两种原代培养方法对血管平滑肌细胞收缩表型的影响

摘要

Objective: To explore the difference between collagenase digestion method and explant-culture method to obtain vascular smooth muscle cells (VSMCs) primary culture on maintaining contractile phenotype. Methods: The 1st, 2nd, 4th, 8th, 12th generation of primary VSMCs were obtained via type II collagenase diges-tion and explant-culture method. VSMCs contractile phenotype protein markers, calopnin and smooth muscle actin-α (SMA-α), as well assecreted protein osteopontin (OPN), were detected by western blot between differ-ent generations. The migration and proliferation ability of VSMCs from two methods were detected by wound-healing assay and MTT assay, and the phenotype protein markers expression by immunolfuorescence and western blot.Results: We found VSMCs obtained from enzyme digestion initially exhibited stronger SMA-α and cal-ponin expression, lower OPN expression and suppressed cellular proliferation and migration compared with that from explant-culture method. However, we observed a positive correlation between cell generation and VSMCs dedifferentiation degree, and a stronger dedifferentiation trend in enzyme digestion cells after 8th generation. Conclusion: Type II collagenase digestion method shows advantages in acquiring primary cells rapidly and maintaining VSMCs contractile phenotype within 4th generation compared with explant-culture method.%目的：探究胶原酶消化法和植块法培养原代血管平滑肌细胞（VSMCs）对细胞收缩表型影响的特点。方法：使用II型胶原酶消化法和植块法分别离体培养原代VSMCs，并获取第1代、第2代、第4代、第8代、第12代VSMCs，Western blot检测不同代数表型标志蛋白α平滑肌肌动蛋白（SMA-α）、调宁蛋白（calponin）和骨桥蛋白（OPN）表达情况；随后检测不同培养方法间表型标志蛋白表达差异，并用划痕实验和MTT实验检测迁移、增殖水平，免疫荧光检测SMA-α胞内表达。结果：消化法最初可获得明显优于植块法的VSMCs收缩表型，表现为SMA-α和calponin高表达，OPN较低表达，增殖、迁移能力相对较弱。培养至第8代后，2种方法的细胞都发生显著的表型改变，增殖、迁移能力增强且消化法细胞呈现更强的去分化表型趋势。结论：II型胶原酶消化法可快速获得良好收缩表型的VSMCs，在第4代前具有优于植块法的收缩表型。