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National Chung Hsing University Institutional Repository - NCHUIR > 獸醫學院 > 獸醫病理生物學所 > 依資料類型分類 > 碩博士論文 >  鈣離子於胸膜肺炎放線桿菌外毒素誘導細胞傷害之角色

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/155169

標題: 鈣離子於胸膜肺炎放線桿菌外毒素誘導細胞傷害之角色
Role of calcium in Actinobacillus pleuropneumoniae exotoxin induced cell damage
作者: 王奕評
Wang, Yi-Ping
Contributors: 宣詩玲
獸醫病理生物學研究所
關鍵字: 胸膜肺炎放線桿菌;外毒素;鈣離子
Actinobacillus pleuropneumoniae;exotoxin;calcium
日期: 2012
Issue Date: 2013-11-21 11:42:59 (UTC+8)
Publisher: 獸醫病理生物學研究所
摘要: 胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae, AP)為豬隻重要呼吸道病原之一,可造成豬隻出血性、壞死性、纖維素性胸膜肺炎。AP所分泌之外毒素Apx屬於repeats in toxin (RTX)毒素家族的一員,為此菌重要的毒力因子之一,在造成豬隻胸膜肺炎的致病機轉中扮演重要的角色。在先前的研究中發現,以另一種RTX毒素,Mannheimia haemolytica的leukotoxin刺激牛肺臟巨噬細胞後,可誘導細胞內鈣離子明顯的提升並造成細胞溶解。但Apx是否誘發豬肺泡巨噬細胞(porcine alveolar macrophage, PAM)細胞內鈣離子提升,以及鈣離子是否調控Apx造成的細胞傷害相關機制,至今尚不明確。因此,本研究以ApxI刺激PAM,探討(1) ApxI是否可以誘發細胞內鈣離子提升,(2)鈣離子於ApxI誘發之細胞膜穿孔傷害、粒線體活性降低及細胞凋亡中所扮演的角色,及(3)鈣離子是否影響ApxI所誘導之p38、JNK活化。首先自4-5週齡SPF豬隻肺臟灌取新鮮PAM,並培養AP血清型第10型細菌分離其上清液製備粗製ApxI外毒素。再以不同濃度ApxI刺激PAM,並以FluoForte®測定細胞內鈣離子螢光強度。結果發現,ApxI可以快速刺激細胞質內的鈣離子濃度提升;而以四種鈣離子抑制劑(amiloride、BAPTA-AM、EDTA、nifedipine)先感作細胞再加入ApxI刺激,發現加入EDTA能有效地抑制細胞內鈣離子濃度上升,顯示細胞內鈣離子提升可能由細胞外鈣離子匯入而來。後續以LDH release assay、XTT assay及Hoechst染劑將細胞核染色方式,以測試上述鈣離子抑制劑是否影響ApxI誘發之細胞膜、粒線體傷害及細胞凋亡。結果發現,四種抑制劑均無法降低ApxI造成之細胞膜損傷程度及細胞凋亡的比率,但以EDTA前處理細胞後可以顯著降低ApxI所誘發的粒線體傷害,顯示細胞內過高的鈣離子會抑制細胞粒線體活性。最後,以西方轉漬法分析,結果發現,ApxI刺激PAM後,p38及JNK分子均發生明顯活化,而加入BAPTA-AM及EDTA感作的細胞,ApxI所誘發p38及JNK的活化均受到明顯的抑制,顯示鈣離子的提升與p38及JNK活化有關。綜合上述,抑制細胞外鈣離子匯入細胞可以降低ApxI所造成的粒線體毒性及ApxI活化p38及JNK,但卻無法降低ApxI所誘發的細胞凋亡及細胞膜損傷。
Actinobacillus pleuropneumoniae (AP) is one of the important respiratory pathogens that causes hemorrhagic, necrotizing, and fibrinous pleuropneumonia in porcine. Actinobacillus pleuropneumoniae exotoxin (Apx) belonging to repeats-in-toxin (RTX) family, is a virulence factor and plays an important role in pathogenesis of pleuropneumonia. Another RTX member, Mannheimia haemolytica leukotoxin has been reported to cause intracellular calcium elevation in bovine alveolar macrophage and cytolysis. It is not clear whether Apx could induce intracellular calcium increase in porcine alveolar macrophage (PAM) and whether calcium signaling participates in Apx-mediated effects. The objectives of this study are to explore (i) whether ApxI induces intracellular calcium increase in PAM, (ii) the role of calcium in ApxI induced cell membrane damage, mitochondrial activity decrease, and in apoptosis, and (iii) the relationship between mitogen-activated protein kinase (MAPK) p38 and JNK and calcium elevation. PAM were collected from 4-5 weeks old SPF piglets. AP serotype 10 was cultured and supernatant collected as crude ApxI preparation. PAM were stimulated by different concentrations of ApxI and intracellular calcium detected with FluoForte assay kit. The results showed that ApxI induced an elevated intracellular calcium concentration in a short time period. Cells were pre-incubated with four different calcium inhibitors, amiloride, BAPTA-AM, EDTA, and nifedipine, followed by ApxI stimulation. The results showed that EDTA inhibited intracellular calcium elevation induced by ApxI, indicating a calcium influx from extracellular space. LDH release, XTT assay, and Hoechst dye staining were used to detect the effects of calcium inhibitors on ApxI-induced membrane damage, mitochondrial activity decrease, and apoptosis, respectively. The results showed that all calcium inhibitors tested did not diminish ApxI-induced cytolysis and apoptosis, but EDTA could significantly prevent ApxI-induced mitochondrial damage. Finally, p38 and JNK activation induced by ApxI was inhibited by pretreatment of BAPTA-AM and EDTA, suggesting a possible role of calcium in p38 and JNK activation. In conclusion, Inhibition of calcium influx from extracellular space could reduce mitochondrial toxicity and p38 and JNK activation, but not apoptosis or cell membrane damage, induced by ApxI.
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