雌激素对成年和老年雌性大鼠血管平滑肌细胞雌激素受体α表达的影响及其机制

Effects of estrogen on expression of ERαin vascular smooth muscle cells of adult and old female rats and the mechanism

目的探讨雌激素对成年和老年雌性大鼠血管平滑肌细胞(VSMC)雌激素受体α(ERα)表达的影响及其机制。方法采用RT-qPCR、Western blot及重硫酸盐修饰后测序(BSP)的方法,检测体外培养的3-4代成年(2-3月龄)和老年(≥20月龄)雌性SD大鼠VSMC在无或有生理剂量(10-10 mol/L、10-8 mol/L)17β-雌二醇(E2)存在下,ERα的表达及其启动子区CpG岛甲基化水平的变化。结果成年雌性大鼠VSMC无论在有或无雌激素存在时均表达ERα,且表达水平随雌激素浓度增加而上升。而老年雌性大鼠VSMC无论是在mRNA水平还是蛋白质水平几乎检测不到ERα表达,即使补充雌激素达最高生理剂量也无法诱导ERα的重新表达,其ERα基因启动区CpG岛呈现高水平甲基化。结论成年大鼠VSMC表达ERα,且生理剂量雌激素对其表达具有诱导作用。而老年大鼠VSMC ERα基因由于CpG岛已发生高度甲基化而抑制,生理剂量雌激素对ERα表达的诱导作用丧失。

Objective To investigate the effects of estrogen on the expression of estrogen receptor alpha(ERα)in adult and old vascular smooth muscle cells(VSMCs)and the underlying mechanism.Methods VSMCs cultured from adult(2-3months)and old(≥20months)female SD rats were incubated in the absence or presence of physiological concentrations(10-10 mol/L,10-8 mol/L)of 17β-estradiol(E2).The expression of ERαwas detected with Real time quantitative PCR(RT-qPCR)and Western blot,and the methylation of CpG island in the ERαgene promoter region was detected by bisulfite sequencing PCR(BSP).Results VSMCs from adult female rats expressed ERαin both absence and presence of E2,and the expression level increased with rising E2 concentrations.But in old female VSMCs,the expression of ERαwas hardly detectable at both mRNA and protein levels in either the absence or the presence of the highest physiological concentration of E2.BSP analysis showed that the CpG island in the ERαgene promoter region of these old cells was highly methylated(>80%).Conclusion The VSMCs of adult female rats can express ERα,with the expression level controlled by E2.But the ERαgene in the old cells is silenced and cannot be re-activated by E2 even at the highest physiological level.This is due to the super methylation of the ERαgene in old female VSMCs.