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目的为证实α1-肾上腺素能受体(α1-AR)是否介导了机械牵张力和/或去甲肾上腺素(NE)对小鼠主动脉血管平滑肌细胞(VSMC)的细胞外信号调节激酶(ERK1/2)的协同激活并加速血管重构。方法静息培养的小鼠血管平滑肌细胞(VSMC)经有或无Prazosin(α1-AR抑制剂)预处理后,给予机械牵张力和/或NE刺激,Western Blotting检测ERK1/2的磷酸化水平,免疫荧光检测ki-67的表达(细胞增殖),RT-PCR检测细胞α1-AR亚型(α1A-,α1B-和α1D-AR)mRNA的表达。分别用三种亚型的α1-AR-siRNA预处理细胞,观察α1-AR各亚型基因沉默对机械牵张力诱导的VSMC ERK1/2磷酸化的影响。结果机械牵张力可分别上调α1B-和α1D-AR mRNA表达,但α1A-AR mRNA未检测到。机械牵张力和/或NE均可激活ERK1/2,引起ERK1/2磷酸化增加,而两种因素共同存在时ERK1/2激活更加明显;ki-67的表达呈现类似效应;这种由机械牵张力和/或NE激活的作用均可被Prazosin部分抑制。α1B-和α1D-AR-siRNA预处理可分别减少α1B-和α1D-AR mRNA的表达,进而相应地导致机械牵张力诱导的ERK1/2磷酸化的抑制。RT-PCR未能检测到经α1A-AR-siRNA预处理后的VSMC表达α1A-AR mRNA,但仍可见机械牵张力诱导的ERK1/2磷酸化被抑制。结论高血压机械牵张力和NE可协同促进VSMC ERK1/2激活及细胞增殖,导致血管重构加速;α1-AR部分介导了这一过程,α1-AR各亚型起着相似作用。本研究可望为深入探讨高血压异常机械力合并交感神经活性亢进协同促进血管重构的致病机制及防治新策略提供实验依据。
Aim To demonstrate whether α1-adrenergic receptor (α1-AR) mediates mechanical stretch and / or extracellular signal-regulated kinases (norepinephrine) in mouse aortic vascular smooth muscle cells (VSMCs) ERK1 / 2) and accelerate vascular remodeling. Methods The resting VSMCs were pretreated with or without Prazosin (α1-AR inhibitor), and then subjected to mechanical stretch and / or NE stimulation. The phosphorylation of ERK1 / 2 was detected by Western Blotting. The expression of ki-67 (cell proliferation) was detected by immunofluorescence and the mRNA expression of α1-AR subtypes (α1A-, α1B- and α1D-AR) were detected by RT-PCR. The cells were pretreated with three subtypes of α1-AR-siRNA to observe the effect of α1-AR subtypes on ERK1 / 2 phosphorylation induced by mechanical strain. Results Mechanical stretch could up-regulate α1B- and α1D-AR mRNA expression, but α1A-AR mRNA was not detected. Mechanical stretch and / or NE could activate ERK1 / 2, causing increased phosphorylation of ERK1 / 2, and ERK1 / 2 activation was more obvious when the two factors coexisted; expression of ki-67 showed a similar effect; Tension and / or NE activation can be partially inhibited by Prazosin. Pretreatment with α1B- and α1D-AR-siRNA reduced the expression of α1B- and α1D-AR mRNA, respectively, which in turn led to inhibition of ERK1 / 2 phosphorylation induced by mechanical strain. RT-PCR failed to detect α1A-AR mRNA expression in VSMCs pretreated with α1A-AR-siRNA, but it was still observed that ERK1 / 2 phosphorylation induced by mechanical stretch was inhibited. CONCLUSION: Hypertension mechanical tension and NE can promote ERK1 / 2 activation and cell proliferation in VSMCs, resulting in accelerated vascular remodeling. Α1-AR partially mediates this process, and α1-AR subtypes play a similar role. This study is expected to provide an experimental basis for further exploring the pathogenesis of hypertension-induced hypertension combined with sympathetic nerve hyperactivity and new strategies for prevention and treatment of vascular remodeling.