【作者】 李世军； 孙宁玲；

【机构】 北京大学人民医院心内科；

【摘要】 目的:研究NO/PKG介导Ca2+/钙调神经磷酸酶(Calcineurin,CaN)信号通路对血管平滑肌细胞(VSMCs)增殖的调控。方法与结果:植块法原代培养Wistar大鼠VSMCs。Western blot测定CaN表达,定磷法通过鉴定游离无机磷酸盐含量测定CaN活性。MTT法测定VSMCs的增殖,丫啶橙/溴化乙锭荧光染色法测定VSMCs的活性。结果显示SNAP和Sp-8-pCPT-cGMPs使苯肾上腺素刺激诱发的VSMCs吸光度分别降低27.32%和36.57%,而Rp-8-pCPT-cGMPs则可使苯肾上腺素刺激诱发的VSMC吸光度增加79.02%。各组药物对于VSMCs活性的作用无显著差异。VSMCs应用维拉帕米预处理后,苯肾上腺素刺激诱发的VSMCs吸光度降低22.05%,并且上述吸光度可以被SNAP或Sp-8-pCPT-cGMPs进一步抑制,但可被Rp-8-pCPT-cGMPs轻微升高。VSMCs应用环胞素A预处理后,苯肾上腺素刺激诱发的VSMC吸光度降低36.67%,但是,上述吸光度不能被SNAP或Sp-8-pCPT-cGMPs进一步抑制或被Rp-8-pCPT-cGMPs升高。苯肾上腺素刺激诱发的CaN表达与活性可被SNAP和Sp-8-pCPT-cGMPs抑制,但可被Rp-8-pCPT-cGMPs增强。结论:NO/PKG调节VSMCs内游离钙离子水平,进而影响CaN表达与活性,从而部分抑制VSMCs增殖而非活性。更多还原

【Abstract】 Objective: Here we studied regulation of VSMC proliferation by NO/PKG mediated via modulating intracellular Ca2+/calcineurin (CaN) signaling pathway. Methods and Results : Primary VSMCs from rat aorta were used as the experimental model. CaN protein and its activity were assayed using immunoblotting and free inorganic phosphate content analysis, respectively. Cell growth was determined by MTT assay, and cell viability was observed using acridine orange and ethidium bromide staining. Our results show that the addition of SNAP and Sp-8-pCPT-cGMPS decrease absorbance of cells stimulated by phenylephrine (PE) by 27.32% and 36.57% respectively, whereas the addition of Rp-8-pCPT-cGMPS increases it by 79.02%. No significant cell viability changes of SMCs were found in the experiment groups. In SMCs pretreated with Ver, absorbance of cells stimulated by PE decreased by 22.05% and was further inhibited by the additional treatment of SNAP and Sp-8-pCPT-cGMPS, but was slightly promoted by the additional treatment of Rp-8-pCPT-cGMPS. In SMCs pretreated with CsA, absorbance of cells stimulated by PE decreased by 36.67%, but it could not be further altered by the additional treatment of SNAP, Sp-8-pCPT-cGMPS and Rp-8-pCPT-cGMPS. Moreover, expression and activities of CaN induced by PE was inhibited by SNAP and Sp-8-pCPT-cGMPS, but was promoted by Rp-8-pCPT-cGMPS. Conclusions: NO/PKG regulates proliferation of vascular SMCs without changes of their viability mediated via modulation of intracellular Ca2+/CaN signaling pathway.更多还原