心脏圆锥动脉干发育相关基因的筛选研究

Screening of Cardiac Conotruncus Developing Related Genes

目的检测剔除心脏神经嵴心脏组织与正常心脏组织之间表达差异基因,筛选心脏圆锥动脉干发育相关基因。方法用电刺激方法建立鸡胚实验模型;用抑制消减杂交法筛选出实验组和对照组心脏组织之间存在的差异表达基因;构建减数杂交文库,用cDNA基因芯片技术验证,并进行测序。结果31.8%克隆在实验组表达显著减少。经检验确定13种不同基因片段,有7种与已知鸡基因序列完全匹配,分别为心脏αactin、DEADboxRNA解旋酶、核糖体蛋白L7a、线粒体基因组、GAPDH、肽延长因子-1;有4种基因片段与人、鼠等种属已知基因存在83%～89%的同源性,分别为核糖体蛋白S3、L10a、SNX1、CIRBP等;有2种在公共数据库未查到同源序列。结论有多种基因参与心脏圆锥动脉干胚胎发育;抑制消减杂交和基因芯片技术是筛选差异表达基因的有效方法。

Objective To determine the conotruncus developing related genes by way of discovering differentially expressed messages between the cardiac neural crest ablated heart and the normal heart during development. Methods Experimental chick embryos were established by using electric stimulation. Suppression subtractive hybridization (SSH) was used to discover messages differentially expressed between two groups. A subtractive library was constructed, and cDNA array was used to identify significant difference, and then sequencing was done. Results 31.8% clones were identified to be significantly decreased in expression in the experimental group. Thirteen kinds of gene fragments were identified. Seven were known as Gallus genes, which were cardiac α-actin, DEAD-box RNA helicase, ribosomal protein L7a, mitochondrion genes, GAPDH, peptide elongation factor-1; 4 gene fragments were homologous to known genes of human sapiens and mouse with homogeneity of 83%~89%, which were ribosomal protein S3, L10a, SNXI, CIRBP, etc; 2 gene fragments could not be identified to be homologous to any known sequence in the public database. Conclusion Many kinds of genes take part in cardiac conotruncus development. SSH and cDNA array are efficient methods to discover the differential expressed genes.