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博碩士論文 etd-0804105-114954 詳細資訊
Title page for etd-0804105-114954
論文名稱
Title
豬胚胎幹細胞分化成神經細胞之發育因子的蛋白體研究
Proteomic studies on development factors of pig embryonic stem cells into neural cells by RA in vitro
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
81
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2005-06-27
繳交日期
Date of Submission
2005-08-04
關鍵字
Keywords
神經細胞、蛋白質體、視網酸、胚胎幹細胞
MALDI-TOF, vimentin, prohibitin, neural cells, ES cells, oct3/4, annexin A10, beta-catenin, neurod1, ZNF482, RA, neurogenin2, annexin A1, proteomic, sox2, FRK
統計
Statistics
本論文已被瀏覽 5675 次,被下載 1935
The thesis/dissertation has been browsed 5675 times, has been downloaded 1935 times.
中文摘要
在本論文中,我們使用研究蛋白質體的技術,分析在豬的胚胎幹細胞 (ES cells) 在分化成類神經細胞 (Neural-like cells) 早期的蛋白質表現情形。豬的胚胎幹細胞由行政院農委會畜產試驗所提供,在離體情形下培養增生,並且加入視網酸 (Retinoic acid)使其分化,我們選擇未分化的豬胚胎幹細胞及加入視網酸後第1、2、4、8 、10 天的豬胚胎幹細胞,利用二維電泳 (Two-dimensional electrophoresis; 2-DE) 分析差異性的蛋白質,再使用基質輔助雷射脫附游離飛行時間 (Matrix-assisted laser desorption ionization-time of flight; MALDI-TOF) 質譜分析,經由MS-Fit工具比對得知蛋白質身分。我們建立了豬胚胎幹細胞的2DE 資料庫,並且選出了六個在早期神經分化過程中,可能參與分化的蛋白質。其中vimentin,prohibitin 和 annexin A10的表現量增加;而 zinc finger protein 482 (ZNF482),fyn-related kinase (FRK) 和 annexin A1的表現量減少。這些差異性的蛋白質也許是影響早期神經分化的重要因子,或是可以成為早期神經分化的標誌。除此之外,利用RT-PCR 分析 mRNA 的表現量,vimentin 和 prohibitin的表現量也增加,但是 anxa1(annexin A1) 則沒有明顯變化。另外,neuroD1和 neurogenin 2在第10天有高表現量,而beta-catenin 在第8天到第10天有高表現量。
Abstract
Proteomic techniques were used to analyze the protein expression profile of the early-stage differentiation of pig embryonic stem cells (ES cells). The pig ES cells were induced to develop to neuronal cells by all-trans retinoic acid (ATRA) in vitro by Tainan Livestock Research Institute. The ES cells were cultured with ATRA and collected at time intervals of 0, 1, 2, 4, 8 and 10 days. The cell lysates were analyzed by two-dimensional electrophoresis, and the differentially expressed proteins are identified by MALDI-TOF. Our data shows that the expression profile of pig ES cells is similar to other mammalian models but with some differences. Preliminary pig ES cells 2D database was set up. Six spots each with up or down-regulation in neurogenesis were identified by MS. These proteins may become the good markers of pig ES cells into neural cells by RA. Among those proteins, vimentin, prohibitin and annexin A10 were up-regulated, zinc finger protein 482 (ZNF482), fyn-related kinase (FRK) and annexin A1 were down-regulated during differentiation of pig ES cells to neural cells. Addtionally, we ultilized RT-PCR technique to investigate mRNA expression during neurogenesis, vimentin and prohibitin was up-regulated, anxa1(annexin A1) was slightly down-regulated, neuroD1 and neurogenin 2 were high expression on day 10, beta-catenin was high expression on day 8 to 10.
目次 Table of Contents
Abstract in Chinese……………………………………………………………… i
Abstract in English……………………………………………………………… ii
Abbriviation……………………………………………………………………... iii
1. Introduction
1.1 Proteomics…………………………………………………………….. 2
1.2 ES cells………………………………………………………………... 3
1.3 Applications of ES cells……………………………………………….. 4
1.4 Retinoic acid (RA)…………………………………………………….. 5
1.5 Functions of CRABPs……………………………………………….... 6
1.6 Functions of RAR and RXR………………………………………….. 7
1.7 Differentiation techniques of ES cells into neural cells………………. 8
1.8 RA induction versus lineage selection………………………………... 9
1.9 Aim of study………………………………………………………….. 11
2. Material and methods
2.1 ES cells Culture……………………………………………………….. 13
2.2 Differentiation of ES cells…………………………………………….. 13
2.3 Protein extraction……………………………………………………… 13
2.4 Two-dimensional electrophoresis (2DE)
2.4.1 First-dimension (IEF)……………………………………... 13
2.4.2 Second-dimension (SDS-PAGE)………………………….. 14
2.5 Protein visualization and image analysis
2.5.1 Silver-staining of 2-D gels (Analytical gels)……………… 14
2.5.2 Coomassie blue-staining of 2-D gels (Preparative gels)…... 15
2.5.3 Image acquisition of 2-D gels……………………………... 15
2.6 Protein isolation and identification by MS
2.6.1 In gel digestion……………………………………………. 15
2.6.2 MALDI-TOF analysis…………………………………….. 15
2.6.3 Peptide mass fingerprinting (PMF)……………………….. 16
2.7 RNA isolation and RT-PCR…………………………………………… 16
2.8 Western blot analysis………………………………………………….. 16
3. Results
3.1 Comparison of proteomic patterns in pig ES-differentiated cells by silver -stained 2-DE………………………………………………………..
19
3.2 Preparative gels of pig ES cells……………………………………….. 20
3.3 Identification of proteins associated with early-stage differentiation of ES cells into neural cells by PMF…………………………………………
20
3.4 RT-PCR analysis in early-stage pES neurogenesis…………………… 20
3.5
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