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標題: 胸膜肺炎放線桿菌外毒素宿主細胞特異性之研究
Effects of Actinobacillus pleuropneumoniae exotoxin on different cells
作者: 張乃云
Chang, Nai-Yun
關鍵字: Actinobacillus pleuropneumoniae
出版社: 獸醫病理生物學研究所
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摘要: 胸膜肺炎放線桿菌Actinobacillus pleuropneumoniae (APP)外毒素Apx為APP強毒力因子之一,該毒素具有溶血性及細胞毒性,於APP感染豬隻可發現毒素所造成嚴重的肺臟病變。本研究主要目的為探討不同種類或不同來源細胞對於Apx之敏感性。首先以APP血清型第10型所分泌毒素ApxI氣管內接種豬隻,利用H&E染色、免疫化學染色、TUNEL、及Hoechst染色等方法探討ApxI對豬肺臟組織所造成之影響。結果顯示,豬隻經ApxI毒素接種後,肺臟組織中可見嗜中性球浸潤及肺臟間質組織增厚,伴隨輕度炎症反應;並於TUNEL標幟後發現較高比例之細胞呈凋亡,而凋亡細胞中吞噬及非吞噬細胞各佔54%與46%。接種經熱不活化ApxI毒素的肺臟組織則可見輕度炎症反應,而生理食鹽水處理組組則無明顯組織病變,且TUNEL染色結果發現熱不活化毒素與生理食鹽水處理組細胞凋亡比例無顯著差異,且細胞凋亡數明顯較ApxI毒素處理組低。為進一步瞭解不同種類細胞對於不同血清型別之Apx毒素之敏感性,本研究利用不同來源之細胞(株)包括豬肺臟巨噬細胞(PAM)、BL-3、BL-3.1、RAW 264.7、PK-15、Vero細胞,比較這些細胞對APP血清型第1及2型外毒素的敏感性。結果發現,BL-3、BL-3.1與PAM對於APP血清型第1及2型菌株所產生的毒素具最高之敏感性,三者間差異不明顯;小鼠巨噬細胞株RAW 264.7對APP血清型第1型分泌毒素有微弱感受性,而腎臟上皮細胞PK-15與Vero則對Apx毒素不具敏感性。此外,本研究利用自殺載體及同源重組互換原理,成功構築APP血清型第10型之apxIA突變菌株,經PCR及基因定序確定其具有正確之基因型,亦利用生長曲線、Biolog生化代謝、溶血試驗、細胞毒性試驗、細胞凋亡試驗及西方轉漬法進行表現型分析。結果顯示,apxIA基因突變菌株與親代株有相似的生長曲線及生化代謝性狀;此外,由於該菌株失去產生ApxI毒素能力,因此突變菌株之培養上清液不具溶血性、細胞毒性,不會誘發PAM凋亡。
Actinobacillus pleuropneumoniae (APP) exotoxin (Apx) is one of the strong virulent factors of APP. Apx possesses hemolytic and cytotoxic activities which cause serious lung damage in APP infected pigs. The aim of this study was to evaluate the sensitivity of different cell types or cell lines originated from different species toward Apx. APP serotype 10-derived exotoxin ApxI was used to intratracheally inoculate piglets; lung sections were subjected to H&E, immunohistochemical, TUNEL, and Hoechst stains in order to assess the effects of ApxI on porcine lung tissue. Lung sections of ApxI treated group had mild neutrophil infiltration and interstitial hyperplaisa. TUNEL staining revealed higher percentage of apoptotic cells in ApxI treated group of which 54% were phagocytic cells. Piglets inoculated with heat-inactivated ApxI showed mild inflammation in lungs while no obvious histopathological changes observed in saline treated piglets. Both groups had lower percentage of apoptotic cells in lung sections as compared to ApxI treated group. To further understand the sensitivities of cell lines from different species toward Apx of different serotypes, porcine alveolar macrophage (PAM), BL-3, BL-3.1, RAW 264.7, PK-15, and Vero cells were used for comparison. Results showed that BL-3, BL-3.1, and PAM had the highest sensitivity toward exotoxins derived from APP serotype 1 (APP1) and 2 (APP2), and no significant difference was found between these cells. RAW 264.7 cells were mildly sensitive toward APP1 exotoxin, and PK-15 and Vero cells were not sensitive toward exotoxins of APP. Further, an APP serotype 10 apxIA mutant was successfully constructed through homologous recombination of APP genomic DNA with a suicide vector carrying apxIA gene inserted with a kanamycin-resistant determinant. The mutant with desired mutation was selected and confirmed by antibiotic resistance, PCR, and DNA sequence analysis. Growth curves, Biolog metabolic profiles, hemolytic assay, cytotoxic assay, TUNEL assay, and Western blot analysis were used to verify the phenotypes of mutant strain. The apxIA mutant had similar growth rate and exhibited similar metabolic profiles compared to the parental strain. In addition, apxIA mutant strain lost its ability to produce ApxI, therefore no hemolytic, cytotoxic, or apoptosis inducing activity was observed in the bacterial culture supernatant.
其他識別: U0005-1407201016365600
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