| 摘要: | 人體的胚胎幹細胞是一種具分化成各種不同細胞類型且同時具有再生能力的多能性細胞(pluripotency)，所以胚胎幹細胞在再生醫學以及開發藥物的應用上有很大的潛力。膜蛋白質已經被發現與調控胚胎幹細胞有關係，可以廣泛地應用在幹細胞的分類和純化或者可以用來監測幹細胞分化的階段。例如:人體胚胎幹細胞的表面蛋白Notch受體，經研究報導指出當Notch受體被活化時，Notch受體被酵素切割後，位於細胞質內的片段會從細胞膜易位到細胞核並且進一步的造成胚胎幹細胞的朝向神經細胞分化。因此，在胚胎幹細胞的細胞膜、細胞質以及細胞核等次細胞蛋白質體進行定量分析，比較胚胎幹細胞分化前後的蛋白質表現亮差異，不僅可以幫助鑑定新的表面蛋白標誌，也可能找到關於調控胚胎幹細胞功能的分子。在此研究上，由於培養胚胎幹細胞並維持胚胎幹細胞的未分化是很困難的，所以在蛋白質體的分析上，我們所能得到的胚胎幹細胞數量是有限的。
為了克服這項限制，此論文發展一個靈敏的定量策略，結合微量樣品的分群方法(fractionation)和蛋白質體的定量分析。目前已發表一項分群技術StageTip (Stop and Go extraction tips)，此技術提供高效率、高回收率以及高重複性的少量樣品分離。我們分析了強陰離子交換與強陽離子交換的StageTip 分群方法，在經由強陽離子交換分離後蛋白質被鑑定的數量從445(沒有分群的結果)增加到765；利用強陰離子交換分離則能將蛋白質數量增加到902個，其中有623 (59.7 %) 蛋白質是強陽離子交換分離與強陰離子交換分離共同能鑑定到的，而強陰離子交換分離能獲得比強陰離子交換分離多出17.9 %的蛋白質數量。接著，我們使用強陰離子交換分離結合等重相對標記絕對定量(iTRAQ)策略並應用在胚胎幹細胞的次細胞蛋白質體分析。結果顯示有6236、4234以及2061個蛋白質在細胞核、細胞質與細胞膜的部分被鑑定到；其中，有3125、1408與865個蛋白質分別在細胞核、細胞質與細胞膜能成功得到分化前後的蛋白質變化量。在具有異常變化量的細胞膜蛋白質中，有部分蛋白質已經有文獻報導過是胚胎幹細胞或分化後的標記分子。除此之外，還有192個表面膜蛋白具有差異表現量，可能是潛在的胚胎幹細胞和分化後的標記分子。我們也觀察到有差異表現的蛋白質會在不同的細胞位置可能發生易位，例如 -catenin 從細胞質易位到細胞核時，可以調控胚胎幹細胞的多能性。因此，我們的策略不僅能幫助發現專一的胚胎幹細胞生物標記，也能勾勒出胚胎幹細胞的再生機制與分化機制。
Human embryonic stem cells (hESCs) are pluripotent cells that are uniquely capable of differentiation and self-renewal and have a great potential in regenerative medicine and drug discovery. Membrane proteins have been investigated to play important roles in regulating ESC functions and expected to be extensively used for stem cell classification and purification and monitored of the differentiation stages. For example, the NOTCH receptor, a cell surface protein of hESCs, will translocate from membrane to nucleus when it is activated and further differentiated toward the neural fates. As a result, we hypothesize that a comprehensive quantitative analysis of the subcellular proteome, including membrane, cytosolic and nuclear fractions between hESCs and differentiated cells, will help to identify not only new cell surface markers but also molecules involving in regulation of ES functions. However, due to the difficulties in maintenance and culturing of hESCs, very limited cell number can be obtained for proteome analysis.
To overcome this challenge, we aim to develop a sensitive quantitation strategy integrating a micro-fractionation method for quantitative proteomic analysis of the low sample amount of stem cells. StageTips (Stop and Go Extraction Tips) have been reported to provide separation of a few micrograms of sample with high efficiency, recovery and reproducibility. After StageTip fractionation, the number of protein increased from 445 to 765 and 902 by using SCX-StageTip and SAX-StageTip fractionation respectively. In addition, we compared the SAX-StageTip and SCX-StageTip using membrane proteins purified from HeLa cells. A total of 623 proteins (59.7 %) are commonly identified. Regarding the identification number, SAX-StageTip fractionation can obtain 17.9% higher number than SCX-StageTip fractionation. We applied SAX-StageTip fractionation with iTRAQ quantitation strategy on hESC subcellular proteomics. 6236, 4234 and 2061 proteins were identified, of which 3125, 1408 and 865 proteins were successfully quantified in nucleus, cytosol and membrane fraction, respectively. Several differentially expressed proteins had been reported as specific hESC markers and EB markers. In addition, there were 192 differentially expressed plasma membrane proteins which may be considered as potential hESC markers or EB markers. Several differentially expressed proteins would translocate from different cellular localization such as -catenin which have been reported to regulate hESC pluripotency with translocation from cytoplasm into nucleus. Our strategy can not only help to discover the specific marker candidates for hESC but may delineate the mechanism of self-renewal and differentiation. |
