8
3.顆粒性白血球的純化 10
4.ROS production 11
5.單株抗體製備 13
6.ELISA screen 16
7.流式細胞儀分析 17
8.細胞與細胞黏著測試 19
9.SDS-PAGE膠體電泳分析 21
10.西方墨點法 24
11.Coomassie blue stain 25
第三章 結果 27
第一部份 細胞刺激對人類臍靜脈內皮細胞的功能測試 27
人類臍靜脈內皮細胞(HUVECs)的分離及培養 27
LPS刺激對HUVECs表現ICAM-1及VCAM-1的影響 28
TNF-a刺激對HUVECs表現ICAM-1及VCAM-1的影響 28
CRP刺激對HUVECs表現ICAM-1及VCAM-1的影響 29
IFN-g刺激對HUVECs表現ICAM-1及VCAM-1的影響 29
合併不同細胞刺激對HUVECs表現ICAM-1及VCAM-1的影響 30
HUVECs表現CRP receptor的情形 31
細胞刺激對於U937與HUVECs黏著的影響 31
第二部份 對抗人類臍靜脈內皮細胞單株抗體的製造與
功能試驗 32
Anti-HUVEC mAbs的製造 32
Anti-HUVEC mAbs對HUVECs表面分子的染色 32
Anti-HUVEC mAbs對其它固著細胞表面分子的染色 32
Anti-HUVEC mAbs對血球細胞或血球細胞株表面分子的染色 33
細胞刺激影響anti-HUVEC mAbs對應分子在HUVECs之表現 33
Anti-HUVEC mAbs對LPS、TNF-a、CRP-treated HUVECs與
U937黏著之影響 34
Anti-HUVEC mAbs對HUVECs產生ROS之影響 35
EN2辨識HUVEC表面分子的鑑定 36
第四章 討論 37
Reference list 43
附圖及附表 46
作者簡歷 73
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