An Jun-yan, Zhang Xiao-lan, Yao Dong-mei, Dun Zhi-na, Xie Shu-rui, Hao Li-sen
Department of Gastroenterology, the Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Shijiazhuang 050000, China.
Zhonghua Gan Zang Bing Za Zhi. 2009 Jul;17(7):509-14.
To investigate the role of focal adhesion kinase (FAK) in adhesion and migration of hepatic stellate cells (HSC).
Two recombinant plasmids expressing short hairpin RNAs (shRNAs) targeting FAK were constructed and one plasmid substantially suppressing FAK expression in HSC was selected. Real-time PCR and Western blot were used to detect the knockdown effects of FAK gene. After 48-hour treatment with FAK shRNA, toluidine blue colorimetric assay was used to detect the cell adhesion. Wound-healing assay and improved Boyden double-chamber were used to detect the cell migration induced by FN.
The recombinant plasmid expressing FAK shRNA was successfully constructed and transfected into HSC. Compared with the controls, the expression of FAK mRNA and protein in HSC treated with FAK shRNA was markedly down-regulated by 76.82% and 72.53%, respectively. The expression of p-FAK (Tyr397) protein was also decreased by 62.71% 48 h posttransfection. The adhesion of HSC was inhibited by 58.69% at 48 h after shRNA transfection. FAK gene silencing could also dramatically inhibit FN-stimulated HSC migration, and the cell migration distance and the cell number of crossing membrane were decreased by 58.27% and 83.70%, respectively.
FAK gene silencing suppresses adhesion and migration of HSC, and FAK may be a potential target for novel anti-fibrosis therapies.
探讨粘着斑激酶（FAK）在肝星状细胞（HSC）粘附及迁移中的作用。
构建两种靶向FAK的短发夹RNA（shRNA）表达重组质粒，筛选出一种能有效抑制HSC中FAK表达的质粒。采用实时定量PCR和蛋白质印迹法检测FAK基因的敲低效果。用FAK shRNA处理48小时后，采用甲苯胺蓝比色法检测细胞粘附情况。采用划痕实验和改良的Boyden双室法检测FN诱导的细胞迁移。
成功构建了表达FAK shRNA的重组质粒并转染至HSC。与对照组相比，用FAK shRNA处理的HSC中FAK mRNA和蛋白的表达分别显著下调76.82%和72.53%。转染后48小时，p-FAK（Tyr397）蛋白表达也降低了62.71%。shRNA转染后48小时，HSC的粘附受到58.69%的抑制。FAK基因沉默也能显著抑制FN刺激的HSC迁移，细胞迁移距离和穿膜细胞数分别减少了58.27%和83.70%。
FAK基因沉默抑制HSC的粘附和迁移，FAK可能是新型抗纤维化治疗的潜在靶点。
