臺灣博碩士論文加值系統

實驗室發現miR-197 會受到EV71 的感染而降低，並且miR-197 的存在是不利於EV71 的複製。使用穩定表現同位素標定胺基酸(SILAC)的方式去標定細胞後，以miR-197 處理的細胞，用質譜儀分析，再搭配TargetScan 資料庫與3’UTR reporter assay 做進一步的鑑定，最後得到9個miR-197 的直接目標基因。我們選定其中的Grb2 作為研究目標，進一步探討Grb2 對EV71 複製的生理意義，在EV71 感染宿主後，miR-197的表現量會隨著感染時間增加而減少，而導致下游的目標基因Grb2 的表現量隨著感染時間增加而上升，並且我們的實驗結果顯示，在抑制Grb2 表現的細胞中，EV71 的增殖在病毒蛋白、病毒RNA 和病毒生成量都有明顯的上升。Grb2 在RTKs 訊息傳導路徑中扮演十分重要的功能，所以我們進一步去探討Grb2 的表現量是否會影響病毒感染宿主後，所引發的訊號傳遞改變。而先前研究報導EV71 感染後ERK 會活化，而ERK 的活化是病毒複製所必須的，而在我們的實驗數據顯示，在EV71感染的晚期ERK 路徑會活化，且抑制Grb2 表現量的組別ERK 的活化是增加的。這些實驗數據顯示EV71 感染後miR-197 的表現量下降，而使下游的目標基因Grb2 的表現量增加，進而調節下游的ERK 路徑活化。

We found that miR-197 was downregulated in response to EV71
infection and the presence of miR-197 was unfavorable for EV71
replication. Nine authentic target genes of miR-197 were identified by
mass-spectrometry- based quantitative proteomic approach coupled with
stable isotope labeling with amino acids in cell culture (SILAC), aided by
TargetScan algorithm and 3’UTR reporter assay. Grb2 (Growth factor
receptor-bound protein 2), one of miR-197 target genes, was chosen to study
its biological role in EV71 replication. MiR-197 expression gradually
decreased after EV71 infection, and that leads to Grb2 expression level was
increased correspondingly in a time-dependent manner. Our results showed
that EV71 replication (in viral protein, viral RNA, and viral production level)
was increased in GRB2 silenced cells compared with scramble siRNA
treated cells. Grb2 was an important adaptor protein in RTKs signaling
pathway, so we further investigated whether grb2 expression level was
affected when viral infection-mediated signaling transduction. Previous
studies support that activation of the ERK pathway as being a necessary
cellular modification to support EV71 proliferation. In our study, ERK was
activated at the late phase of viral infection, and the phosphorylation of
ERK was enhanced in siGrb2-treated cells after EV71 infection. These data
indicated that EV71-induced downregulation of miR-197 may constitute a
new mechanism that increases the expression of Grb2 to modulate ERK
activation that was required for EV71 replication.

指導教授推薦書
口試委員審定書
誌謝------------------------------------------------------------------------------iii
縮寫表-------------------------------------------------------------------------- iv
中文摘要----------------------------------------------------------------------- v
英文摘要-----------------------------------------------------------------------vi
目錄-----------------------------------------------------------------------------vii
圖目錄--------------------------------------------------------------------------ix
表目錄-------------------------------------------------------------------------- x
附錄目錄--------------------------------------------------------------------------xi
第一章、緒論
一、腸病毒的簡介-----------------------------------------------------------1
1.1 腸病毒的分類-------------------------------------------------------1
1.2腸病毒的傳染途徑和症狀-----------------------------------------1
1.3腸病毒的結構--------------------------------------------------------2
1.4腸病毒的生活史-----------------------------------------------------3
1.5病毒與宿主受體之間的關係--------------------------------------5
1.6病毒與宿主間的訊號傳遞之關係--------------------------------6
二、MicroRNA的介紹------------------------------------------------------8
2.1 MicroRNA的作用機制-------------------------------------------8
2.2 MicroRNA與病毒之間的關係----------------------------------9
三、GRB2介紹--------------------------------------------------------------11
3.1 Grb2的結構---------------------------------------------------------11
3.2 Grb2的功能--------------------------------------------------------11
3.3 Grb2與病毒之間的關係-----------------------------------------12
3.4 Gab蛋白與Grb2蛋白之間的關係-----------------------------14
第二章、研究目的----------------------------------------------------------17
第三章、材料與方法-------------------------------------------------------19
3.1 材料------------------------------------------------------------------19
3.2 實驗方法------------------------------------------------------------21
第四章、實驗結果----------------------------------------------------------28
第五章、討論----------------------------------------------------------------43
參考文獻----------------------------------------------------------------------50
附錄----------------------------------------------------------------------------83

圖目錄
圖一、Grb2為miR-197的目標基因-----------------------------------------64
圖二、探討Grb2再宿主細胞中的表現量--------------------------------------66
圖三、探測Grb2 siRNA最低有效的抑制濃度和不同廠牌siRNA之比較----------------67
圖四、探討抑制內生性的Grb2的表現量對於EV71病毒複製的影響--------------------68
圖五、探討大量表達Grb2對病毒複製的影響---------------------------70
圖六、利用FACS的方式分析EV71的receptor是否會受到抑制Grb2的表現量而影響EV71的Binding-------------72
圖七、探討EGFR訊息傳遞與MEK/ERK活化之間的關係----------73
圖八、EV71感染對EGFR訊息傳遞的影響------------------------------74
圖九、EV71 感染對宿主細胞的Gab family訊息傳遞的狀況--------75
圖十、探討抑制ERK路徑的活化對病毒複製的影響-------------------76
圖十一、探討在EV71病毒感染下，抑制內生性的Grb2是否會影響ERK路徑活化 --------------------------77

表目錄
表一、實驗所需要的抗體名稱、廠牌、貨號和稀釋倍數。--------78
表二、實驗所需要的引子。------------------------------------------------79
表三、實驗所需的siRNA序列。-----------------------------------------80
表四、實驗所需的質體列表。---------------------------------------------81
表五、用TargetScan資料庫比對在Grb2 3’UTR上的miR-197的Target site。(GeneBank accession: NM_002086)---------------------------------82

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