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研究生:郭子雷
研究生(外文):Tzu-Lei Kuo
論文名稱:腸病毒七十一型感染之人類橫紋肌肉瘤早期膜蛋白質體分析
論文名稱(外文):Proteomic Analysis of Membrane Fraction of Rhabdomyosarcoma Cells in Response to Early Enterovirus 71 Infection
指導教授:黃弘文黃弘文引用關係
指導教授(外文):Hurng-Wen Huang
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物醫學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:59
中文關鍵詞:膜蛋白質體腸病毒七十一型
外文關鍵詞:EV71membrane proteomic
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腸病毒為台灣小兒感染性疾病之中常見病因,一般感染為無症狀或輕微類似
感冒症狀,但因腸病毒感染而轉變重症者卻會有極高致死率,以 Enterovirus 71
最具危險性,會引起手口足病、腦炎及中樞神經疾病並且在1998年在台灣造成大
流行。一般認為小核糖核酸病毒科的病毒,經由與特定細胞上的病毒受體結合,
例如:小兒麻痺病毒與克沙奇病毒分別以細胞表面上人類小兒麻痺病毒接受體
(human poliovirus receptor, PVR; CD155) 和克沙奇病毒及腺病毒受體
(coxsackievirus and adenovirus receptor, CAR) 接合後,改變病毒本身的蛋
白質結構 (protein conformation) 、幫助病毒脫去病毒外殼 (uncoating) ,
且有研究指出內皮細胞受到克沙奇病毒感染後會表現更多 CAR 和 DAF 當作病
毒受器。但到目前為止並未明確了解腸病毒71型的接受器,為了瞭解腸病毒71
型初期感染途徑。我們利用人類橫紋肌肉瘤細胞感染腸病毒71型六小時後,淬取
其膜蛋白並配合二維電泳分析找出差異性蛋白質。在此我們發現有些熱休克蛋白
如 HSP60、70 大量表現,其可幫助細胞渡過病毒感染所造成的壓力而不會死亡。
另外脂質相關蛋白表現量發生改變,這可能指出腸病毒感染會改變膜上脂質的組
成,使細胞更容易接受病毒顆粒。另一方面發現有醣化蛋白表現增加,其也可能
會影響病毒與細胞表面的吸附。這些蛋白質的改變對腸病毒七十一型的初期感染
研究中扮演的角色具有進一步研究的價值。
Enterovirus 71 (EV71) infection is one of epidemic disease in children commonly
in Taiwan. The clinical manifestation of EV71 infection may include acute respiratory
disease, hand foot and mouth disease, herpangina, myocarditis, aseptic meningitis,
acute flaccid paralysis, brainstem or cerebellar encephalitis. EV71 infection usually
occurs through the fecal–oral route, leading to viremia and invasion of the skin and
mucosa. Infection is initiated by attachment to putative receptor, which induces
conformational changes in the virus that facilitate translocation of the viral RNA into
the cytoplasm. Some cell surface molecules have been primarily identified for
enterovirus which like poliovirus receptor (CD155), coxsackievirus and adenovirus
receptor, Decay accelerating factor (CD55) belong Echoviruses but no EV71 receptor
has yet been found. Rhabdomyosarcoma cells were used as a model for EV71
infection. We use two-dimensional gel electrophoresis to analyse membrane fraction
from rhabdomyosarcoma cells infected with EV71 at 6 h post infection. Twenty-eight
differentially expressed protein spots were identified. Some lipid-associated protein
slightly change after EV71 infection that may indicate EV71 infection will change
membrane structure of rhabdomyosarcoma cells. And some O-linked glycosylation
proteins were also upregulated after EV71 infection. It is interesting to reveal the role
of these proteins in early EV71 infection and cell response.
中文摘要 I
英文摘要 II
英文縮寫表 III
壹、序言(Introduction)
1.1 腸病毒流行病學 1
1.2 腸病毒71 型的病毒特性 2
1.3 病毒進入細胞的方式 4
1.4 腸病毒受體 5
貳、實驗流程(Experiment flowchart) 7
參、 材料與方法(Materials and Method)
3.1 RD 細胞繼代培養 8
3.2 RD 細胞解凍 8
3.3 RD 細胞冷凍保存 9
3.4 腸病毒培養與感染 10
3.4.1 病毒大量培養 11
3.4.2 病毒感染 12
3.4.3 病毒價效測定 Viral titration 12
3.5. 膜蛋白萃取與定量 13
3.5.1 細胞膜蛋白萃取 14
3.5.2 TCA/Acetone 沉澱 14
3.6. 二維電泳法 15
3.6.1 Isoelectric focusing electrophoresis 聚電焦電泳 15
3.6.2 Immobiline DryStrip Equilibration 17
3.6.3 聚丙烯酼胺膠體電泳Sodiumdodecylsulfate Polyacrylamide Gel
Electrophoresis (SDS-PAGE)
17
3.6.4 Silver stain 銀離子染色 19
3.6.5 Coomassie brilliant blue R-250 stain 20
3.6.6 Gel imaging analysis 膠體影像分析及統計分析 20
3.6.7 In gel digestion 21
3.6.8 MALDI-TOF 質譜操作 22
3.7 免疫沉澱
23
肆、 實驗結果(Result)
4.1 膜蛋白萃取技術建立與分析 24
4.2 腸病毒感染差異 25
4.3 二維電泳膠片之分析結果 25
4.4 軟體比對分析及統計 26
4.5 差異性蛋白質身分鑑定 26
4.6 腸病毒外殼蛋白VP1 免疫沉澱結果 28
伍、 實驗討論(Discussion)
5.1 腸病毒七十一型與RD 細胞株 29
5.2 腸病毒七十一型的細胞毒性 30
5.3 膜蛋白質體學與疾病治療的討論 31
5.4 差異性蛋白質與討論 33
陸、 結論(Conclusion) 37
柒、 參考文獻(Reference) 38
捌、 圖表(Figures and Tables Countents)
Figure 1-A. 萃取RD 細胞膜蛋白為細胞膜層與細胞質層之西方點墨分析 43
Figure 1-B. EV71 感染前後RD 細胞膜蛋白初步差異性電泳分析 44
Figure 1-C. EV71 感染前後膜蛋白電泳膠量化分析 45
Figure 2. 二維電泳分析EV71 感染前後之RD 細胞膜蛋白差異 46
Figure 3. RD 細胞株感染EV71 二維電泳膠圖之表現量上升蛋白點 47
Figure 4. 二維電泳鑑定蛋白質點3D 分析 48
Figure 5. Anti-EV71 VP1 antibody 免疫沉澱分析感染EV71 後之RD 細胞 49
Table 1. RD 細胞株感染腸病毒七十一型後表現改變之蛋白質身分鑑定一覽表 50
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