臺灣博碩士論文加值系統

Jak/STAT訊息鏈調控多種生理反應，包含細胞增生、幹細胞維持、免疫反應和造血作用。當Jak/STAT訊息鏈失去妥善調控，可能會引發癌症和免疫相關的疾病。果蠅的Jak/STAT訊息鏈在演化上與哺乳動物同源且較為簡單，所以果蠅是良好的模式生物來研究Jak/STAT訊息鏈的功能。果蠅的Jak/STAT訊息鏈調控許多重要發育過程和生理反應。在果蠅的Jak/STAT訊息鏈中，三個Upd-like配體(Upd、Upd2、Upd3)及兩個Dome受體(Dome、Domelike)的下游，僅只有一個Jak和一個STAT，但可以調控許多發育過程及生理反應。最近的研究中發現，Dome和Domelike會形成同雙體及異雙體。在我的實驗中，利用免疫共沉澱法發現Upd-like蛋白也會形成同雙體及異雙體。我們推測在Jak/STAT訊息鏈中Upd-like蛋白形成雙體，可能會影響Upd-likes在組織中的分布，或改變和Dome受體的親和力，進而執行不同功能。另一方面，我發現在Upd-like蛋白中，Upd和Upd3不論在組織中的分布情形及長距離活化Jak/STAT活性的能力都很相近，而Upd2的特性較為不同。我也發現在果蠅眼碟表達不同組合的Upd-like配體時，會對複眼表型產生不同的影響。在觀察同時表達Upd-like配體和Dome受體對複眼發育的影響時，我發現表達Upd3時，複眼變大的性狀對於Dome受體的劑量很敏感，此外我也發現分別表達Upd和Upd3配合不同的Dome受體會造成果蠅複眼不同的性狀。此研究使我們對於果蠅Jak/STAT訊息鏈中配體和受體的作用機制有更深一層的了解，也透過對於果蠅這個模式生物的研究，我們能更了解哺乳動物Jak/STAT訊息鏈可能的作用機制。

The Jak/STAT pathway plays a crucial role in physiological and developmental processes in mammals, such as immune response, hematopoiesis, and adipogenesis. Since the Jak/STAT signaling is evolutionarily conserved and Drosophila has only one Jak and one STAT, Drosophila is a simple and non-redundant system to investigate function and regulation of Jak/STAT pathway in vivo. Three ligands, Unpaired (Upd), Upd2, Upd3 and two receptors, Domeless (Dome) and Domelike, have been identified in Drosophila. Upd is regarded as an essential factor to affect cell proliferation, cell migration and differentiation whereas Upd3 is implicated in immune response. The function of upd2, however, remains obscure. On the other hand, it is suggested in recent studies that Dome and Domelike can form homodimers and heterodimers in vivo. Hence, we are interested in how such a simple Jak/STAT signaling regulates multiple distinct developmental processes. We proposed that different combinations of Upd-likes and Dome receptors would have different affinity and induce different biological effects. In my study, I found Upd-like proteins have different properties. Both Upd and Upd3 are N-linked glycoproteins, but Upd2 is not. The localization of Upd2 in vivo is also different from that of Upd and Upd3. Upd and Upd3 can influence Jak/STAT activity in a long-range manner, but Upd2 cannot. From the Co-immunoprecipitation assay, I found Upd-like proteins can form homodimers and heterodimers in S2 cells. The adult eye phenotype of Upd/Upd3 combination is different from Upd or Upd3. Also, different combinations of Upd-likes and Dome receptors have different effects on Drosophila eye development. This study may help us to further understand the molecular mechanism of ligands-receptors interaction in Drosophila Jak/STAT pathway.

