臺灣博碩士論文加值系統

　　血管中白血球的黏著在許多心血管疾病的發展過程扮演一個相當重要的角色。已有許多文獻指出，在動脈硬化潰瘍部位上的內皮細胞，會異常表現各類的黏著分子。而當這些黏著分子被抑制住時，可以看到白血球及脂肪的堆積在動脈硬化潰瘍部位有減少的結果出現。此外，氧化的低密度脂蛋白(ox-LDL)會刺激內皮細胞上各類的黏著分子表現量增加。因此，ox-LDL可能藉著增加內皮細胞黏著分子表現使內皮細胞功能失調，進而促進白血球的黏著及穿越內皮細胞進入血管內層堆積的反應，最後使動脈硬化潰瘍發展加劇。這篇論文主要的目的，是想觀察及量化白血球在LDL處理後之內皮細胞黏著程度的變化。為了達成此一目的，我們運用一個扇形流體室，其特性為管腔中的流體剪力呈線性變化。我們將人類臍靜脈內皮細胞培養在玻片上，再組裝於流體室中，之後讓白血球沉降及黏著，以測試白血球的黏著力。由正常男子的週邊血液分離出白血球，注入流體室中，經一段時間沉降後，通入一固定流率之緩衝液，在不同流體剪力的沖刷下，觀察白血球黏著之情形。結果顯示，受到ox-LDL前處理後的內皮細胞，在測試的各個剪力強度下的白血球黏著總數及攤平之白血球黏著數目皆大幅上升。反之，若以自然態的低密度脂蛋白前處理內皮細胞，則不影響白血球的黏著。如果在白血球注入流體室前，先處理以VCAM-1抗體，則會使得白血球粘著總數及攤平之白血球數目下降，尤其是後者降幅更大。此外，以ICAM-1抗體及或thrombomodulin lectin domain處理白血球也看到相同的結果。我們的結論是，當內皮細胞受到ox-LDL(尤其是氧化八小時的ox-LDL)的前處理後，白血球與內皮細胞之間的黏著強度會增強；這可能是因為處理後的內皮細胞表現了較多的 VCAM-1、ICAM-1及P-selectin之故。本研究之結果，不僅確認黏著分子在白血球黏著於內皮細胞上所扮演的重要角色，對LDL調控此種重要的細胞間交互作用也有更深一層的瞭解。

　　Intravascular adhesion of leukocytes plays an important role in the pathogenesis of various vascular diseases. Previous studies have indicated that the endothelium overlying atherosclerotic lesions express various adhesion molecules. Blocking of these adhesion molecules reduced leukocytes and lipid accumulation in the lesion area. Various forms of low-density lipoprotein (LDL), especially oxidized LDL (ox-LDL), have been reported to increase the expression of adhesion molecules in endothelial cells. Therefore, ox-LDL may contribute to the development of arterial lesions by altering the function of endothelium, promoting the adhesion of leukocytes and their migration from vascular lumen to intima. This study was undertaken to obtain the quantitative information regarding the adhesiveness between leukocytes and lipoprotein-treated endothelial cells. To address this subject, we used a tapered flow chamber that has a linear variation of shear stress across it flow channel. Cultured human umbilical vein endothelial cells (HUVECs) on a glass slide served as the testing surface of the chamber to allow sedimentation and adhesion of leukocytes. Peripheral leukoocytes collected from normal male subjects were loaded into this chamber and their adhesiveness to HUVECs was accessed by flushing them with a buffer under specific local shear stresses. Our results showed that the numbers of leukocytes and spreaded leukocytes which adhered to oxLDL-pretreated HUVECs increased significantly at all shear stress levels tested. In constrast, native LDL-pretreatment was ineffective. When VCAM-1 antibody was added to a leukocyte suspension before filling into the chamber, the number of adherent leukocytes on ox-LDL-pretreated HUVECs decreased significantly. This was mainly caused by the absence of spreaded leukocytes.Similar results were obtained by the addition of ICAM-1 antibody or thrombomodulin lectin domain. We concluded that the adhesiveness between leukocytes and endothelial cells was enhanced by ox-LDL-pretreated HUVECs. Moreover, the expression of VCAM-1, ICAM-1 antibody and P-selectin on HUVECs may play a role in the enhanced adhesiveness between leukocytes and endothelial cells. This study should provide valuable information regarding not only the adhesion between leukocytes and endothelial cells, but also the roles of LDL in modulating this important cell-cell interaction.

目錄

圖目錄------------------------------ 5
附圖表目錄-------------------------- 7
致謝-------------------------------- 8
中文摘要---------------------------- 9
英文摘要--------------------------- 10
壹、簡介---------------------------- 13
一、動脈硬化是一種發炎性的疾病--------- 13
二、內皮細胞受損是形成動脈硬化的起始---- 14
三、發炎和黏著分子的關係-------------- 15
四、脂蛋白與動脈硬化的關係------------ 17
五、低密度脂蛋白調控內皮細胞上黏著分子的表現
--------------------------------- 19
貳、研究動機------------------------ 21
參、材料---------------------------- 22
肆、方法---------------------------- 27
一、脂蛋白的製備--------------------- 27
二、所有白血球與、單核球的純化--------- 38
三、人類臍靜脈內皮細胞之分離與培養------ 42
四、內皮細胞以脂蛋白做前處理----------- 46
五、白血球黏著力測定------------------ 47
六、細胞免疫染色--------------------- 48
伍、結果---------------------------- 51
一、低密度脂蛋白的純化與氧化低密度脂蛋白的鑑定
-------------------------------- 51
二、不同氧化程度之低密度脂蛋白作用後內皮細胞形態上的改變
-----------------------52
三、利用不同氧化程度之低密度脂蛋白對於內皮細胞的處理與單核球之黏著作用尋找白血球黏著力分析之條件--------------------------- 53
四、不同氧化程度之低密度脂蛋白的處理對於內皮細胞與白血球之間黏著作用的影響--------- 54
五、黏著分子之抗體對於經過不同氧化程度之低密度脂蛋白前處理之內皮細胞與白血球之間黏著作用的影響---------------------------- 55
六、不同氧化程度的低密度脂蛋白的處理對於內皮細胞黏著分子表現的響
----------------- 60
陸、討論---------------------------- 63
一、低密度脂蛋白的純化與鑑定----------- 64
二、不同氧化程度之低密度脂蛋白對於內皮細胞之前處理
---------------------------- 68
三、血球的純化---------------------- 71
四、白血球黏著力分析----------------- 72
五、黏著分子之免疫螢光染色------------ 77
柒、結論---------------------------- 80
捌、未來展望------------------------ 82
玖、參考文獻------------------------ 84
拾、圖------------------------------ 94
拾壹、附圖表----------------------- 112

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