胚胎干细胞分化为表皮样细胞的基因表达谱差异研究

 摘 要] 目的：用全基因表达谱芯片技术筛选小鼠胚胎干细胞（ESC）定向分化为表皮样细胞(Epidermal-like cells, ELC)的差异表达的基因并对其进行分析，以进一步阐明ESC分化为ELC的分子机制。方法：利用人羊膜建立体外诱导体系，将小鼠ESC诱导定向分化为ELC，做3次生物学重复，分别取未分化的ESC和诱导分化的ELC提取总RNA进行扩增、Cy3标记，与NimbleGen 135k 小鼠全基因表达谱芯片（含44170个基因）杂交，荧光扫描，筛选出差异表达基因，并进行GO分析和pathway分析，用实时定量PCR对结果进行验证。结果：芯片筛选结果显示，分化后的ELC与ESC间的差异表达的基因多达4856个（Cut off：2）。基因本体（Gene Ontology，GO)分析显示与生物学过程（Biological Process,BP）相关的差异表达基因最多的是细胞过程（cellular process，CP），1931个；与亚细胞组分（Cellular Component, CC）相关的差基因中最多的是细胞和细胞组分（Cell, Cell part）相关基因，2391个；与分子功能（Molecular Function, MF）相关的差异基因中最多是的结合功能(binding)基因，1869个。Pathway分析显示，与DNA复制（DNA replication）通路相关的差异基因有23个；与蛋白酶体（Proteasome）通路相关的24个；剪接体（spliceosome）通路相关有47个；细胞周期（Cell cycle）相关的有44个。结论：ESC体外诱导定向分化为ELC过程中，基因表达谱发生了巨大的变化，提示细胞分化是一个众多基因参与的复杂的生物学过程，这些基因在分化过程所起的作用和作用机制尚需大量的实验研究阐明。

Genome-wide Expression Profiles Analysis of ES Cells and ES Derived Epidermal-like Cells

【Objective】 To screen and analyze different expressed genes between mouse embryonic stem cells(ESC) and their differentiated epidermal-like cells(ELC) with the whole genomic expression profile chip,and explore the molecular mechanisms of differentiation further.【Methods】 Mouse ESC was induced differentiation into ELC in vitro by human amnion repeated for three times.Total RNA of ESC and ELC was extracted,and then amplified by RT-PCR.The product cDNA was labeled with Cy3 and hybridized on NimbleGen 135k mouse whole gene chip(containing 44,170 genes).Fluorescence scanning,Gene Ontology(GO) and pathway analysis were used to screen the different expressed genes.The analytic result was validated by Real-time Quantitative PCR.【Results】 A total of 4,856 genes showed differentially expressed between ESC and ELC(Cut off: 2).GO analysis showed that the differential expressed genes between ESC and ELC mainly involved cellular process(CP) genes of 1931,Cell/Cell part genes of 2391,and binding function genes of 1869,which belong to biological process(BP),cellular component(CC),and molecular function(MF),respectively.The pathway analysis showed that the differential expressed genes mainly involved genes related to DNA replication,proteasome pathway,spliceosome pathway,and cell cycle with the number of 23,24,47,and 44,respectively.【Conclusion】 The change of expression profile between ESC and ELC indicated that large variety of genes were involved in this complex cellular differentiation process,and how these gene worked and co worked to regulate the differentiation procedure need further investigations.