臺灣博碩士論文加值系統

家禽里奧病毒(ARV)是常見的感染家禽的病毒，使雞隻產生關節炎、呼吸道疾病與營養吸收不良症，導致經濟上嚴重的損失。過去研究指出，ARV S1133病毒株可以在Vero細胞中引起細胞凋亡，利用此系統，我們探討病毒引發細胞凋亡之訊息傳遞途徑。我們證明ARV感染細胞後，細胞中p53表現量、p53 serine 46磷酸化、p53-driven reporter活性以及bax皆有增加。p53顯性突變抑制物 (dominant negative mutant) 能減緩ARV S1133引起的細胞凋亡及bax表現。此外， Src tyrosine 418的磷酸化在病毒感染後增加，C-terminal Src kinase (Csk) 及Src抑制劑SU-6656，能抑制ARV S1133誘發之p53活化、bax表現及細胞凋亡。本研究證明ARV S1133可經由活化Src及下游之p53使感染細胞進行細胞凋亡。
p38抑制劑SB203580及JNK抑制劑SP600125能保護細胞，減緩由ARV S1133感染導致的細胞凋亡，ARV S1133能使p38及JNK磷酸化程度增加，負責磷酸化p38及JNK上游之MAPKK， MKK3/MKK6及SEK1/MKK4之磷酸化亦呈增加，MAPK下游轉錄因子ATF-2及AP-1次單位c-Jun磷酸化亦呈增加，chromatin precipitation結果證明磷酸化之c-Jun 能結合至p53啟動子，顯示p38及JNK路徑能被ARV S1133所活化。本研究說明ARV S1133活化Src及MAPK之細胞凋亡訊息，ARV S1133可藉由活化c-Jun使p53表現增加，但Src及-MAPK之訊息傳導是否有交互作用仍需進一步研究。

The avian reovirus (ARV) is one of the common viruses that infects poultry and results in severe economic losses. ARV S1133 strain induced apoptosis in Vero cells, but the related mechanism is still unknown. In the current studies, we demonstrated that ARV S1133 induced apoptosis in a dose-, and time-dependent manner. Upon ARV infection, the p53 protein levels, serine-46 phosphorylation of p53, p53-driven reporter activity and bax expression were elevated significantly. Dominant negative p53 alleviated the ARV S1133-induced apoptosis and the bax expression. In addition, Src tyrosine-418 phosphorylation was elevated following ARV S1133 infection. In the presence of C-terminal Src kinase (Csk) and Src specific inhibitor SU-6656, the ARV S1133-induced p53 activation, bax expression and apoptosis extent were diminished. Taken together, these studies revealed ARV initiated a proapoptotic signaling by linking Src to p53.
p38 inhibitor and JNK inhibitor SP600125 were able to protect cells from ARV S1133-induced apoptosis. Phosphorylation level of p38 and JNK, as well as two correspond MAPKK, MKK3/MKK6 and SEK1/MKK4 were increased following ARV S1133 infection. In addition, two downstream transcription factors, ATF-2 and c-Jun phosphorylation were increased. Chromatin precipitation demonstrated phosphorylated c-Jun binds to p53 promoter. These data indicated the p38 and JNK pathways were active in ARV S1133-infected Vero cells. Taken together, the current research demonstrated that ARV activates Src and MAPK proapoptotic signals, the cross talk between these two pathways will be elucidated, and, phosphorylated c-Jun binding to p53 promoter may account for the accumulation of p53 upon ARV S1133 infection.

中文摘要………………………………………………………………..2
英文摘要………………………………………………………………..3
文獻探討
一. 家禽里奧病毒………………………….………………………...7
二. 細胞凋亡(apoptosis) ……………………………………………..8
材料與方法
化學藥品與試劑………………… …...……………………………..18
實驗方法
1. 細胞培養(Cell culture) ………………………………..……. .22
2. 病毒增值…………………………………………………….. 22
3. 冷凍細胞(Frozen cells) …………………………………….. 22
4. 解凍細胞(Cells reconstitution) ……………………………..22
5. 勝任細胞的製備…………………………….………………..23
6. 細菌的轉型(Transformation) …..…………………………..23
7. 細胞的轉染(Transfection) ……....…………………………..23
8. beta-半乳糖酵素試驗(��-galatosidase test)................................24
9. 蛋白質定量 (Protein concentration assay)……………….. 24
10. 冷光酵素測定(Luciferase assay)…………………………... 25
11. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) ……………………………………………….. 25
12. 西方墨點分析 (Western blot) …………………………….. 26
13. flow cytometry analysis…………………...………………...27
14. Enzyme-linked immunosorbent assay (ELISA)…………..27
15. Chromatin immunoprecipitation assays (Chip assay) ………………………………… ………………… 28
16. Cell Death Detection ELISAPLUS…………………………..29
結果
一. ARV time- and dose-dependent induction of apoptosis………31
二. Inhibition of ARV-mediated cytopathic effect and apoptosis by p53mt135,genistein, SU-6656, and Csk………………………32
三. Activation of p53-dependent apoptosis by ARV……………...35
四. ARV enhancement of Src phosphorylation…………………... 37
五. Diminished ARV-mediated p53 activation upon Src inhibition………………………………………………………39
六. Requirement for viral gene expression and replication for Src
and p53 activation, and induction of apoptosis………………
…………………………………………..……………………. 40
七. Activation of p53 and induction of Bax by ARV-encoded
structural protein �� ……………………………………………44
八. SP600125 and SB203580 protect cells from ARV-induced
apoptosie………………………………………………………46
九. Activation and JNK/SAPK and p38 MAPK by ARV………..47
十. Activation and MKK3 and SEK1/MKK4 MAPKK by ARV..…48
十一. Activation of transcription c-Jun and ATF-2 by ARV……48
十二. Binding of c-Jun to p53 promoter…………………………49
討論……………………………………………………………………51
參考文獻 ………………………………………………………………54

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