关键词: 肝星状细胞, 第10号染色体缺失的磷酸酶和张力蛋白同源物基因, RNA干扰, 细胞粘附

Abstract: Objective To investigate the down regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene by adenovirus mediated short hairpin RNA ( shRNA) on the adhesion in activated hepatic stellate cells（HSC）in vitro and the related signal transduction mechanism. Methods The recombinant adenovirus (Ad-shRNA/PTEN) with shRNA targeting PTEN and expressing green fluorescent protein (GFP) were transient transfected into the cultural activated HSC in vitro. The experimental group as follows: 1) Control group, viral medium was replaced by DMEM at virus transfection step. 2) Ad-GFP group, HSC were infected with adenovirus expressing GFP alone. 3) Ad-shRNA/PTEN group, HSC were infected with adenovirus both taking shRNA targeting PTEN and expressing GFP. PTEN mRNA expression was detected by real-time fluorescent quantitation PCR, and western blot was used for detecting protein expressions of PTEN, focal adhesion kinase (FAK) and phosphorylated FAK (Thr397) ［p-FAK（Tyr397）］in HSC. The toluidine blue stain method and MTT colorimetric method were used to determine the adhesion ability of HSC. Results When HSC were infected by adenovirus for 48 hours, PTEN protein and mRNA expressions in Ad-shRNA/PTEN group significantly decreased (P＜0.05), compared to control group and Ad-GFP group, and the expressions of p-FAK (Tyr397) in Ad-shRNA/PTEN group were significantly higher than those in control group and Ad-GFP group (P＜0.05). The adhesion cell number and the adhesion rate of HSC in Ad- shRNA/PTEN group significantly increased, compared with control group and Ad-GFP group (P < 0.05). Conclusions The down-regulation of PTEN expression can promote the adhesion by increasing the activation of FAK signaling transduction in activated HSC in vitro.

Key words: hepatic stellate cells, Phosphatase and tension loss of chromosome 10 protein homologue gene, RNA interference, Cell adhesion