Department of Physiology, School of Medicine, CHA University, Seongnam, Gyeonggi, Republic of Korea.
Pancreas. 2011 Mar;40(2):271-80. doi: 10.1097/mpa.0b013e3182003335.
This study was aimed to investigate important proteins associated with endoderm differentiation by pancreatic derivation protocol from human embryonic stem cells (hESCs).
Comparative proteomic analysis of endoderm cells differentiated from hESCs by activin A and low serum was performed. Proteins with altered expression levels during endoderm differentiation were investigated by 2-dimensional gel electrophoresis (2-DE) with mass spectrometric analysis.
Thirty-four protein spots with significantly changed intensities were identified. These were functionally annotated based on gene ontology. The messenger RNA expression levels of 5 genes, APC, CCNB3, HSPA9, CCT2, and YWHAE, were correlated with 2-DE analysis. We further validated the protein expression levels of adenomatous polyposis coli (APC) and cyclin B3 (CCNB3) by using Western blot analysis and immunocytochemistry. They are involved in the regulation of cell cycle, thus, cyclins and cyclin-dependent kinases, which regulate the cell cycle, were examined. Cyclin A1, cyclin D2, and cyclin E2 were upregulated, and other cyclins and cyclin-dependent kinases were downregulated in endoderm cells.
The increase in expression of APC and CCNB3 suggests that these proteins will be important markers for identifying endoderm cells differentiated from hESCs, and they can play important roles in the differentiation of endoderm cells from hESCs or in human endoderm development for pancreas.
本研究旨在通过人胚胎干细胞（hESC）的胰腺衍生方案，研究与内胚层分化相关的重要蛋白。
通过激活素 A 和低血清诱导 hESC 分化为内胚层细胞，进行比较蛋白质组学分析。通过二维凝胶电泳（2-DE）和质谱分析，研究内胚层分化过程中表达水平发生变化的蛋白质。
鉴定出 34 个蛋白斑点，其表达强度显著改变。根据基因本体论对这些蛋白进行功能注释。5 个基因（APC、CCNB3、HSPA9、CCT2 和 YWHAE）的信使 RNA 表达水平与 2-DE 分析相关。我们进一步通过 Western blot 分析和免疫细胞化学验证了腺瘤性结肠息肉病基因（APC）和细胞周期蛋白 B3（CCNB3）的蛋白表达水平。它们参与细胞周期的调节，因此，我们检查了调节细胞周期的细胞周期蛋白和细胞周期蛋白依赖性激酶。细胞周期蛋白 A1、细胞周期蛋白 D2 和细胞周期蛋白 E2 上调，而其他细胞周期蛋白和细胞周期蛋白依赖性激酶下调。
APC 和 CCNB3 表达增加表明这些蛋白将成为鉴定 hESC 分化的内胚层细胞的重要标志物，它们可能在 hESC 分化为内胚层细胞或人类内胚层发育为胰腺中发挥重要作用。
