臺灣博碩士論文加值系統

摘要
家禽里奧病毒 (avian reovirus, ARV) 感染雞隻後會造成病毒性關節炎 (viral arthritis, VA) 或營養不良吸收症候群 (malabsorption syndrome, MAS)。為了探討病毒性關節之分子致病機轉，本研究以造成雞隻病毒性關節炎的ARV病毒株S1133為對象，調查此病毒感染對於宿主基因表現之影響。我們將ARV的S1133病毒株接種於1日齡雞隻，於接種不同天數後萃取各組織的RNA，依據病毒S4基因序列合成引子對，利用反轉錄-聚合鏈反應 (reverse transcription-polymerase chain reaction, RT-PCR) 調查病毒組織趨性。結果顯示病毒於接種後2-6天分布於全身各臟器，第8-16天後漸集中於腳掌及跗關節部位，至第18天之後則僅侷限於腳掌及跗關節部位。以原位基因雜合法 (in situ hybridization, ISH) 進一步觀察，病毒主要分布於腳掌及跗關節之真皮層及軟骨部位。
繼而以PCR偵測一些關節炎相關因子如骨形態發生蛋白2 (bone morphogenetic protein 2, BMP2)、BMP受體 (BMP receptor, BMPR)、基質金屬蛋白2 (matrix metalloproteinae 2, MMP2)、膜三型MMP (membrane type 3-matrixmetalloproteinase, MT3-MMP)、表皮生長因子 (epidermal growth factor, EGF)、EGF受體 (EGF receptor, EGFR)、需鈣蛋白 (calpain) 及caspase等基因表現之變化。發現ARV的感染會誘導BMP2及MMP2於腳掌及跗關節部位的提前表現，利用ISH觀察，BMP2主要表現在關節部位的板 (epiphyseal plate)，而MMP2則是表現於關節部位的2級鈣化區 (secondary center of ossification)、板中的增殖區 (zone of proliferation)、及肥大區 (zone of hypertrophy) 的軟骨處。BMPR、MT3-MMP、EGF、EGF receptor、calpain及caspase的表現則並未受到ARV感染的影響。
最後，我們運用抑制性扣減雜合法 (suppression subtractive hybridization, SSH) 尋找ARV感染所誘導表現的宿主基因，由此發現U2核內小核糖核蛋白輔助因子 (U2 small nuclear ribonucleoprotein particle auxiliary factor , U2AF) 之基因在ARV感染過程有差異表現。進一步以RT-PCR去驗證，確認病毒感染後12小時至第8日期間，會持續誘導U2AF基因於關節組織表現。U2AF蛋白質與ARV或是與關節炎的相關性並無前人報導過，此為首度發現，值得深入研究。

