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研究生:張金鉅
研究生(外文):Chin-Chu Chang
論文名稱:多形核白血球反向穿越培養的人類臍靜脈內皮細胞層之過程
論文名稱(外文):The Reverse Transmigration of Polymorphonuclear Leukocytes across a Cultured Human Umbilical Vein Endothelial Monolayer
指導教授:任卓穎
指導教授(外文):Chauying J. Jen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:87
中文關鍵詞:內皮細胞多形核白血球穿越內皮細胞層反向穿越內皮細胞層趨化物質
外文關鍵詞:formly-Met-Leu-Phetransendothelial migrationendothelial cellspolymorphonuclear leukocytereverse transendothelial migration
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在血液循環的白血球能接受發炎訊號的刺激而進行穿越內皮細胞層(transendothelial migration)的動作。此外,單核球在分化的過程中則會反向穿越內皮細胞層(reverse transendothelial migration)而成為樹突狀細胞。而位於血液與周邊組織間的屏障,是內皮細胞藉由接合蛋白所連接而成,其具有調控白血球穿越的功能。由本實驗室最近的研究顯示,多形核白血球(PMN)穿越內皮細胞層時,會引發毗鄰穿越位置的內皮細胞內鈣離子訊號;更進一步,內皮細胞間的接合蛋白於多形核白血球穿越時,會呈現出重新分佈的現象。因此,本研究主要是想瞭解在有趨化物質(fMLP)濃度梯度的誘導下,多形核白血球在反向穿越內皮細胞層時的現象。本實驗中,人類臍靜脈內皮細胞(HUVECs)是養殖在0.4﹪的膠原凝膠薄層上,將此長滿內皮細胞的膠原凝膠薄層浸泡於fMLP中並組裝至流體室內,依序灌流不含fMLP的緩衝液、含多形核白血球的細胞懸浮液及再次灌流含有fMLP的緩衝液。在此條件下,多形核白血球會首先穿越內皮細胞層到達膠原凝膠薄層,並再反向穿越至內皮細胞層的頂端。多形核白血球整個穿越與反向穿越內皮細胞層的過程,會以相位差顯微鏡進行觀察,並在不同的時間點將試樣固定後,以掃描式電子顯微鏡拍攝記錄。此外,也藉由免疫螢光染色的方式觀察VE-cadherin分子在此過程中的動態變化。於本研究中,我們發現(1)穿越過內皮細胞層的多形核白血球會在內皮細胞底部進行橫向的遷移,並離原始穿越位置有相當遠的距離;(2)多形核白血球也能夠以原始的穿越位置進行反向穿越內皮細胞層;(3)多形核白血球的細胞體取代偽足而首先露於內皮細胞層之上;(4)多形核白血球會於反向穿越內皮細胞層後伸展出其偽足;(5)位於多形核白血球反向穿越位置的內皮細胞接合蛋白(VE-cadherin、PECAM-1)呈現出不連續的分佈;(6) VE-cadherin分子會於多形核白血球反向穿越內皮細胞層時,以側推的方式被推開。綜括本研究之結果,應該可對多形核白血球反向穿越內皮細胞層時的相互關係有更深一層的瞭解。
Leukocytes in the circulating bloodstream undergo transendothelial migration in response to inflammatory stimuli. In addition, complicated trafficking behaviors including reverse transmigration occur in monocytes and dendritic cells as part of their differentiation process. Endothelial cells (ECs), the main barrier between blood and tissue, are connected by intercellular junctions, which presumably control leukocyte transmigration. Recent studies from our laboratory have shown that polymorphonuclear leukocyte (PMN) transmigration is accompanied with endothelial [Ca2+]i signaling that only happens in ECs adjacent to the transmigration site. Moreover, the inter-endothelial junction proteins relocate to allow the passage of PMN. This study is to examine in detail the reverse transmigration process of PMN across a monolayer of cultured ECs in response to a gradient of chemoattractant formly-Met-Leu-Phe (fMLP). Human umbilical vein endothelial cells (HUVECs) were cultured on a thin layer of 0.4﹪collagen gel until confluence. The EC monolayer was immersed in fMLP, mounted on a flow chamber and sequentially perfused with a fresh buffer, a PMN suspension, and an fMLP-containing buffer again. PMNs initially transmigrated across the EC monolayer to the collagen gel and reverse transmigrated to the apical side of the EC monolayer. The entire process of PMN transmigration and reverse transmigration across the EC monolayer was traced under phase contrast optics. Specimens were fixed at various times and processed for examination under a scanning electron microscope. In addition, immunostaining technique was used to observe the dynamic movement of VE-cadherin of interest. We found that (1) transmigrated PMNs usually moved laterally underneath the endothelial monolayer before undergoing reverse transmigration at locations some distances away from their original transmigration sites;(2) The process of PMN reverse transendothelial migration could also occur in the original transmigration site;(3) PMN cell bodies, instead of lamellipodia, emerged at the EC apical surface first;(4) PMNs spread out their pseudopodia after reverse transendothelial migration;(5) both EC junction proteins, VE-cadherin and PECAM-1, showed discontinuities at the PMN reverse transmigration site;(6) VE-cadherin molecules were pushed aside by PMNs undergoing reverse transmigration. Results from this study should shed some light on the understanding of endothelial responses during the reverse transmigration of PMN.
