体外培养的平滑肌细胞多梳基因Bmi-1的表达变化及其与细胞增殖和细胞周期的关系

The Dynamic Expression of Polycomb Gene Bmi-1 in Cultured VSMC in Vitro and Its Correlation with Vascular Smooth Muscle Cell Proliferation and Cell Cycle

目的探讨多梳基因Bmi-1在体外培养的血管平滑肌细胞中的表达变化及其与细胞增殖和细胞周期的关系,以了解在平滑肌细胞表型转换及增殖过程中的早期基因事件。方法逆转录聚合酶链反应检测1～4代大鼠平滑肌细胞及不同浓度的丙戊酸钠作用于第4代血管平滑肌细胞24、48、72和96 h后Bmi-1 mRNA的表达。倒置显微镜下观察正常细胞及各组药物作用后细胞数量和形态的变化。四甲基偶氮唑盐法分析各组药物作用后细胞增殖活力的改变。流式细胞术检测细胞周期和细胞凋亡。结果体外培养的第1代大鼠血管平滑肌细胞有少量的Bmi-1 mRNA的表达,在传代过程中Bmi-1 mRNA的表达量逐渐增加,到第4代达到较高水平;经0.5～4.0mmol/L丙戊酸钠作用于第4代平滑肌细胞24、48、72和96 h后,Bmi-1 mRNA的表达量随浓度的增加和时间的延长而呈下降趋势。各浓度丙戊酸钠干预组随作用时间及浓度的增大均出现了生长抑制。经丙戊酸钠处理后各组出现细胞周期G2/M期阻滞及细胞凋亡,且随药物浓度的升高而增大。结论平滑肌细胞体外增殖及由收缩表型向合成表型的转化过程中出现平行的Bmi-1基因表达上调。丙戊酸钠可通过降低Bmi-1的表达明显抑制血管平滑肌细胞生长,并具有细胞周期G2/M期阻滞及促凋亡作用。

Aim To investigate the relationship between the dynamic expression of polyeomb gene Bmi-1 in cultured vascular smooth muscle cell （ VSMC ） in vitro and the VSMC proliferation and cell cycle, so as to illustrate the potential early mechanism in VSMC proliferation and phenotype transition. Methods The Bmi-1 mRNA transcription in cultured VSMC in vitro from the first to fourth passage, and in the valproate acid sodium （VPA） -treated VSMC for 24, 48, 72 and 96 h were measured by RT-PCR. The morphological changes and the proliferation rate of VSMC were examined by invert microscope and methyl thiazolyl tetrazolium （MTT） assay. The apoptosis and cell cycle were analyzed by flow cytometry. Results The expression of Bmi-1 in first passage of cultured VSMC was mild, and then increased progressively along with the passage. The treatment of VSMC by VPA induced down-expression of Bmi-1 in a dose-dependent and time-dependent manner. The morphological examination and MTT assay in VPA-treated groups also disclosed significant inhibition in proliferation rate of VSMC. The analysis of flow cytometry revealed increased apoptosis rate and G2/M phase blockage in cell cycle of VSMC in a VPA dose-dependent manner. Conclusion The up-expression of Bmi-1 gene was parallel with the VSMC proliferation and phenotype transition in vitro. The histone deacetylase inhibitor VPA could induce down-expression of Bmi-1. It is suggested that the VPA could inhibit the VSMC proliferation by way of down-expression of polyeomb gene Bmi-1, block the cultured VSMC in G2/M phase and pro-apoptosis effect.