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標題: 豬β2整合素基因轉殖小鼠對胸膜肺炎放線桿菌之感受性探討
Sensitivity of porcine β2 integrin transgenic mice to Actinobacillus pleuropneumoniae
作者: 洪榮昭
Jung-Chao Hung
關鍵字: 胸膜肺炎放線桿菌;β2整合素;基因轉殖小鼠;Actinobacillus pleuropneumoniae;β2 integrin;transgenic mice
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胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae, AP)感染豬隻呼吸道所引起的纖維素性、出血性及壞死性胸膜肺炎會造成豬隻死亡。AP所分泌外毒素,包含ApxI~ApxIV,Apx依結構特色被歸類為Repeat in Toxin(RTX)家族,RTX經由與白血球上的β2整合素(CD11/CD18)結合,造成細胞毒殺作用。過去研究指出,ApxIII對豬隻CD18(porcine CD18, pCD18)具物種專一性。本研究為探討pCD18與AP致病機轉之相關性,以C57BL/6JNarl小鼠構築表現pCD18基因轉殖(Transgenic, TG)小鼠,並分析TG小鼠對AP之感受性。首先,分離小鼠腹腔灌洗細胞與脾臟細胞,並以流式細胞儀分析其中巨噬細胞、B細胞及T細胞族群比例,發現TG與Wild type(WT)小鼠相似,但TG小鼠表現pCD18比例明顯高於WT小鼠,且母鼠表現量顯著高於公鼠。此外,以AP血清型第1型(AP1)和第2型(AP2)菌株進行腹腔內攻毒實驗。結果顯示,攻毒後TG母鼠較快出現死亡,TG與WT母鼠於AP1的半致死劑量並無差異,而TG母鼠對AP2的半致死劑量比WT母鼠低7.5倍,說明TG母鼠對AP菌株具較高敏感性。另為探討AP所分泌Apx對小鼠免疫細胞所造成的影響,本研究自WT與TG母鼠分離脾臟單核細胞(SMCs),分別與AP10所分泌之ApxI或AP2所分泌之ApxII/III進行感作,結果發現無論ApxI或ApxII/III對TG母鼠SMCs皆造成較嚴重的細胞毒性。為進一步探討pCD18表現比例與Apx致病性關係,自TG母鼠分離腹腔灌洗細胞與SMCs,利用磁珠抗體分選高純度pCD18陽性細胞與無標記的陰性細胞,分別與1 ~ 10 CU ApxII/III進行感作。結果顯示,ApxII/III對pCD18陽性細胞具劑量效應,毒素濃度越高造成更高比例之細胞膜損傷,其中對腹腔灌洗細胞造成更嚴重的細胞損傷情形。綜合上述結果,本研究已建立pCD18基因轉殖小鼠對AP疾病動物模式,證實pCD18於AP致病性和Apx外毒素細胞毒殺作用中扮演重要的角色,此基因轉殖小鼠具有應用於AP致病機轉探討或作為效力檢定工具之潛力。

Actinobacillus pleuropneumoniae (AP) causes fibrinous, hemorrhagic, and necrotizing pleuropneumonia in pigs. The exotoxins secreted by AP, including ApxI to ApxIV, are classified as members of the Repeats-in-Toxin (RTX) family based on their structural characteristics. It has been demonstrated that RTX toxins target specifically to β2 integrin (CD11/CD18) and lead to cell death. Previous studies indicate that the porcine CD18 (pCD18) mediates ApxIII-induced species-specific toxicity on pig leukocytes. The aim of this study was to investigate the role of pCD18 in AP pathogenicity. A line of transgenic (TG) C57BL/6JNarl mouse expressing pCD18 was generated and studied. To characterize the expression of pCD18, we isolated the peritoneal lavage cells (PLCs) and splenocytes, and analyzed the percent of macrophages, B cells, and T cells among them. A similar percentage of cell population was found in both wild type (WT) and TG mice. PLCs and splenocytes derived from TG mice, especially from TG female mice, had a higher expression level of pCD18 as compared to WT mice or TG male mice. Therefore, female TG mice were used in the subsequent experiments. To investigate the susceptibility of TG and WT mice to AP, mice were intraperitoneally inoculated with AP serotype 1 (AP1) or 2 (AP2), and the mortality assessed and compared. It was found that the death of TG mice occurred at time points earlier than that of WT mice after challenge. The LD50 of AP1 in TG mice was equivalent to that in WT mice, while the LD50 of AP2 in TG mice was 7.5 times lower than that in WT mice. We inferred that the higher susceptibility of TG mice to AP may result from the expression of pCD18. To test this hypothesis, PLCs and splenic mononuclear cells (SMCs) of TG mice were collected and purified using magnetic beads coupled with antibodies recognizing porcine CD18. The susceptibility of pCD18+-cells towards Apx toxins were assessed and compared to pCD18--cells using the lactate dehydrogenase assay. The results showed that pCD18+-cells were more susceptible toward ApxI and ApxII/III comparing to pCD18--cells. Apx toxins induced significant levels of cytotoxicity in a concentration-dependent manner in pCD18+-cells, which signifies an indispensable role of pCD18 in Apx cytotoxic effect. In conclusion, we have successfully generated the TG mice expressing pCD18 and demonstrate the role of pCD18 in AP pathogenicity and in Apx cytotoxicity. The TG mice might be a valuable tool for the study of AP pathogenicity and for the evaluation of vaccine efficacy in the future.
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