AIM: To study the molecular mechanisms of cellular repressor of E1A - stimulated genes ( CREG ) on the proliferation of human vascular smooth muscle cells ( VSMCs ) in vitro.METHODS: The pLNCX2 - CREG plasmid and the pSM2 - siCREG plasmid were transfected into VSMCs to produce the cell clones of over - expression and down - expression of CREG, respectively.BrdU assay and FACS cell cycle analysis were performed to detect the proliferation of the cells.The expression and localization of insulin - like growth factor Ⅱ receptor( IGF2R ) in the hVSMCs were detected by Western blotting and immunocytochemistry.The expression and secretion of insulin - like growth factor Ⅱ ( IGFⅡ ) were measured by RT - PCR and ELISA.Alexa 488 - labeled rhIGFⅡ was used to investigate the endocysis of the cells.The blockade of IGFⅡ internalization was conducted by treating the cells with both neutralized antibody of IGF2R and recombinant IGF2R peptide to detect the effect of IGFⅡ on HVSMCs growth.Furthermore, Western blotting and signal pathway inhibitor were used to analysis the activation of PI3K/Akt and ERK on VSMCs proliferation.RESULTS: Compared with the control cells, Western blotting identified that the expression of CREG was increased in VSMCs - CREG cells and was decreased in VSMCs - siCREG cells.Meanwhile, the over - expression of CREG in the cells inhibited the proliferation of VSMCs and enhanced the distribution of IGF2R in cellular membrane.Furthermore, over - expression of CREG also accelerated the endocytosis of IGFⅡ in VSMCs - CREG cells, and attenuated the secretion of IGFⅡ into cell medium.Blockade experiments showed that enhancement of IGFⅡ secretion promoted the proliferation of HVSMCs.PI3K/Akt and ERK signal pathways mediated the effect of IGFⅡ on the VSMCs.CONCLUSION: CREG inhibits the proliferation of VSMCs through interfering with the internalization of IGF2R - IGFⅡ.%目的:探讨E1A激活基因阻遏子(CREG)抑制人血管平滑肌细胞(VSMCs)增殖的机制.方法:应用逆转录病毒载体pLNCX2-CREG和pSM2-siCREG分别转染VSMCs,G418和puromycin筛选得到稳定转染细胞VSMCs-CREG和VSMCs-siCREG;Western blotting检测转染前后细胞CREG的表达;BrdU和流式细胞术检测CREG表达及对VSMCs增殖的影响.Western blotting和免疫荧光染色检测细胞胰岛素样生长因子Ⅱ受体(IGF2R)的表达;ELISA和RT-PCR检测胰岛素样生长因子Ⅱ(IGFⅡ)的表达;Alexa 488标记重组IGFII内吞实验观察CREG表达对IGFII内吞的影响;重组IGF2R和anti-IGF2R中和抗体阻断实验观察IGF2R-IGFII内吞作用对细胞增殖的影响;Western blotting检测细胞增殖信号分子PI3K/Akt和ERK表达,抑制剂阻断研究分析上述信号分子表达变化在细胞增殖中的作用.结果:实验分别得到VSMCs-CREG和VSMCs-siCREG的细胞克隆,VSMCs-CREG中CREG表达增加,而VSMCs-siCREG中CREG表达减少.VSMCs-CREG细胞增殖较正常对照组明显受抑,VSMCs-siCREG细胞增殖显著增加.VSMCs-CREG细胞中IGF2R蛋白在细胞膜上的表达及分布均显著增加,而VSMCs-siCREG细胞中IGF2R在膜上分布明显下降.CREG过表达促进IGF2R对IGFII的内吞作用,抑制了IGFII的分泌.IGFII分泌增加启动了CREG沉默后VSMCs的增殖,PI3K/Akt和MAPK/ERK信号协同参与了IGFII对VSMCs增殖的调控作用.结论:CREG通过调控IGF2R在细胞膜的分布,加速IGFII内吞作用,抑制了体外培养VSMCs的增殖.
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