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研究生:鐘玉娟
研究生(外文):Yu-Chuan Chung
論文名稱:探討內毒素誘發血管平滑肌細胞凋亡的過程中:一氧化氮和鉀離子所扮演的角色
論文名稱(外文):Lipopolysaccharide-Induced Apoptotic Process in Vascular Smooth Muscle Cells:Roles of Nitric Oxide and Potassium Ions
指導教授:吳劍男
學位類別:碩士
校院名稱:國防醫學院
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:2002
畢業學年度:90
語文別:中文
論文頁數:70
中文關鍵詞:內毒素鉀離子通道細胞凋亡
外文關鍵詞:lipopolysaccharidepotassium channelapoptosis
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鉀離子在調控細胞質離子的恆定上扮演極重要的角色。細胞內鉀離子生理恆定的維持,主要是藉由細胞膜上的 Na+-K+ —ATPase的運轉主動將鉀離子往細胞內運送及鉀離子通道的開閉,使鉀離子外流的方式調控;兩者作用平衡可維持細胞容積和防止細胞凋亡發生。內毒素會使一氧化氮大量增加進而引發敗血症,造成血管平滑肌舒張,細胞膜鉀離子導度增加,甚至對升壓劑產生低反應性,但真正造成的機轉如何仍不是十分清楚。所以,我們假設內毒素造成敗血症經由活化鉀離子通道,大量鉀離子流出造成血管平滑肌細胞凋亡。因此,本實驗目的探討內毒素引發血管平滑肌細胞凋亡的過程中,一氧化氮和鉀離子所扮演的角色。實驗結果顯示: LPS誘導平滑肌細胞產生NO與時間和LPS濃度的關係成正相關,但細胞存活度 (MTT吸光值)會降低,且在作用48小時後用TUNEL 染色呈陽性反應,平滑肌細胞的DNA會呈現斷裂的凋亡現象。AG,一種選擇性的一氧化氮合成抑制劑,能完全抑制LPS所誘導的NO產量、細胞凋亡及增加細胞的存活度。SNAP,一種NO donor,所釋放的NO產量在20小時會使TUNEL 染色呈陽性反應,平滑肌細胞的DNA會呈現斷裂的凋亡現象。高鉀溶液會部份抑制LPS所誘導的NO產量、降低細胞的存活度及蛋白質的含量,並且在高鉀溶液中TUNEL 染色下細胞的DNA亦會呈現斷裂的凋亡現象更為顯著。TEA,一種非選擇性的鉀離子通道抑制劑,能部份抑制LPS所誘導的NO產量,但會降低細胞的存活度。CTX,一種大傳導型鈣離子活化的鉀離子通道抑制劑,無法抑制LPS所誘導的NO產量及增加細胞的存活度。GB,一種ATP敏感性的鉀離子通道抑制劑,能部份抑制LPS所誘導的NO產量及細胞凋亡,且會增加細胞的存活度。綜合以上的結果顯示,LPS所誘導的細胞凋亡,除了一氧化氮之外,其中ATP-敏感性鉀離子通道(KATP channels)也扮演重要的角色。

Intracellular K+ plays an important role in controlling the cytoplasmic ion homeostasis for maintaining cell volume and apoptotic enzymes in the cytosol and nucleus. Cytoplasmic K+ concentration is mainly regulated by K+ uptake via Na+-K+-ATPase and K+ efflux through K+ channels in the plasma membrane. The vascular hyporeactivity to vasoconstrictor agents has been acknowledged to be induced by lipopolysaccharide (LPS) in septic shock. Also, LPS lead to a marked increase in the potassium conductance in the plasma membrane. However, the underlying mechanism are poorly understood. We hypothesized the possibility that activation of K+ channels and the subsequent K+ efflux through plasmalemmal may be one of the major causes for the LPS-induced apoptosis in vascular smooth muscle cells (VSMCs). Thus, we examined the roles of nitric oxide and potassium ions in the apoptotic process induced by LPS in VSMCs. Our results showed that LPS induced a time and concentration-dependent release of nitric oxide from VSMCs up to 48 hours. In addition, higher concentrations of LPS decreased the reduction of 3-(4, 5-dimethylthiazol-2-yl)2, 5-diphenyl-tetrazolium bromide (MTT), an index of mitochondria function by VSMCs. In situ nick end-labeling assay revealed that incubation for 48 hours with LPS induced a positive staining of fragmented nuclei. SNAP(10-4 M), an NO donor, increased BrdUTdT-positive cells . Aminoguanidine, an inhibitor of nitric oxide synthase, could fully inhibit nitric oxide release, apoptosis and enhanced the reduction of MTT induced by LPS. Elevation of extracellular K+ concentrations from 5 to 20 mM did not affect nitric oxide release, apoptosis induced by LPS, but the reduction of MTT, and the protein content were still significant decreased as compared with those in normal potassium medium. Tetraethylammonium (TEA), a non-selective inhibitor K+ channels, partially decreased nitric oxide release, but still decrease the reduction of MTT induced by LPS. Charybdotoxin ( CTX ), a large conductance Ca2+ activated K+ channels blocker, did not affect in the presence of LPS. Glibenclamide (GB), an ATP sensitive K+ channels blocker, partially decreased both nitric oxide release and apoptosis but enhanced the reduction of MTT induced by LPS. These results suggest that LPS-mediated apoptosis in VSMCs is partially due to activation of ATP-sensitive potassium channels in plasma membrane.

目錄……………………………………………………………I
圖次……………………………………………………………II
中文摘要………………………………………………………IV
英文摘要………………………………………………………VI
第一章緒言……………………………………………………1
第二章材料與方法……………………………………………8
第三章結果……………………………………………………16
第四章討論……………………………………………………29
第五章結論……………………………………………………33
第六章參考資料………………………………………………34

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