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標題: 胸膜肺炎放線桿菌RTX毒素對培養的內皮細胞之毒性
Toxicity of Actinobacillus pleuropneumoniae RTX-Toxins(Apx) to endothelial cells
作者: 陳威霖
Chen, Wei-Lin
關鍵字: 胸膜肺炎放線桿菌
Endothelial cell
出版社: 獸醫微生物學研究所
摘要: 胸膜肺炎放線桿菌,是導致豬放線桿菌胸膜肺炎的病原菌。主要造成的典 型病變為,纖維素性、出血性、壞死性胸膜肺炎。胸膜肺炎放線桿菌的莢 膜及內毒素可能是產生致病性的毒力因子,但是近來許多的報告,指出本 菌所分泌之外毒素中的胸膜肺炎放線桿菌RTX毒素(Actinobacillus pleuropneumoniae RTX-Toxins; Apx),亦可能是導致肺臟出血病變的主 要毒力因子。由於前人的報告是以養菌的上清液,進行內皮細胞的毒性試 驗,並以添加中性紅來測試內皮細胞的存活率。本實驗則是要以穿透式電 子顯微鏡,研究是否為上清液中的Apx,造成血管內皮細胞的傷害。本實 驗收集臨床及人工接種的放線桿菌胸膜肺炎病例,以光學顯微鏡及穿透式 電子顯微鏡,觀察肺臟的出血病變區,並利用硫酸銨及膠體層析法純化出 Apx,接種到六格培養盤的內皮細胞中,分別加入10、50和100ug/ml 等不 同濃度Apx,於接種後1、2、3、4和5小時用戊二醛固定細胞、利用電子顯 微鏡觀察內皮細胞形態變化。結果顯示,由組織切片、超薄切片,觀察豬 放線桿菌胸膜肺炎臨床病例及實驗病例,發現肺泡腔內含有炎症滲出液、 纖維素、血球及細菌。在組織切片下所見的出血病變,亦可於超薄切片電 子顯微鏡觀察下發現,是由於肺臟微血管構造的傷害,紅血球漏出至肺泡 腔,形成出血。而在培養的內皮細胞接種Apx的結果上可見單獨存在的內 皮細胞,圓形化的細胞,細胞膜缺損的細胞,核膜缺損的細胞,粒線體擴 張,粒線體濃縮,粗內質網擴張,以致於細胞核消失的死亡細胞。由以上 的結果可知Apx確實可以造成內皮細胞的傷害以及細胞死亡而造成出血的 病變。
Actinobacillus pleuropneumoniae is the causative agent of pig's fibrino-hemorrhagic or local extensive necrotizing pleuropneumonia. Capsule and endotoxin are probable virulence factors of this organism, but it has been postulated that other potent toxins also contribute to virulence. 3 potential toxic virulent factors are the heat-labile Actinobacillus pleuropneumoniae RTX-Toxins(Apx)of the all serotypes. Some reported papers indicated that supernatant and crude Apx could induce cytotoxicity of alveolarmacrophage, type Ⅱ pneumocyte and endothelial cell. The aim of this study wasto evaluate the cytotoxicity of purified Apx.The collected clinical and artifical inoculated cases were observed by paraffin and ultrathin sections. The Apx added to challenge endothelial cells,was purified from supernatant of cultured Actinobacillus pleuropneumoniae serotype 1 and filtrated through Sephadex G-100 TM. In order to confirm that Apx did induce the cytotoxicity of endothelial cells, this study was performed that cultured endothelial cells challenged with 10, 50 and100μg/ml Apx, then fixed with 4﹪glutaraldehyde at 1, 2, 3, 4 and 5 hour later, and observed the morphologic changes with transmission electron microscope. There were found exudate, fibrin, erythrocytes and bacteria in the lumen of alveoli. The damaged pulmonary vessels were filled with erythrocytes, some passed through vessel wall into alveolar space. The Apx inoculated endothelial cells became single rounding cells from a spindle shape, cell membrane and nuclear envelop ruptured, mitochondria and rough endoplasmic reticulum vacuolated, dense mitochondria and dead cells.According to the results, it could be concluded that gel filtration purified Apx from Actinobacillus pleuropneumoniae serotype 1 did exhibit the cytotoxicity of endothelial cells.
Appears in Collections:微生物暨公共衛生學研究所



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