目錄
目錄...............................................................................................1
圖表目錄.......................................................................................4
中文摘要.......................................................................................6
英文摘要.......................................................................................8
前言.............................................................................................10
哺乳動物中的Jak/STAT訊息傳遞鏈....................................................................10
果蠅為研究Jak/STAT訊息傳遞鏈的良好模式生物............................................11
果蠅Jak/STAT訊息傳遞鏈的成員........................................................................12
果蠅Jak/STAT訊息傳遞鏈的配體：Upd...............................................................14
果蠅Jak/STAT訊息傳遞鏈的配體：Upd2.............................................................15
果蠅Jak/STAT訊息傳遞鏈的配體：Upd3.............................................................16
果蠅Jak/STAT訊息傳遞鏈的受體：Dome............................................................18
果蠅Jak/STAT訊息傳遞鏈的受體：Domelike.......................................................19
Dome及Domelike受體和Upd-like配體預測可能的結合區域..........................21
問題及假說...........................................................................................................22
實驗材料.....................................................................................24
引子清單.............................................................................................................24
果蠅株.................................................................................................................27
化學藥品.............................................................................................................29
抗體.....................................................................................................................31
實驗方法.....................................................................................33
細胞培養.............................................................................................................33
細胞短暫轉染(Transient transfection)................................................................33
S2細胞蛋白萃取.................................................................................................34
蛋白質濃度定量.................................................................................................34
免疫共沉澱法(Co-immunoprecipitation)...........................................................35
西方點墨法(Western blot)..................................................................................36
S2 R+細胞免疫染色.............................................................................................37
S2細胞RNA萃取................................................................................................38
反轉錄聚合酶連鎖反應.....................................................................................38
聚合酶連鎖反應.................................................................................................39
轉型作用(Transformation)..................................................................................39
小量質體萃取.....................................................................................................40
中量質體萃取(Viogene Midi kit)........................................................................40
免疫組織染色.....................................................................................................41
細胞外免疫組織染色.........................................................................................42
掃描式電子顯微鏡.............................................................................................43
實驗結果.....................................................................................46
Upd和Upd3為氨基連接的醣化蛋白(N-linked glycoprotein).............................47
Upd-like蛋白在S2細胞中可形成同雙體及異雙體............................................49
Upd-like蛋白在果蠅眼碟組織內的分布.............................................................52
Upd-likes表達於眼碟分化中區域對Jak/STAT訊息傳遞鏈活性的影響.............55
Upd-likes表達於眼碟未分化區域對Jak/STAT訊息傳遞鏈活性的影響.............57
Upd-likes表達於眼碟未分化區域對成蟲複眼表型的影響................................59
表達Upd-likes配上Dome或Domelike受體對Jak/STAT訊息鏈的影響..............61
表達Upd-likes和Dome或Domelike受體對果蠅成蟲複眼表型的影響.............62
討論.............................................................................................64
果蠅Jak/STAT訊息傳遞鏈中三種Upd-like蛋白特性的比較..............................64
Upd-like蛋白形成同雙體及異雙體後可能的功能.............................................66
Upd-like蛋白分別表現在眼碟已分化和未分化區域，對Jak/STAT訊息傳遞鏈活性有不同影 響...................................................................................................67
不同的Upd-like配體與Dome受體之間有不同的交互作用..............................68
Upd-like蛋白配上Dome或Domelike受體造成不同的交互作用.......................69
果蠅Jak/STAT訊息傳遞鏈可能和MAPK訊息傳遞鏈有交互作用......................70
參考文獻.....................................................................................72

圖表目錄
圖表
圖一、Upd及Upd3皆為氨基連接的醣化蛋白(N-linked glycoprotein)......................77
圖二、共同免疫沉澱法所利用之Actin-Gal4及UAS-upd-likes質體..........................78
圖三、Upd-like蛋白在S2細胞中會形成同雙體(Homodimers).................................79
圖四、Upd-like蛋白在S2細胞中會形成異雙體(Heterodimers)................................80
圖五、GFP抗體不會直接辨認Upd-HA、Upd2-flag及Upd3(共同免疫沉澱法控制 組).....................................................................................................................81
圖六、Upd-like蛋白分泌至細胞外後於果蠅眼碟內的分布情形..............................82
圖七、Upd-likes表達於眼碟分化中細胞的區域對Jak/STAT訊息傳遞鏈活性的影 響.......................................................................................................................84
圖八、Upd-likes表達於眼碟未分化細胞的區域對Jak/STAT訊息傳遞鏈活性的影 響.......................................................................................................................86
圖九、Upd-likes表達於眼碟未分化區域對成蟲複眼表型的影響............................88
圖十、在果蠅眼碟未分化區域表達Upd-likes和Dome或Domelike受體對Jak/STAT訊息鏈的影 響...................................................................................................89
圖十一、表達Upd-likes和Dome受體對果蠅成蟲複眼表型的影響.........................90
圖十二、表達Upd-likes和Domelike受體對果蠅成蟲複眼表型的影響...................91

附圖
附圖一、果蠅Jak/STAT訊息傳遞鏈及其活性的報告者10XSTAT92E-GFP-NLS..........92
附圖二、果蠅複眼的發育過程及專一表達於果蠅眼碟的Gal4果蠅株ey-Gal4及GMR- Gal4....................................................................................................93

附圖三、Jak/STAT訊息傳遞鏈調控果蠅多種發育過程及生理反應之可能的分子機制模式 圖.....................................................................................................94
附圖四、表達upd2於果蠅眼碟邊緣的區域會活化Jak/STAT訊息鏈及造成成蟲複眼變 大.........................................................................................................95
個人資料表........................................................................................96

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