黃義侑臺灣大學:醫學工程學研究所曾莉萍Tseng, Li-PingLi-PingTseng2010-06-022018-06-292010-06-022018-06-292009U0001-0207200913473600http://ntur.lib.ntu.edu.tw//handle/246246/184654利用微脂粒包覆活的(living)或去活的(killing)新城病毒(NDV)，以點鼻(intranasal)方式接種雞隻，發現在雞隻鼻腔/氣管沖洗液與血清中都有產生特異性NDV抗體。給予雞隻接種三種不同成份的巨型多層微胞微脂粒，其中使用磷脂醯膽鹼(PC)或磷脂醯絲胺酸(PS)組成的微脂粒，在動物實驗中黏膜分泌型抗體(s-IgA)與血清抗體(IgG)及功能性抗體力價(HI titer)表現量在兩次接種後抗體均提升，攻毒後保護率也達80-90%。在微脂粒加入脂多醣體(LPS)佐劑，接種於雞隻後，在利用PC微脂粒組會增加黏膜抗體與PS微脂粒組增加會血清中抗體力價。更進一步，添加銀耳多醣(tremella)以增加微脂粒的黏度，延長微脂粒在黏膜時間，點鼻接種後黏膜分泌型抗體與血清中抗體均顯著提升，攻毒後有100%的保護率。微脂粒佐劑對於免疫細胞之機轉由共軛焦顯微鏡觀察到，PC組成份微脂粒被雞脾臟巨噬細胞胞吞量較多。PC微脂粒與PS微脂粒均活化雞脾臟巨噬細胞產生NO釋放，其中PC微脂粒NO的釋放可被Bay 11-7085抑制與U0126抑制，表示PC微脂粒在早期活化雞脾臟巨噬細胞是以NF-κB磷酸化與ERK上游的MEK磷酸化的路徑。最後我們利用獲得免疫接種微脂粒或銀耳多醣微脂粒的雞隻，發現下游的p-ERK磷酸化的現象存在，其中微脂粒p-ERK磷酸化量較多而銀耳多醣微脂粒p-ERK磷酸化量較少，p-ERK的磷酸化主要促進免疫反應走向體液免疫的Th2路徑，因此，銀耳多醣微脂粒組造成的免疫反應是完整的細胞免疫的Th1與體液免疫的Th2路徑的生成。Liposomal-NDV vaccine intranasal administration to chicken can induce specific-NDV antibody at mucosal and serum. The adjuvant effect of multi-lamellar vehicles (MLVs) liposomes formulated with three phospholipids including phosphatidylcholine-liposomes (PC-Lip), phosphatidylserine-liposomes (PS-Lip), and stearylamine-liposomes (SA-Lip) was compared with that for virus alone using the inactivated Newcastle disease virus (NDV) as model antigen. PC-Lip and PS-Lip induced more NDV antibody titer after secondary immunizations. In response to virulent viral challenge, all control animals died, whereas 80~90% of animals which still survived. Added LPS with liposomal-NDV vaccine which enhanced chickens produced antibody in serum and nasal/tracheal lavages. Furthermore, we combined tremella with liposomal-NDV vaccine to enhance the muco-adhesive property and prolong liposomes on the mucosal surface. Chickens received T-PC-Lip resulted in a significant increase in HI titer also elicited a significant mucosal secretary immunoglobulin A (s-IgA) response in tracheal lavages and a serum IgG response. After virulent virus challenge, the protection rate of T-PC-Lip vaccine showed 100% survival rate. The molecular mechanism of liposomal-adjuvant was study both in vitro and in vivo. Confocal laser scan microscopy showed that PC-Lip were uptaken into chicken spleen macrophages. PC-Lip and PS-Lip stimulated chickens spleen macrophages produce NO. In response to PC-Lip stimulated, Bay 11-7085 and U0126 inhibited macrophages produced NO. PC-Lip was shown to be involved in both activation of NF-κB and upstream ERK of MEK pathway. Moreover, PC-Lip and T-PC-Lip were shown to be activated ERK pathway in vovo. In conclusion, T-PC-Lip is an active adjuvant for chickens resulting in trigger Th1 pathway of cellular immunity and Th2 pathway of humoral immunity.口試委員會審定書 ...................................................................................................... i 謝 ............................................................................................................................. ii 文摘要 .................................................................................................................... iii 文摘要 .................................................................................................................... iv 目錄 .....................................................................................................................…vi 目錄 ....................................................................................................................... vii 一章 緒言及文獻回顧 ............................................................................................1 .1 黏膜免疫之介紹 ...................................................................................................1 .2 動物用黏膜接種之疫苗.........................................................................................2 .3 佐劑之介紹 ...........................................................................................................3 .4 顆粒性疫苗傳輸系統 ...........................................................................................6 .5 微脂粒佐劑 ...........................................................................................................6 .6 脂多醣體與黏著性多醣體 .................................................................................10 .7 雞隻黏膜感染病毒 .............................................................................................11 二章 論文研究目的 ..............................................................................................13 三章 材料與方法 ..................................................................................................14 .1 實驗動物 .............................................................................................................14 .2病毒 .......................................................................................................................14 .3實驗試藥及分析試劑 ...........................................................................................14 .3.1試劑藥品..............................................................................................................14 .3.2抗體 ....................................................................................................................15 .3.3塑膠、玻璃製品 ................................................................................................16 . 4微脂粒包覆病毒抗原 ..........................................................................................16 .4.1 微脂粒配方........................................................................................................16 .4.2 製備流程 ..........................................................................................................17 .4.3 粒徑、界面電位與包覆率 ..............................................................................17 .4.4 相轉移溫度........................................................................................................18 .4.5 螢光微脂粒 ......................................................................................................18 .4.6 雙佐劑微脂粒 ..................................................................................................19 .4.7 銀耳多醣微脂粒和三仙膠微脂粒 ..................................................................19 .5免疫接種、採血和鼻與氣管沖洗液 ...................................................................20 .6血球凝集抑制試驗與酵素免疫連結分析法 .......................................................21 .7攻毒實驗 ...............................................................................................................22 .8雞脾臟細胞分離 ...................................................................................................22 .9共軛焦顯微鏡 .......................................................................................................22 .10 RT-PCR偵測病毒 ..............................................................................................23 .11材料對細胞之毒性偵測 ....................................................................................24 .12偵測細胞培養液中NO .......................................................................................24 .13西方點墨法偵測蛋白質表現 ............................................................................25 四章 結果 .............................................................................................................27 五章 討論 .............................................................................................................40 六章 結論 .............................................................................................................48 考文獻 ...................................................................................................................49 ...............................................................................................................................64 ...............................................................................................................................72 表論文列表 ...........................................................................................................93application/pdf2315678 bytesapplication/pdfen-US微脂粒佐劑黏膜新城病毒一氧化氮黏膜黏著銀耳多醣liposomeadjuvantmucosalNDVnitric oxidemucoadhesiveTremella[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/184654/1/ntu-98-D93548009-1.pdf