臺灣博碩士論文加值系統

白血球穿越血管內皮細胞的現象是一種重要的免疫反應。 當個體組織受到損傷時，血液中的白血球會受到受傷處組織所釋放出化學吸引劑的誘導，向著患處前進，清除外來抗原。其過程中，需經過擔任阻絕與調節血管內外物質通透工作的血管內皮細胞。這一層直接接觸血液的單層細胞，會因為物理性或者化學性刺激而產生功能上的變化，用以調節局部的生理恆定。 我們在實驗中試圖觀察及瞭解白血球穿越血管內皮細胞現象的過程與訊息傳遞，說明兩種不同細胞之間的交互作用。 在這個研究之中，我們使用人類臍靜脈內皮細胞以及單核球當作實驗材料，將內皮細胞養殖長滿在以膠原蛋白凝結而成的膠體上，浸潤以formy-Met-Leu-Phe (fMLP)，並且以鈣離子螢光劑Fura PE3或Fura2 指示內皮細胞內鈣離子的濃度。將此膠體置於流體室中，再以新鮮的緩衝液沖洗造成一個化學吸引劑梯度的環境，之後通入單核球，使單核球與內皮細胞接觸，並有滾動(Rolling)、黏著(Adherence)、以及穿越(Transmigration)的現象發生，以及內皮細胞內鈣離子濃度的上升，各以相位差顯微鏡與螢光顯微鏡穿插記錄之。 另外，我們也使用掃瞄式電子顯微鏡觀察實驗後的內皮細胞與單核球形態，先以相位差顯微鏡對樣本拍照進行相對位置標定(Mapping)，再經過多步驟樣本固定以及對膠體樣本的浮動處理後鍍金完成，可以觀察到單核球在穿越內皮細胞前、中、後，形態上的變化，以及相位差影像與電子顯微鏡影像的異同。

Transmigration of leukocytes through endothelial cell (EC) monolayer is an important step in our immune responses. In this study, human umbilical vein ECs were cultured on a thin layer of collagen gel until reaching confluence. The specimen was immersed in formy-Met-Leu-Phe (fMLP), labeled with the fluorescent Ca2+ indicator fura-2 or fura PE3, and mounted on a flow chamber. After monocytes were applied into the chamber, their transendothelial migrations were recorded under phase contrast optics and the accompanying endothelial [Ca2+]i changes were monitored by taking fluorescent ratio images. At the end of an experiment, specimens were silver-stainedfor observing cellular boundaries and further processed for scanning electron microscopic observation. In the absence of fMLP, the transmigration percentage reached about 50% in 90 min at 37℃. The fMLP pretreatment greatly enhanced this value. Transmigrating monocytes started with the formation of several filopodia and the migration towards endothelial boundaries (especially the tricellular corners), followed by the insertion of a leading filopodium between adjacent endothelial cells, and finally by the movement of whole cell body across the monolayer. After monocyte transmigration, some platelets usually remain on the EC surface near the transmigration site. These platelets were adherent to monocytes but could not pass through the EC monolayer with the transmigrated monocytes. The entire process usually lasted more than 10 min, and the transendothelial migration usually spent about 2 minutes. EC [Ca2+]i elevation were observed in ECs adjacent to the transmigration site when monocyte cell bodies forming filopodia. In conclusion, monocyte transmigration is a complex process that appears to be accompanied with drastic changes in morphology and [Ca2+]i elevations in adjacent ECs.

中文摘要-----------------------------------------------------------------------------------1
英文摘要-----------------------------------------------------------------------------------2
誌謝-----------------------------------------------------------------------------------------4
目錄-----------------------------------------------------------------------------------------5
圖目錄--------------------------------------------------------------------------------------6
前言-----------------------------------------------------------------------------------------7
材料與方法-------------------------------------------------------------------------------10
結果----------------------------------------------------------------------------------------27
討論----------------------------------------------------------------------------------------32
參考文獻----------------------------------------------------------------------------------40
圖表----------------------------------------------------------------------------------------42

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