劉振軒臺灣大學：獸醫學研究所何勝裕Ho, Sheng-YuSheng-YuHo2007-11-282018-07-092007-11-282018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/59970結節性多發動脈炎（polyarteritis nodosa, PAN），又稱為結節性動脈周圍炎（periarteritis nodosa）或結節性全動脈炎（panarteritis nodosa）。本病係指不同原因所致之動脈炎，以侵犯小動脈及中型動脈為主；由於血管壞死炎症反應而呈結節狀。探討其原因發現腦部腦膜血管常常有結節樣出血病灶，而腦部實質部位有腦軟化壞死現象，其位置是運動神經經過之處，在組織病理學發現常常有血管炎的病變，而國內外探討其致病機制皆不多，且在細菌性引起之病因又以豬鏈球菌被提及最多，但做相關探討卻有限。自2004年9月至2005年12月期間，自南部某屠宰場收集120頭淘汰母豬腦部組織臨床採集病例，其中多以行動障礙問題（locomotor problems）的豬隻為主（113/120, 94.2％）、其包括癱瘓、後肢無力、跛腳、行動不便、神經症狀等。發現有6個豬腦部組織有動脈炎（arteritis）或動脈周圍血管炎（periarteritis）的病灶，佔了6/120（5％），且發現皆在軟膜蜘蛛膜（leptomeninges）部分動脈壁變厚及變硬且帶有出血病灶。臨床細菌學分析此120個腦組織並沒有分離到豬第二型鏈球菌；但在某一農業試驗場豬隻成功分離到野外型的豬第二型鏈球菌，且利用PCR及API 20 STREP system加以鑑定。利用傳統免疫組織化學染色和非生物素過氧化酵素免疫組織化學染色方式分別在豬鏈球菌性腦炎、陽性對照豬第二型鏈球菌包埋塊及豬結節性多發動脈炎病灶區，皆有陽性訊號出現。再利用小鼠腦部血管內皮細胞株（CRL-2299）來探討豬第二型鏈球菌感染後的免疫反應；利用RT-PCR定性分析macrophage inflammatory protein-2（MIP-2）基因表現，和real-time PCR半定量分析MIP-2 mRNA表現，皆可以發現豬第二型鏈球菌在濃度104和105 CFU/ml共同培養12小時後，MIP-2 mRNA表現皆有顯著增加。利用ELISA方法分析上清液中MIP-2蛋白質的表現，發現細胞在豬第二型鏈球菌濃度103、104和105 CFU/ml共同培養12小時後MIP-2蛋白質皆有顯著增加的現象（P＜0.05）。本實驗再度配合組織病理學發現：（1）感染此菌的豬隻可以發現化膿性腦膜炎；（2）免疫組織化學染色方法可以染出巨噬細胞內及病灶區皆有鏈球菌感染的陽性訊號。本研究希望再針對豬第二型鏈球菌對於結節性多發動脈炎之慢性病灶區加以探討；分析當血管內皮細胞受到豬第二型鏈球菌感染時的第四型膠原蛋白表現，利用西方墨點分析法（western blot）實驗方法發現細胞在101和103 CFU/ml共同培養24小時後，可以使第四型膠原蛋白（collagen type IV）表現出顯著增加的現象（103 CFU/ml, P＜0.05）；又再度以免疫組織化學染色在豬結節性多發動脈炎病灶區發現陽性訊號出現，可以和實驗結果互相配合。藉由臨床檢體採集分析和實驗室分析探討方法，本實驗可以間接說明豬第二型鏈球菌的感染和豬腦結節性多發動脈炎致病因子有一定程度的關係。The term polyarteritis nodosa（PAN）has been used to denote periarteritis in several species of animals. The condition was first comprehensively documented in 1866 in man Kussmaul and Maier. The lesions of the disease develop in small and medium sized arteries anywhere in the body and the reaction involves all layers of the vessel wall. In domestic animals are seen sporadically. In most cases, the cause of polyarteritis nodosa remains unknown, but viruses and microbes have been considered as etiologic or contributing factor. According to some references, many authors believe the Streptococcus suis type 2 is an important factor but still isn’t demonstrated. Between September in 2004 and December in 2005, a total of 120 pig brains with 113 locomotor disturbances were collected from the slaughterhouse. 6 of 120 brain samples（6/120, 5％）showed arteritis or periarteritis lesion varying degrees of leptomeninges and irregular, thickened leptomeninges with congested blood vessels were observed in the gross lesion. One PAN-like lesion showed IHC positive for S. suis type 2. The expression of macrophage inflammatory protein–2 (MIP-2) mRNAs analyzed by reverse transcriptase–PCR (RT-PCR) in mouse brain microvascular endothelial cells (CRL-2299) by co-culture with different S. suis type 2 concentration（101 to 105 CFU/ml）for 12 hours. The expression of MIP-2 mRNAs significantly increased as compared with untreated cells. The expression of MIP-2 mRNAs analyzed by real-time PCR also significantly increased as compared with untreated cells. MIP-2 peptide levels were measured by the ELISA. The protein levels of the supernatant from CRL-2299 were measured after exposure to 101 to 105 CFU/ml S. suis type 2 for 12 h and in the case of blocking experiments. The MIP-2 peptide significantly increased as compared with untreated cells（P＜0.05; P＜0.01）. The collagen type IV was analyzed by western blot. The CRL-2299 treated with S. suis type 2 101 and 103 CFU/ml for 24 hours revealed 200-kD protein bands. The protein significantly increased as compared with untreated cells (P＜0.05). Based on the results of IHC and expression of MIP-2 and collagen type IV, S. suis type 2 infection may play a role to the formation of PAN.目錄............................................................................................................. I 圖次............................................................................................................. IV 表次............................................................................................................. VI 中文摘要..................................................................................................... VII 英文摘要..................................................................................................... IX 第一章 前言............................................................................................. 1 第二章 文獻探討..................................................................................... 3 第一節 血管炎的介紹與分類............................................................ 3 2.1.1 血管炎的診斷...................................................................... 6 2.1.1.1 嗜中性白血球細胞質抗體（ANCA）...................... 7 2.1.1.2 嗜中性白血球細胞質抗體與血管炎的關係.............. 8 2.1.2 血管炎的治療...................................................................... 8 第二節 豬結節性多發動脈炎............................................................ 9 第三節 豬第二型鏈球菌感染症........................................................ 10 2.3.1 病因探討.............................................................................. 10 2.3.2 傳播途徑.............................................................................. 12 2.3.3 致病機序.............................................................................. 13 2.3.4 臨床症狀與病變.................................................................. 13 第四節 豬結節性多發動脈炎與感染豬鏈球菌後致病機制之關係 14 2.4.1 感染豬鏈球菌後與結節性多發動脈炎致病機制之探討 14 2.4.2免疫反應之探討................................................................... 15 第三章 實驗材料與方法.................................................................................. 