应用抑制消减杂交和cDNA表达谱芯片筛选肝星形细胞持续活化的相关基因

Combination of suppresion subtractive hybridization and cDNA microarray for identification of the hepatic stellate cell continual activation related genes

目的:利用抑制消减杂交(suppression subtractive hybridization,SSH)和cDNA表达谱芯片筛选早期活化的肝星状细胞与大鼠正常肝脏组织、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构建的大鼠轻度和重度肝纤维化中两个差异cDNA文库中挑选1 000条上调显著的基因,与正常大鼠4 136条基因克隆制作成一张芯片,筛选持续期活化的肝星状细胞相关基因。结果:获得与HSC持续活化相关的上调基因633条,下调基因715条,其中血清和糖皮质激素调节蛋白激酶(SGK)在正常大鼠基因克隆和2、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。

Objective To screen and identify the differentially expressed genes in the continually activated hepatic stellate cells, the normal hepatic tissues, the mild liver fibrosis tissues, as well as the serious liver fibrosis tissues using suppression subtractive hybridization （SSH） and cDNA microarray. Methods One cDNA chip was made by gathering clones of three differentially expressed cDNA libraries derived from normal hepatic tissues, mild liver fibrosis tissues and serious liver fibrosis tissues. Then the clones were hybridized with cDNA probes extracted from the continually activated hepatic stellate cells. Results Six hundred and thirty-three cDNAs that showed up-regulated and 715 cDNAs that showed down-regulated were obtained. It demonstrated that serum and glucocorticoid sensitive kinase （SGK） gene was up-regulated in the normal hepatic tissues, the mild liver fibrosis tissues and the serious liver fibrosis tissues. Conclusions The combination of SSH and cDNA microarray is rapid and effective in screening and identification of the differentially expressed genes in different samples. SGK gene seems to play a key role in the continual activation of hepatic stellate cells.