An efficient and simple method to obtain aortic media for primary culture of rat vascular smooth muscle cells (RVSMCs) is developed. The main steps to obtain aortic media include isolation of rat aortic artery, removal of the fat tissue and branches, separation of longitudinal cutting edge, and peeling off the adventitia. Then, aortic media was used to obtain RVSMCs by our tissue explants method and the enzyme digestion method. The removal efficiency of the intima and adventitia was confirmed by hematoxylin–eosin and immunohistochemical staining. Morphology and immunofluorescent staining were used to identify cells and cell purity. RVSMCs at the 3rd and 8th passages were isolated by our tissue explants method; the enzyme digestion method and the traditional tissue explants method were compared respectively. Western blotting and gel contraction assay were used to investigate the phenotype and contraction ability of RVSMCs obtained by the different methods. Compared with the other methods, RVSMCs isolated by our method showed higher purity and demonstrated “contractile” phenotype with retained contraction ability for more passages. And the aortic media obtained showed no visible damage with few endothelial cells and fibroblasts remained. An efficient and simple method was established to obtain rat aortic media for primary culture of RVSMCs with high purity, “contractile” phenotype characteristics, and more stable during subculturing.

开发了一种有效且简单的方法来获得用于大鼠血管平滑肌细胞 (RVSMCs) 原代培养的主动脉培养基。获得主动脉中膜的主要步骤包括大鼠主动脉的分离、脂肪组织和分支的去除、纵向切割边缘的分离和外膜的剥离。然后，通过我们的组织外植体法和酶消化法，使用主动脉介质获得 RVSMC。通过苏木精-伊红和免疫组织化学染色证实了内膜和外膜的去除效率。形态学和免疫荧光染色用于鉴定细胞和细胞纯度。我们的组织外植体方法分离了第3代和第8代的RVSMCs；分别对酶消化法和传统组织外植体法进行了比较。使用蛋白质印迹和凝胶收缩测定来研究通过不同方法获得的 RVSMC 的表型和收缩能力。与其他方法相比，通过我们的方法分离的 RVSMCs 显示出更高的纯度，并表现出“收缩”表型，并保留了更多传代的收缩能力。获得的主动脉中层没有可见的损伤，内皮细胞和成纤维细胞很少。建立了一种高效、简单的方法来获得用于 RVSMC 原代培养的大鼠主动脉培养基，该培养基具有高纯度、“收缩性”表型特征，并且在传代过程中更稳定。通过我们的方法分离的 RVSMCs 显示出更高的纯度，并表现出“收缩”表型，并保留了更多传代的收缩能力。获得的主动脉中层没有可见的损伤，内皮细胞和成纤维细胞很少。建立了一种高效、简单的方法来获得用于 RVSMC 原代培养的大鼠主动脉培养基，该培养基具有高纯度、“收缩性”表型特征，并且在传代过程中更稳定。通过我们的方法分离的 RVSMCs 显示出更高的纯度，并表现出“收缩”表型，并保留了更多传代的收缩能力。获得的主动脉中层没有可见的损伤，内皮细胞和成纤维细胞很少。建立了一种高效、简单的方法来获得用于 RVSMC 原代培养的大鼠主动脉培养基，该培养基具有高纯度、“收缩性”表型特征，并且在传代过程中更稳定。