19 第一節 實驗設計及流程圖................................................................ 19 第二節 材料來源................................................................................ 20 第三節 形態病理學檢查.................................................................... 21 3.3.1 肉眼及組織病理學檢查...................................................... 21 3.3.2 免疫組織化學染色技術...................................................... 23 3.3.3 非生物素過氧化酵素免疫組織化學染色技術.................. 25 第四節 豬第二型鏈球菌之鑑定........................................................ 26 3.4.1標準菌株來源....................................................................... 26 3.4.2 細菌分離與鑑定流程.......................................................... 26 3.4.2.1 分離之鏈球菌菌株鑑定............................................. 26 3.4.2.2 API 20 STREP system生化鑑定.............................. 27 3.4.2.3 豬鏈球菌PCR診斷.................................................... 28 第五節 分析MIP-2及第四型膠原蛋白實驗方法............................ 30 3.5.1 CRL-2299細胞培養............................................................. 30 3.5.1.1細胞來源...................................................................... 30 3.5.1.2繼代培養...................................................................... 31 3.5.1.3細胞計數...................................................................... 32 3.5.1.4實驗細胞之準備.......................................................... 32 3.5.2 細菌菌數計算...................................................................... 32 3.5.3 細胞形態學的影響……………………………………….. 33 3.5.4 細胞上清液收集.................................................................. 33 3.5.5 反轉錄酶-聚合酶鏈鎖反應偵測MIP-2基因表現............ 34 3.5.5.1細胞RNA之製備....................................................... 34 3.5.5.2 RNA定量.................................................................... 35 3.5.5.3 RNA反轉錄反應........................................................ 35 3.5.5.4引子之設計.................................................................. 35 3.5.5.5 PCR反應..................................................................... 36 3.5.6 即時定量PCR偵測MIP-2基因表現............................... 36 3.5.7 細胞培養上清液中MIP-2蛋白質濃度的測定................. 37 3.5.8 西方墨點分析法測定第四型膠原蛋白.............................. 37 3.5.8.1 蛋白質抗原的製備..................................................... 38 3.5.8.2 蛋白質抗原的電泳分離............................................. 39 3.5.8.3 將電泳分離的蛋白質抗原從凝膠上轉移到PVDF膜 40 3.5.8.4 覆蓋PVDF膜上非特異性的結合位置........................ 40 3.5.8.5 結合上蛋白質抗體collagen type IV…………………. 41 3.5.8.6 偵測................................................................................ 41 第四章 結果......................................................................................................... 42 第一節 臨床檢體分析........................................................................... 42 4.1.1 臨床症狀................................................................................. 42 4.1.2 細菌分離................................................................................. 42 4.1.3 肉眼病理................................................................................. 42 4.1.4 組織病理學............................................................................. 43 4.1.5 免疫組織化學染色................................................................. 44 第二節 實驗室分析............................................................................... 44 4.2.1 細胞形態學分析..................................................................... 44 4.2.2 MIP-2目標基因的表現…………………………………….. 44 4.2.3 細胞培養上清液中MIP-2蛋白質濃度的測定……………. 45 4.2.4 第四型膠原蛋白的表現……………………………………. 45 4.2.5 免疫組織化學染色結果......................................................... 45 附圖……………………………………………………………………………….. 46 第五章 討論......................................................................................................... 57 第一節 臨床檢體分析........................................................................... 57 5.1.1 臨床症狀分析......................................................................... 57 5.1.2 細菌分離探討......................................................................... 57 5.1.3肉眼病理結果探討.................................................................. 58 5.1.4 組織病理學結果探討............................................................. 58 5.1.5 免疫組織化學染色和非生物素過氧化酵素免疫組織化學染色之比較............................................................................. 59 第二節 實驗室分析............................................................................... 61 5.2.1 細胞形態學分析..................................................................... 61 5.2.2 MIP-2基因表現於反轉錄酶-聚合酶鏈鎖反應與即時定 量PCR之分析.......................................................................... 62 5.2.3 細胞培養上清液中MIP-2蛋白質濃度的測定探討............. 63 5.2.4 西方墨點分析法測定第四型膠原蛋白探討......................... 64 第三節 結論........................................................................................... 65 第四節 未來工作及展望....................................................................... 67 附表.......................................................................................................................... 68 參考文獻.................................................................................................................. 73 附錄1. ..................................................................................................................... 89 附錄2. ..................................................................................................................... 914710499 bytesapplication/pdfen-US豬腦結節性多發動脈炎豬第二型鏈球菌Pigcerebral polyarteritis nodosaStreptococcus suis type 2thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59970/1/ntu-94-R93629018-1.pdf