臺灣博碩士論文加值系統

Gurken是TGF-alfa在果蠅的同源蛋白，在卵子發育中期時活化EGFR，其下游訊息建立背腹體軸。以螢光顯微技術觀察Grk蛋白質的分佈，發現它集中在卵母細胞核的周圍，也是將來背部前端的位置。因此，由不對稱分佈來推測Grk只會活化鄰近卵泡細胞的EGFR，使得接受訊息的一邊分化成背部；但是由實驗發現，若D-Cbl (EGFR的負向調控因子)失去功能時，在腹部卵泡細胞有EGFR下游基因kekkon的表現，但在grk突變株中則無此現象。因此我們推測在腹部卵泡細胞中Grk或許可直接或間接活化EGFR，活化後的Grk-EGFR複合體或許會藉由內噬路徑，最後送至lysosome中分解來調降EGFR活性。在此我們利用電子顯微鏡直接觀察Grk是否散佈到腹部卵泡細胞，並且將Grk接上HRP分子，利用HRP追蹤Grk在卵泡細胞中是否進入內噬路徑。首先，我們發現從時期8開始，HRP-Grk集中在背部卵母細胞的tER-Golgi unit，代表著絕大部分的Grk是由背部運輸出去。接著觀察卵泡細胞發現，時期8到早期9時，在背部及腹部的卵泡細胞都有HRP-Grk的分佈，代表Grk可直接活化腹部卵泡細胞的EGFR；同時也發現它會集中在lysosome及MVB中，代表在腹部Grk可能由分解來中止訊息傳遞。另一方面，初步觀察發現側邊的卵泡細胞有較活躍的分解機制，或許為Grk濃度梯度形成的機制之一。

Gurken is a specific ligand for Drosophila epidermal growth factor receptor (EGFR) during oogenesis. It has been known to act as a morphogen and observed to distribute dynamically and asymmetrically in developmental egg chambers by fluorescence microscopy. In middle stages, Grk seems to only localize in the dorsal-anteriror corner of the oocyte and activate EGFR signaling in adjacent follicle cells, therefore defines the polarity of dorsal-ventral axis. However, some evidences suggest EGFR have biological activity in ventral follicle cells, so we are interested in whether Grk also disperse to the ventral side but where the EGFR signaling is down-regulated by endocytosis of EGFR-Grk complex. Here, we use HRP-Grk fusion protein to observe Grk directly in the endocytic pathway of follicle cells by immuno-electron microscopy (IEM). Firstly, we found the HRP-Grk concentrates in the dorsal-anterior tER-Golgi units of the oocyte from stage 8 to late 9 oocytes, this means HRP-Grk is transported directionally. Interestingly, HRP-Grk could be observed in the dorsal and ventral follicle cells in stage 8 to early 9 egg chambers under the transmission electron microscope, this means HRP-Grk triggering EGFR signaling at both sides. Especially, they concentrate in the lysosome and multivesicular body (MVB) suggesting the EGFR signaling is down-regulated through ligand-receptor complex endocytosis. In addition, the preliminary data shows that the lysosome area in lateral follicle cells are larger than in dorsal follicle cells that suggesting degradative machinery may be more active in lateral side which perhaps contribute to Grk gradient formation.

目 錄
指導教授推薦書 ……………………………………………………
口試委員會審定書 …………………………………………………
授權書 ……………………………………………………………… iii
誌謝 ……………………………………………………………… iv
中文摘要……………………………………………………………… v
英文摘要……………………………………………………………… vi
目錄 ………………………………………………………………… vii
第一章 背景介紹……………………………………………………. 1
第二章 材料和方法 ……..…………………………………………... 12
1. 果蠅株…………………………………………………..…….. 12
2. 抗體……………………………………………………..…….. 12
3. 免疫螢光染色…………………………………………..…….. 12
4. 組織包埋與冷凍超薄切片…………………………………… 13
5. 電子顯微鏡的免疫染色……………………………………… 14
第三章 實驗結果…………………………………..…………………. 16
第四章 討論……………………………………..……………………. 22
參考文獻……………………………………………………………… 27

圖表目錄……………………………………………………………… 31
圖一、HRP-gurken轉殖基因………………………………………… 31
圖二、以螢光顯微鏡檢視HRP-Gurken融合蛋白之分佈………….. 32
圖三、卵巢管(ovariole)利用冷凍超薄切片厚切(厚度0.5m)時的結構..……………………………………………………………….. 33
圖四、發育中期的果蠅卵室在電子顯微鏡下的構造……………….. 35
圖五、野生型(OreR)果蠅之時期晚期8到早期9的卵，利用HRP體做免疫染色，由於野生型果蠅無HRP，可作為負對照組。... 37
圖六、HRP-Gurken轉殖果蠅之時期8的卵，分別只加Protein A-gold 和二抗的對照組，證明抗體無非專一性結合。………………….. 39
圖七、HRP-Gurken轉殖果蠅之發育至中期的卵，可看到HRP-Grk在卵母細胞中的tER-Golgi unit也會呈現不對稱的分佈。…...... 40
圖八、HRP-Gurken轉殖果蠅之晚期8到早期9的卵，在背部和腹部的卵泡細胞中都可看到HRP-Grk融合蛋白分佈在lysosome和MVB的結構中，由此可知HRP-Grk確實會被內噬進入。…. 42
圖九、HRP-Gurken轉殖果蠅之發育至晚期9的卵，側邊卵泡細胞比背部卵泡細胞有較大的lysosome面積。……………………... 44
表一、HRP-Gurken轉殖果蠅之發育至中期的卵，HRP-Grk融合蛋白在背部和腹部的卵泡細胞中其分佈在lysosome和MVB的統計。……………………………………………………………….. 45

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