联合应用SSH和cDNA表达谱芯片筛选肝星状细胞早期活化的相关基因

目的利用抑制消减杂交(suppressionsubtractivehybridization,SSH)和cDNA表达谱芯片筛选早期活化的肝星状细胞(hepaticstellatecell,HSC)和大鼠正常肝脏组织、轻度肝纤维化组织、重度肝纤维化组织中差异表达的基因。方法从SSH构建的大鼠轻度肝纤维化、重度肝纤维化2个差异cDNA文库中挑选1000条上调显著的基因,与正常大鼠4136条基因克隆制作成一张芯片,筛选早期活化的肝星状细胞相关基因。结果获得与HSC早期活化相关的上调基因有202条,下调基因有80条,其中血清和糖皮质激素调节蛋白激酶(serumandglucocorticoidsensitivekinase,SGK)在正常大鼠基因克隆和2周及8周SSH上调差异基因中均呈上调信号。结论联合应用SSH和cDNA表达谱芯片是筛选和鉴定不同样本中差异表达基因的快速、经济和有效的方法;SGK可能作为多种细胞信号传导通路和细胞磷酸化级联反应的一个功能性交汇点,参与了肝星状细胞的早期活化和信号传导。

Identification of initiative activation related genes of hepatic stellate cell by combining SSH and cDNA microarray

Objective To identify initiative activated genes of hepatic stellate cell in normal hepatic tissues,slight liver fibrosis tissues and severe liver fibrosis tissues by using suppression subtractive hybridization (SSH) and cDNA microarray. Methods Initiative activated genes of hepatic stellate cell was identified by selecting 1 000 genes with obvious up-regulation from two cDNA libraries of slight liver fibrosis and severe liver fibrosis to clone with 4 136 genes from normal rats for making a chip. Results We obtained 202 cDNAs with up-regulated expression and 80 cDNAs with down-regulated expression. The glucocorticoid sensitive kinase (SGK) gene showed to be up-regulated among normal hepatic tissues,slight liver fibrosis tissues and serious liver fibrosis tissues. Conclusions The combination of SSH and cDNA microarray is a rapid and effective method to screen and identify differentially expressed genes in different samples. As a functional converge site for signal transduction pathway of many cells and cascade reaction of cell phosphorylation,SGK gene plays a key role in initiative activation and signal transduction of hepatic stellate cells.