c-Fos基因RNA干扰对大鼠血管平滑肌细胞表型及迁移的影响

摘要

Objective To investigate the influence of RNA interference targeting c-Fos gene on the phenotypic transition and migration of vascular smooth muscle cells (VSMCs). Methods VSMCs were transfected with siRNA targeting c-Fos gene by liposome. Effects of c-Fos siRNA on mRNA expression of c-Fos and α-SM-actin were examined by RT-PCR while the levels of a-SM-actin, bFGF and ET-1 were determined by Western blot. We observed the differentiation of VSMCs by immunofluorescence and examined the migration ability of VSMCs with a modified Boyden-chamber assay simultaneously. Results After transfected with siRNA targeting c-Fos gene, expression of c-Fos was decreased compared to the control groups (normal control and negative siRNA groups). The expression and mRNA level of α-SM-actin were increased in the c-Fos siRNA-transfected VSMCs. The shape of c-Fos siRNA-transfected VSMCs turned into spindle-like. C-Fos siRNA slowered VSMC migration and reduced the number of migrating VSMCs. Expressions of bFGF and ET-1 were significantly decreased after c-Fos siRNA transfection. Conclusion c-Fos gene silence through RNA interference can induce the phenotypic transition of VSMCs and inhibit their migration.%目的 以RNA干扰抑制血管平滑肌细胞(VSMC)c-Fos基因,探讨c-Fos基因表达抑制后c-Fos siRNA对平滑肌细胞表型转换及迁移的作用.方法 设置正常对照组、阴性siRNA组及阳性c-Fos siRNA转染组.应用RT-PCR半定量法检测VSMC中c-Fos和α平滑肌肌动蛋白(α-SM-actin )mRNA的水平,采用Western blot检测α-SM-actin、碱性成纤维细胞生长因子(bFGF)、内皮素1(ET-1)蛋白活性的变化,应用免疫荧光染色检测平滑肌分化标志蛋白α-SM-actin,同时应用Transwell检测平滑肌细胞迁移能力.结果 siRNA转染后阳性c-Fos siRNA转染组c-Fos基因表达水平降低,而正常对照组与阴性siRNA组比较c-Fos基因表达水平无统计学差异.siRNA转染使c-Fos表达减弱后,VSMC中α-SM-actin mRNA和蛋白表达较正常对照组及阴性siRNA组显著上调,bFGF、ET-1表达减弱,VSMC迁移速度减慢.结论 RNA干扰介导的c-Fos基因沉寂可在诱导VSMC分化的同时抑制VSMC迁移.