Rac1对 L02肝细胞株上皮细胞间质转化及增殖和凋亡的影响

摘要

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 of 5ng/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 Rac1G12V 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 Rac1T17N 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.