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系統識別號 U0002-0908201118523300
中文論文名稱 斑馬魚第四型酪胺酸磷酸水解酶對早期胚胎細胞移動能力之影響
英文論文名稱 Functional analysis and its effects on cell migration of zebrafish ptp4a3 during early embryogenesis
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 99
學期 2
出版年 100
研究生中文姓名 鄭凱文
研究生英文姓名 Kai-Wen Cheng
學號 698180030
學位類別 碩士
語文別 中文
口試日期 2011-07-14
論文頁數 63頁
口試委員 指導教授-陳曜鴻
委員-林明德
委員-陳銘凱
委員-陳曜鴻
中文關鍵字 斑馬魚  第三型酪胺酸磷酸水解酶  細胞移動 
英文關鍵字 zebrafish  ptp4a3  cell  migration 
學科別分類 學科別自然科學化學
中文摘要 第三型酪胺酸磷酸水解酶(ptp4a3)在各類的研究中指出與細胞的生長發育、增生以及癌細胞的轉移有關。然而其在胚胎早期發育上的功能卻仍不清楚,在本篇論文中我們利用斑馬魚做為模式物種來研究ptp4a3在早期胚胎發育上的生物功能。藉由原位雜交法我們可以發現斑馬魚的ptp4a3在單細胞時期就有所表現,而在胚胎的原腸期其訊號表現於胚環及胚盾上,在胚胎發育的後期其訊號則遍佈全身;包括腦部、體節、咽弓、嗅基板、視網膜以及blood island等。由這些觀察結果我們可以得知ptp4a3在斑馬魚早期胚胎上的重要性。此外,我們利用顯微注射施打了反股的morpholino藉以觀察ptp4a3缺失所造成的畸形。包括了胚胎外觀上由圓形變的狹長、卵黃因受力而腫脹向外凸出以及尾芽部分因細胞堆積形成的囊泡狀。這些缺陷的比例隨著morpholino的劑量而增減且能藉由注射ptp4a3的mRNA來進行補救回覆。為了進一步剖析ptp4a3表型的分子機制,我們使用了細胞移動的探針(gsc, hgg1, ntl)進行研究,且由結果發現了gsc及hgg1的訊號在胚胎腦部原基處難以聚集表現,而ntl訊號則是難以由胚盾處向上延伸。此外phalloidin的染色顯示了在下列兩種胚胎主要細胞的明顯缺陷,分別為胚胎細胞邊緣處的eYSL由纖維性肌動蛋白(F-actin)所構成的點狀寬帶以及EVL上細胞周圍的環狀F-actin訊號。由這些觀察結果可知ptp4a3會影響細胞移動且在斑馬魚早期胚胎發育的原腸期中扮演了非常重要的角色。
英文摘要 Protein tyrosine phosphatase 4a3 plays a role in the regulation of cell growth, proliferation and metastasis, but its function during early embryogenesis is still unclear. In this thesis, we used zebrafish model to study the biological function of ptp4a3 during early development. Whole mount in situ hybridization revealed that zebrafish ptp4a3 transcript was first observed at 1-cell-stage and extended its expression to germ ring and embryonic shield during gastrula period. Later, ptp4a3 signals were detected at head, somite, blood island, pharyngeal arch, olfactory placode, retina as well as at head muscle precursors. These observations highlight the importance of ptp4a3 during early development. Furthermore, we injected antisense morpholino (MO) to knockdown ptp4a3 expression for studying ptp4a3 loss-of-function phenotypes. Ptp4a3-morphant displayed several classic gastrulation defects, such as longitudinal shapes, swelling yolk and bubble-like tail bud vesicles. These defects were does-dependent and can be rescued by injection of ptp4a3 mRNA. To further dissect the molecular mechanism underlying ptp4a3-MO-induced gastrulation defects, three cell migration markers (gsc, hgg1 and ntl) were investigated. Results showed that gsc and hgg1failed to express at the head primordium, and ntl was difficult to extend from the embryonic shield. In addition, phalloidin stain showed that a significant reduction of the F-actin punctate band of the eYSL and the peripheral F-actin ring within the EVL in the ptp4a3-morphant. On the basis of these observations, we conclude that ptp4a3 affects cell migration and plays an important role during gastrulation period of zebrafish embryogenesis.
