人胚胎造血的agm区基质细胞基因表达谱分析.docVIP

人胚胎造血的AGM区基质细胞基因表达谱分析 nbsp;nbsp;nbsp;nbsp;nbsp;nbsp;nbsp; 作者：付劲蓉，陈惠芹，张绪超，黄绍良，周敦华，黄科 【摘要】nbsp; 　　为了筛选在主动脉-性腺-中肾（AGM）区基质细胞中差异表达的基因，以药物流产的孕30-35天的人胚胎AGM区来源基质细胞为“实验方”，以源自意外而致流产的孕4-5月的水囊引产的胎儿肝组织的基质细胞为“驱动方”，建立了在人AGM区基质细胞中差异表达基因的消减杂交文库。进一步用基因芯片技术对所建立的抑制消减杂交文库进行筛选，并挑选其中明显差异表达的18个克隆进行序列测定和同源性分析。结果表明： 在所构建的AGM抑制消减杂交文库中，经过PCR鉴定有特异性扩增带且扩增带分子量在200-700 bp的克隆有211个，阳性率高达76.4％。经过基因芯片筛选，挑选出ratio值大于5的克隆共18个进行序列测定显示，在测序的18个明显差异表达的阳性克隆中，4个为未知基因，14个为已知基因，其中这些已知基因分别参与了细胞迁移、分化、生长、细胞周期、信号转导及血管发生等细胞重要生命过程。这些基因大多数在胚胎分化造血发育中的作用尚未见报道。结论 　筛选AGM区基质细胞中差异表达的基因为进一步研究胚胎造血发育的分化调控的分子机理提供了必要的理论基础。 【关键词】nbsp; AGM区；胎肝；基质细胞；抑制消减杂交；基因芯片 　　Screening andnbsp; Cloning the Genes Differentially Expressed in Human Embryonic AGM-Derived Stromal Cells 　　Abstractnbsp; To screen and separate the genes differentially expressed in human embryonic aorta-gonad-mesonephros(AGM)-derived stromal cells，a subtracted library was generated through the suppression subtractive hybridization using the cDNAnbsp; of human embryonic AGM-derived stromal cells as target and human fetal liver(FL)-derived stromal cells as drivers. Then a high though screening technique，gene chip，was used to screen the differentially expressed genes in the established subtractive library. Approximately 18 of the resulting subtracted cDNA clones were partially sequenced and analyzed by blastn in the GenBank database. The results showed thatnbsp; 211 Clones were selected and identified from the established subtractive library，the positive ratio was amount to 76.4%. 18 over-expressed genes were screened by gene chip with more than anbsp; 5-fold difference expression levels between AGM and FL-derived stromal cells，and were selected to sequence，results of sequencing indicated thatnbsp;nbsp; the 18 sequences was compared to known sequences in the GenBank database， and among the sequenced clones，14 sequences were considered as part of the known genes，and 4 sequences representing previously unknown genes. The known gens were repo