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研究生:黃佳紋
研究生(外文):Chia-Wen Huang
論文名稱:氯胺酮對於小鼠大腦內皮細胞通透度影響之研究
論文名稱(外文):EFFECTS OF KETAMINE ON PERMEABILITY OF MOUSE CEREBRAL ENDOTHELIAL CELLS
指導教授:陳瑞明陳瑞明引用關係
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學科學研究所
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:2008
畢業學年度:96
語文別:中文
論文頁數:56
中文關鍵詞:氯胺酮
外文關鍵詞:ketamine
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在臨床上,氯胺酮是一種常見的靜脈麻醉藥劑,被使用於診斷或不需肌肉鬆弛之手術,尤其適用於短時間之小手術或全身麻醉時誘導之用。它可以直接作用在中樞神經系統,非競爭性的拮抗NMDA (N- methyl-D-aspartate)接受器,達到止痛的效用。另外,近幾年已被列為三級毒品的氯胺酮,也是坊間風行的濫用藥物之一。在血液循環系統與中樞神經系統之間,存在一道血腦障壁(blood brain barrier, BBB),它是由一連串的大腦血管內皮細胞(cerebral endothelial cells, CECs)藉由緊密排列成tight junction (TJ)所組成,負責保護中樞神經免於免疫細胞及其他分子的侵犯,同時維持腦與脊髓所需的體內生理恆定 (homeostasis),本篇研究的藥物氯胺酮乃經由血液循環通過這層屏障進入並影響中樞神經系統 。
為了釐清氯胺酮對於腦血管內皮細胞的影響,我們從小鼠大腦微血管製備大腦血管內皮細胞,並處理臨床濃度100 μM的氯胺酮,結果發現在處理藥物後,腦血管內皮細胞的增生受到抑制,可能與細胞週期實驗中發現的G1-arrest現象有關,但即使在處理時間長達三天後,仍然沒有造成腦血管內皮細胞顯著的凋亡。而在細胞屏障功能的偵測方面,我們使用dextran的通透與跨內皮細胞膜電阻來評估,結果顯示單層大腦內皮細胞的通透度隨著氯胺酮濃度的增加而升高,且隨著藥物處理的時間增長而升高。藉由共軛焦顯微鏡擷取的影像,我們也發現細胞骨架與TJ蛋白的結構隨著藥物處理時間增長而遭到愈嚴重的破壞,並且伴隨著細胞內ATP的含量下降,追溯其上游粒線體膜電位也隨著處理時間增長而降低。依照結果推論,臨床濃度的氯胺酮會輕微的抑制大腦內皮細胞的增生,但不會造成細胞凋亡,並且會藉由抑制ATP產生而使肌動蛋白聚合不完全,進而導致TJ無法緊密結合,最後造成細胞屏障功能的缺失,通透度上升。
Clinically, ketamine (KET) is usually used as an anesthetic agent due to its ability to inhibit central and peripheral catecholamine reuptake and to direct stimulation of the central nervous system (CNS). Between blood circulation and CNS, the blood brain barrier (BBB) plays an important role in micro-environmental regulation. It mainly consists of cerebral endothelial cells (CECs) and astrocytes that form compact tight junctions as a selective physical barrier, which restricts transport of immunocytes and most molecules from blood circulation to the CNS .
A recent study shows that treatments of neurons with clinical KET dosages can result in time-dependent decreases in neuron growth and increases in the amount of apoptotic cells. But the influence of KET in CECs still remains unknown. We therefore investigated the toxicity effects of KET on CECs, and found that 100 µM of KET inhibited the proliferations of CECs, and arrested cell cycle at G1 phase. However, this treatment did not cause apoptosis of CECs after being exposed to KET for 24, 48, and even 72 hrs. These results led to an investigation on functional effects of KET on CECs and we found that a therapeutic concentration of KET increased the permeability of CEC monolayer, and destroyed the structures of not only actin filaments but also tight junctions. KET also appeared to decrease the mitochondrial membrane potential, and as a result decreased the amount of ATP in CECs.
In conclusion, a clinically relevant concentration of KET did not induce apoptosis of CECs, but did cause cell dysfunctions of CECs by increasing cell permeability through inhibited ATP synthesis and suppressed polymerization of actin filaments. As a result, tight junctions were loosed and no longer intact.
目錄……………………………..………………….…………………..…I
縮寫表……………………...……………..…….…………………..…III
圖目次…………………..…………………...………….………….……IV
中文摘要…………………………………....………………...………….V
英文摘要……………………………………...……....………...……VII
第一章 緒言…………………………………...……….…...……………1
第一節 血腦障壁與中樞神經系統…………………………………...1
第二節 氯胺酮………………………………………………………...6
第三節 研究假說與研究目標……………………………………….10
第四節 實驗設計及說明………………………………………..…...12
第二章 材料與方法………………………………………...…………..17
第一節 實驗材料……………………………………….……………17
第二節 小鼠大腦血管內皮細胞之製備………………………….…17
第三節 藥物處理………………………………………………….....19
第四節 細胞增生率之測定………………………………………….19
第五節 細胞週期及細胞凋亡之測定……………………………….20
第六節 細胞通透度之測定………………………………………….21
第七節 肌動蛋白纖維之螢光化學染色…………………………….22
第八節 Tjght junction蛋白之免疫螢光染色………………………..23
第九節 三磷酸腺?ㄖt量之測定…………………………………….24
第十節 粒線體膜電位之測定……………………………………….25
第十一節 統計分析……..…………………………………………..25
第三章 結果……………………………………….……………………26
第一節 氯胺酮抑制大腦內皮細胞增生…………………………….26
第二節 氯胺酮對大腦內皮細胞的細胞週期的影響……………….26
第三節 氯胺酮會造成大腦內皮細胞層的通透度上升…………….28
第四節 氯胺酮會抑制細胞骨架結構–肌動蛋白的聚合………….29
第五節 氯胺酮會破壞TJ結構………………………………………30
第六節 氯胺酮導致細胞能量ATP的減少……………….………...30
第七節 氯胺酮降低粒線體膜電位………………………………….31
第四章 討論……………………………………….……………………32
參考文獻……………………………………….………………………..37
圖表……………………………………….……………..………………45
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