Rac1对L02肝细胞株上皮细胞间质转化及增殖和凋亡的影响

Effects of Rac1 on epithelial-mesenchymal transition,and proliferation and apoptosis of L02 cells in vitro

目的检测Rac1在TGF-β1诱导的L02细胞上皮间质转化中的作用,及其对细胞增殖和凋亡的影响。方法应用不同活性的Rac1质粒pExRed-NLS Flag(空载体组)、pExRed-NLS Flag Rac1(野生型Rac1组)、pExRed-NLS Flag Rac1T17N(显性负调控Rac1组)、pExRed-NLS Flag Rac1G12V(持续活化型Rac1组)瞬时转染L02细胞,经5 ng/ml TGF-β1处理细胞。采用免疫印迹法检测融合蛋白Flag-Rac1表达,采用细胞免疫荧光及免疫印迹法检测Ck8和Vimentin表达,采用细胞划痕实验及Transwell法检测细胞迁移能力。使用不同浓度的Rac1特异性抑制剂NSC23766处理L02细胞,采用CCK-8法检测细胞增殖,采用Annexin V-FITC/PI双染法检测细胞凋亡。结果四组质粒均成功瞬时转染到L02细胞中;与空载体组和野生型Rac1组比,持续活化型Rac1转染细胞Vimentin蛋白表达水平显著增高,CK8蛋白表达水平降低,细胞迁移能力增加;与空载体组和野生型Rac1组比,显性负调控Rac1转染细胞Vimentin蛋白表达水平降低,CK8蛋白表达水平增高,细胞迁移能力降低(P<0.05);在NSC23766处理L02细胞后,细胞增殖被抑制,但各处理组细胞凋亡无明显差异。结论 Rac1可促进TGF-β1诱导的L02肝细胞株上皮间质转化和细胞增殖,但对细胞凋亡无明显影响。

Objective To investigate the effects of Rac1 on epithelial mesenchymal transition（EMT）induced by transforming growth factor β1（TGF-β1）,and on cell proliferation and apoptosis of L02 cells in vitro.Methods Rac1 plasmids with different activity including pExRed-NLS Flag（vector）,pExRed-NLS Flag Rac1（wild type）,pExRed-NLS Flag Rac1T17N（dominant negative mutant） and pExRed-NLS Flag Rac1G12V（constitutively active mutant） were transiently transfected into L02 cells,followed by stimulation of exogenous TGF-β1 at dose of5ng/ml;Exogenous Flag-Rac1 fusion protein was determined by Western blot analysis;Immunofluorescence and Western blot were used to evaluate epithelial markers of Ck8 and mesenchymal markers of vimentin;Cell motility was assessed by transwell assay and wound healing assay;L02 cells were treated with different concentrations of Rac1 inhibitor（NSC23766）,and the influence of Rac1 on cell proliferation and apoptosis were detected by CCK-8 assay or Annexin V-FITC/PI double staining,respectively. Result All kinds of plasmids were successfully transiently transfected into L02 cells;Compared with the vector group and the wild type group,constitutively active mutant pExRed-NLS Flag Rac1G12 V significantly increased the expression of vimentin,decreased the expression of CK8 and enhanced cell motility,whereas the effects of dominant negative mutant pExRed-NLS Flag Rac1T17 N were just the opposite of above results（P 0.05）;Disruption of Rac1 activity with NSC23766 inhibited cell proliferation（P〈0.05） without increasing cell apoptosis. Conclusions Rac1 promotes the EMT process and cell proliferation induced by TGF-β1 in L02 cells without affecting cell apoptosis in vitro.