目次
中文摘要………………………………………………………… Ⅰ
英文摘要…………………………………………………………. Ⅲ
中英文縮寫對照表………………………………………………. Ⅴ
目次………………………………………………………………. Ⅵ
表次………………………………………………………………. Ⅸ
圖次………………………………………………………………. Ⅸ
第一章 緒言……………………………………………………. 1
第二章 文獻探討………………………………………………. 3
第一節 家禽里奧病毒簡介…………………………………… 3
1-1 家禽里奧病毒分離的背景……………………………. 3
1-2 ARV與哺乳類里奧病毒之差異………………………. 3
1-3 ARV所造成的疾病……………………………………. 3
1-4 ARV的感染途徑………………………………………. 4
1-5 ARV的血清型及致病型………………………………. 4
1-6 ARV的檢驗……………………………………………. 5
1-7 預防控制………………………………………………. 5
第二節 ARV的一般特性……………………………………… 6
2-1 ARV的型態與結構……………………………………. 6
2-2 ARV的物理及化學性質………………………………. 6
2-3 病毒的增殖……………………………………………. 6
2-4 病毒的基因體…………………………………………. 7
2-5 ARV蛋白質……………………………………………. 8
第三節 骨形態發生蛋白與關節炎之相關性………………… 9
第四節 基質金屬蛋白與關節炎之相關性………………… 10
第五節 表皮生長因子與關節炎之相關性…………………… 12
第六節 Caspase與關節炎之相關性…………………………... 12
第七節 需鈣蛋白與關節炎之相關性……………………… 13
第八節 原位基因雜合法的原理及其應用…………………… 14
第九節 抑制性扣減雜合法 (suppression subtractive hybridization, SSH) 的原理及其應用…………… 16
第十節 U2核內小核糖核蛋白輔助因子 (U2 small nuclear ribonucleoprotein particle auxiliary factor, U2AF)…. 17
第三章 材料與方法……………………………………………… 19
一、病毒株………………………………..……………………… 19
二、動物試驗…………………………………………………….. 19
三、細胞培養…………………………………………………….. 20
四、細胞製片…………………………………………………….. 20
五、組織切片…………………………………………………….. 20
六、酵素連結免疫吸附法 (enzyme-linked immunosorbant
assay, ELISA) 偵測ARV抗體力價……………………….. 21
七、萃取組織核糖核酸 (RNA)…………………………………. 21
八、ARV的S1、S2、S3、S4基因片段選殖……………………. 22
8-1 S基因引子序列..………………………………………. 22
8-2以反轉錄-聚合鏈反應 (reverse transcription-polymerase chain reaction，RT-PCR) 增幅病毒S基因…………………………………………. 23
九、雙步驟RT-PCR (two-step RT-PCR) 偵測關節炎相關的
基因表現……………………………………………………… 23
9-1 互補股DNA (complementary DNA, cDNA) 合成….… 23
9-2 PCR增幅雞的cDNA基因……………………………… 24
十、核酸探針的製備……………………………………………. 25
10-1 PCR製備雙股DNA (ds DNA) 探針………………... 25
10-2 缺口轉譯法 (nick translation) 製備ds DNA探針…. 26
十一、原位基因雜合法 (in situ hybridization, ISH)………26
十二、抑制性扣減雜合法 (suppression subtractive
hybridization, SSH)……………………………………… 27
12-1 分離mRNA……….…………………………………. 27
12-2 合成第一股cDNA (first-strand cDNA)……………... 27
12-3 合成第二股cDNA (second strand cDNA)…………... 28
12-4 Rsa I 限制酵素切割…………………………………. 28
12-5轉接子 (adaptor) 的連接………………………….. 28
12-6 第一次雜合反應 (first hybridization)………………. 29
12-7 第二次雜合反應 (second hybridization)…………… 30
12-8 PCR增幅……………………………………………... 30
12-9 PCR產物的純化……………………………………... 31
十三、重組質體之構築………………………………………… 31
13-1 DNA接合作用 (DNA ligation)……………………… 31
13-2 轉形作用 (transformation)……………………….…. 31
13-3 菌落挑選及質體的抽取……………………………... 32
第四章 結果………………………………………..............33
一、雞隻接種病毒後之臨床症狀觀察…………………….. 33
二、組織切片H＆E染色後觀察…………………………… 33
三、血清中抗ARV抗體的變化………………………….. 34
四、RT-PCR分析病毒組織趨性………………………….. 34
五、RT-PCR觀察關節組織基因表現.……………………. 35
六、利用原位基因雜合法(in situ hybridization, ISH)
偵測細胞及組織切片中病毒的分布………………………. 35
七、利用ISH偵測組織中BMP2的分布………………... 36
八、利用ISH偵測組織中MMP2的分布……………….. 36
九、利用抑制性扣減雜合法 (suppression subtractive hybridization, SSH) 偵測ARV感染後誘導細胞表現的基因………………………………………………… 37
第五章 討論……………………………………………………… 57
參考文獻…………………………………………………………… 63
表次
表一、RT-PCR偵測ARV病毒株S1133在各臟器之分布…………. 38
表二、以原位基因雜合法 (ISH) 檢測ARV病毒株S1133…………39
表三、以RT-PCR偵測U2AF1 基因於ARV感染雞隻腳掌及跗關節的表現………………………………………………………... 40
圖次
圖1、抑制性扣減雜合法 (SSH) 所使用的的引子對序列圖…… 41
圖2、抑制性扣減雜合法 (SSH) 原理圖…….………………… 42
圖3、感染ARV第6天後雞隻的跗關節腫脹情形…………………. 43
圖4、感染ARV第6天雞隻軟腳蹲坐、行動不便情形……………..43
圖5、H＆E染色觀察組織切片病變 (一)………………………… 44
圖6、H＆E染色觀察組織切片病變 (二)……….…….………… 45
圖7、血清中抗ARV抗體的變化…………………………………….46
圖8、RT-PCR偵測ARV S4基因在各臟器之分布……………………47
圖9、PCR觀察腳掌、跗關節的BMP2表現情形………………….. 48
圖10、PCR觀察腳掌、跗關節的MMP2表現情形………………... 49
圖11、ISH偵測ARV感染之Vero細胞………………………….…. 50
圖12、ISH偵測ARV在關節組織之分布 (一)……………………..51
圖13、ISH偵測ARV在關節組織之分布 (二).…………………….52
圖14、ISH偵測關節組織中BMP2之分布…………………………. 53
圖15、ISH偵測關節組織中MMP2之分布 (一)…………………… 54
圖16、ISH偵測關節組織中MMP2之分布 (二)…………………… 55
圖17、RT-PCR觀察腳掌及跗關節中U2AF1基因表現情形………. 56

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