目 錄
中文摘要…………………………………………………………..……1
英文摘要………………………………………………………………..3
致 謝…………………………………………………………………….5
目 錄…………………………………………………………………….6
圖目錄…………………………………………………………………..9
附圖目錄………………………………………………………………11
壹、序言…………………………………………………………….…12
黏著連鎖效應……………………………………………………...12
滾動……………………………………………………………13
活化與穩固黏著………………………………………………14
穿越內皮細胞層………………………………………………16
內皮細胞層下遷移……………………………………………17
內皮細胞接合蛋白與白血球之關係……………………………...18
內皮細胞接合蛋白……………………………………………18
接合蛋白調控白血球之穿越…………………………………21
研究動機………………………………………………………...…23
貳、材料與方法…………………………………………………...….25
一、儀器設備……………………………………………………….25
二、材料…………………………………………………………….26
三、方法…………………………………………………………….31
人類臍靜脈內皮細胞之分離與培養…………………………31
人類臍靜脈內皮細胞之繼代培養……………………………32
蓋玻片之清洗與滅菌…………………………………...…….32
0.4﹪膠原凝膠薄層之製備…………………………………...32
0.4﹪膠原凝膠薄層之內皮細胞……………………………...33
流體室各元件之組裝…………………………………………33
趨化物質濃度梯度之形成……………………………………34
多形核白血球之分離與純化…………………………………35
在相位差顯微鏡系統下觀察多形核白血球反向穿越內皮細胞層………………………………………………………………36
在螢光顯微鏡系統下觀察多形核白血球反向穿越內皮細胞層
…………………………………………………………………37
螢光顯微鏡之樣品觀察………………………………………38
掃描式電子顯微鏡之樣品前處理……………………………39
臨界點乾燥……………………………………………………39
樣品金屬覆膜與保存…………………………………………40
掃描式電子顯微鏡之樣品觀察………………………………40
參、結果………………………………………………………….…..42
一、利用流體室製造趨化物質之濃度梯度……………….…….42
二、利用相位差顯微鏡連續記錄多形核白血球反向穿越內皮細胞
層的動態變化………………………………………………...43
多形核白血球穿越內皮細胞層………………………………43
多形核白血球反向穿越內皮細胞層…………………………43
多形核白血球穿越與反向穿越內皮細胞層之位置選擇……43
多形核白血球穿越與反向穿越內皮細胞層之時間比較……44
多形核白血球反向穿越內皮細胞層之端點變化……………44
三、利用掃描式電子顯微鏡觀察多形核白血球反向穿越內皮細胞
層時的形態構造……………………………………………...44
反向穿越內皮細胞層的多形核白血球之形態構造…………44
已反向穿越內皮細胞層的多形核白血球之形態構造………45
四、利用螢光顯微鏡觀察多形核白血球反向穿越內皮細胞層時接
合蛋白分子的分佈現象…………..…………………………..45
五、利用螢光顯微鏡連續記錄活體之多形核白血球反向穿越內皮
細胞層時的分子反應……………………………………..…..46
肆、討論……………………………………………………….……...47
伍、參考資料………………………………………………………….52
圖………………………………………………………………………..65
附圖………………………………………………………………….…81
縮寫表…………………………………………………………………85
作者簡介………………………………………………………………87
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