論文目次 授權書
口試委員審議通過簽名單
謝誌
中文摘要----------------------------------------------------------------------------I
英文摘要--------------------------------------------------------------------------II
目錄-------------------------------------------------------------------------------III
圖表目錄----------------------------------------------------------------------VII

第一章 前言-----------------------------------------------------------------------1
1-1 酪胺酸磷酸水解酶(protein tyrosine phosphatase, PTPs)---------1
1-2 第四型酪胺酸磷酸水解酶(protein tyrosine phosphatase 4a)-----2
1-3 第四型酪胺酸磷酸水解酶與人類之關係-------------------------------3
1-4 第四型酪胺酸磷酸水解酶對癌症細胞的影響-------------------------3
1-5 致癌基因與發育-------------------------------------------------------------4
1-6 斑馬魚胚胎早期發育-------------------------------------------------------5
1-7 以斑馬魚為模式物種研究第四型酪胺酸磷酸水解酶對胚胎發育
的影響------------------------------------------------------------------------7
第二章 材料與方法--------------------------------------------------------------8
2-1 野生型斑馬魚(AB strain)的飼養--------------------------------------8
2-2 斑馬魚胚胎收集-------------------------------------------------------------8
2-3 斑馬魚胚胎固定、脫水、保存--------------------------------------------9
2-4 斑馬魚胚胎RNA的萃取--------------------------------------------------9
2-5 反轉錄聚合酶連鎖反應(RT-PCR)-----------------------------------10
2-6 勝任細胞(Competent cell)之備製------------------------------------10
2-7 聚合酶連鎖反應(Polymerase Chain Reaction, PCR)-------------11
2-8 DNA純化(purification)-------------------------------------------------12
2-9 接合反應(ligation)------------------------------------------------------12
2-10 轉型反應(transformation)--------------------------------------------13
2-11 質體萃取(Isolation of plasmid DNA)------------------------------14
2-12 合成RNA探針(Riboprobe)-----------------------------------------14
2-13 原位雜交法(Whole-mount in situ hybridization)-----------------15
2-14 顯微注射(Microinjection)--------------------------------------------18
2-15 樣品包埋及冷凍切片(Embedding and Cryosection)------------19
2-16 協同注射(co-injection)-----------------------------------------------19
2-17 mRNA之備製------------------------------------------------------20
2-18 Phalloidin呈色法--------------------------------------------------23
2-19 顯微照相系統--------------------------------------------------------23
第三章 結果------------------------------------------------------------------25
3-1 斑馬魚的ptp4a 胺基酸序列比對與演化樹分析--------------------25
3-2 以原位雜交法(whole mount in situ hybridization)確認各時期斑
馬魚胚胎之ptp4a3訊號表現位置-------------------------------------26
3-3 透過冷凍切片更確實的觀察ptp4a3訊號於斑馬魚體內的位置-29
3-4不同劑量的morpholino注射後對斑馬魚胚胎造成的影響------30
3-5抑制ptp4a3基因表現對於斑馬魚胚胎發育的影響----------------31
3-6 利用gsc、hgg1、ntl探針觀察ptp4a3抑制後對斑馬魚胚胎發育造
成的影響--------------------------------------------------------33
3-7 利用phalloidin螢光染色觀察斑馬魚胚胎變異情形----------36
3-8 利用共同注射ptp4a3 mRNA觀察變異胚胎恢復之情況-----37
第四章 討論---------------------------------------------------------------------38
4-1 ptp4a3基因在脊椎動物上具有高度保留性-----------------------38
4-2 透過原位雜交法推論ptp4a家族三者間的關係--------------------39
4-3利用原位雜交法與顯微注射推論ptp4a3可能之功能------------40
4-4 抑制ptp4a3的表現廣泛的影響了各種細胞移動的能力-----------42
4-5 由phalloidin染色來觀察ptp4a3的缺失對原腸期中EVL、YSL
所造成的變異及後續發育可能產生的影響--------------------------44
4-6 ptp4a3 mRNA的共同注射成功補救了部分受到影響的變異胚胎
--------------------------------------------------------------------------------45
第五章 參考文獻------------------------------------------------------------46
圖表--------------------------------------------------------------------------------51

圖表目錄
圖一 斑馬魚ptp4a與果蠅PRL-1胺基酸序列比對示意圖-------------51
圖二 斑馬魚ptp4a3與其他各物種ptp4a3(PRLs)胺基酸序列比對示
意圖------------------------------------------------------------------------52
圖三 演化樹與親緣關係示意圖---------------------------------------------53
圖四 利用原位雜交確認ptp4a3在各時期(一細胞至30小時)斑馬
魚胚胎內的表現位置---------------------------------------------------54
圖五 利用原位雜交確認ptp4a3在各時期(36小時至72小時)斑馬
魚胚胎內的表現位置---------------------------------------------------55
圖六 利用冷凍切片觀察原位雜交後之斑馬魚(36小時)體內訊號正
確位置---------------------------------------------------------------------56
圖七 利用冷凍切片觀察原位雜交後之斑馬魚(48小時)體內訊號正
確位置---------------------------------------------------------------------57
圖八 利用冷凍切片觀察原位雜交後之斑馬魚(72小時)體內訊號正
確位置---------------------------------------------------------------------58
圖九 ptp4a3 morpholino顯微注射及共同注射ptp4a3 mRNA後之斑馬
魚變異外觀--------------------------------------------------------59
圖十 利用gsc、hgg1、ntl RNA探針以原位雜交法觀察斑馬魚胚胎變
異情形-------------------------------------------------------------60
圖十一 利用phalloidin染色法來觀察斑馬魚胚胎上EVL與eYSL變
異情形----------------------------------------------------61
表一 斑馬魚ptp4a1、ptp4a2、ptp4a3與果蠅PRL-1胺基酸序列比對
相同程度百分比---------------------------------------------------------62
表二 斑馬魚ptp4a3與其他各物種ptp4a3(PRL)胺基酸序列比對相
同程度百分比------------------------------------------------------------62
表三 不同劑量之ptp4a3 morpholino顯微注射對斑馬魚胚胎之影響
-------------------------------------------------------------------------62
表四 不同劑量之ptp4a3 morpholino顯微注射對斑馬魚胚胎變異程度
之影響---------------------------------------------------------------------63
表五 ptp4a3 morpholino/mRNA共同注射對斑馬魚胚胎之影響
-----------------------------------------------------------------------63
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