两种原代培养方法对血管平滑肌细胞收缩表型的影响

The influence of two vascular smooth muscle cell primary culture methods on cellular contractile phenotype

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。

Objective： To explore the difference between collagenase digestion method and explant-culture method to obtain vascular smooth muscle cells （VNMtgs） primary culture on maintaining contracmc pncnutyl3c. 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-et （SMA-a）, 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 immunofluorescence and western blot. Results： We found VSMCs obtained from enzyme digestion initially exhibited stronger SMA-u 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.