转录因子Foxo1对RAW264.7细胞迁移产生的影响

Effects of transcription factor Foxo1 on migration in RAW264.7 cells

目的观察Foxo1基因过表达对鼠源性单核巨噬细胞RAW264.7中单核细胞趋化蛋白-1(MCP-1)及其受体CCR2表达的影响,探讨转录因子Foxo1在早期心血管疾病识别及预防中的作用。方法实验分为未转染的对照组、Foxo1基因转染组及空质粒转染组,Foxo1基因转染组的转染方法为:运用脂质体转染方法将pc DNA-Foxo1真核表达载体瞬时转染至RAW264.7细胞中。空质粒转染组的转染方法为:运用脂质体转染方法将pc DNA-3.1质粒瞬时转染至RAW264.7细胞中。通过Western blot检测细胞中Foxo1的过表达情况。采用实时定量聚合酶链反应(RT-PCR)及蛋白免疫印迹法分别检测各组细胞中MCP-1及CCR2的表达情况。采用transwell小室法检测各组细胞的迁移能力。结果 pc DNA-Foxo1重组质粒转染48 h后,RAW264.7细胞中Foxo1蛋白的表达量为(1.812±0.067),明显高于空质粒转染组的(1.092±0.088)和未转染组的(1.107±0.095),差异均有统计学意义(均P<0.05)。Foxo1基因转染组RAW264.7细胞中MCP-1 mRNA表达量为(3.331±0.587),明显高于空质粒转染组的(1.125±0.114)和未转染组的(1.121±0.065),差异均有统计学意义(均P<0.05)。Foxo1基因转染组RAW264.7细胞中MCP-1蛋白的表达量为(2.670±0.117),明显高于空质粒转染组的(1.128±0.107)和未转染组的(1.037±0.096),差异均有统计学意义(均P<0.05)。Foxo1基因转染组RAW264.7细胞中CCR2的mRNA的表达量为(2.099±0.331),明显高于空质粒转染组的(1.027±0.095)和未转染组的(1.018±0.177),差异均有统计学意义(均P<0.05)。计数Transwell小室膜下表面细胞数,结果显示Foxo1基因转染组RAW264.7细胞穿过小室的数量为(39.670±2.333)个,明显高于未转染组的(22.000±2.309)个,差异有统计学意义(P<0.05)。结论转录因子Foxo1过表达通过促进MCP-1及CCR2的表达从而增强RAW264.7细胞的迁移,参与巨噬细胞的炎症反应,推测Foxo1在心血管疾病早期识别及预防中具有重要作用。

Objective To investigate the effect of overexpression of Foxo1 gene on the expression of monocyte chemoattractant protein-1（MCP-1） and its receptor CCR2 in murine mononuclear macrophages（RAW264.7）,and to explore the role of transcription factor Foxo1 in early recognition and prevention of cardiovascular disease.Methods pc DNA-Foxo1 eukaryotic expression vector was transiently transfected into RAW264.7 cells by liposome transfection.The transfection methods of empty plasmid group were as follows：the pc DNA-3.1 plasmid was transiently transfected into RAW264.7 cells by liposome transfection.The overexpression of Foxo1 was detected by Western blot.The expression of MCP-1 and CCR2 were detected by real-time quantitative polymerase chain reaction（RT-PCR） and Western blot.The migration ability of each group was measured by transwell chamber assay.Results After transfection of pc DNA-Foxo1 recombinant plasmid for48 h,the expression of Foxo1 protein in RAW264.7 cells was（1.812±0.067）,which was significantly higher than that in blank plasmid group（1.092±0.088） and untransfected group（1.107±0.095）.The difference was statistically significant（all P〈0.05）.The expression of MCP-1 mRNA in RAW264.7 cells of Foxo1 gene transfection group was significantly higher than that of blank plasmid transfection group（1.092±0.088） and untransfected group（1.107±0.095）.（3.331±0.587）,significantly higher than that of empty plasmid transfection group（1.125±0.114） and untransfected group（1.121±0.065）.The expression of MCP-1 protein in RAW264.7 cells of Foxo1 gene transfection group was（2.670±0.117）,significantly higher than that of empty plasmid transfection group（1.128±0.107） and untransfected group（1.037±0.096）.The expression of CCR2 mRNA in RAW264.7 cells of Foxo1 gene transfection group was（2.099±0.331）,significantly higher than that of empty plasmid transfection group（1.027±0.095） and untransfected group（1.018±0.177）,and the difference was statistically significant（all P〈0.05）.The number of submembrane surface cells in Transwell compartment was counted.The results showed that the number of RAW264.7 cells in Foxo1 gene transfected group was（39.670±2.333）,significantly higher than that in untransfected group（22.000±2.309）,the difference was statistically significant（P〈0.05）.Conclusion The overexpression of transcription factor Foxo1 enhances the migration of RAW264.7 cells by promoting the expression of MCP-1 and CCR2,and participates in the inflammatory reaction of macrophages.It is suggested that Foxo1 plays an important role in the early recognition and prevention of cardiovascular disease.