两种血管外膜成纤维细胞培养方法的比较与经验总结

Comparison and experience of two methods for the culture of adventitial fibroblasts

目的比较两种血管外膜成纤维细胞(AFs)培养方法的差异并总结经验。方法获取大鼠主动脉血管外膜组织,切成小块,一部分采用组织贴块法进行原代培养,另一部分使用消化酶消化获取细胞,使用相差显微镜观察细胞形态、免疫组化鉴定细胞内特定蛋白表达、细胞计数计算生长曲线等实验手段,并比较两种方法之间的差异。结果贴块法和消化法均能成功获得AFs;经免疫组化检测,两种方法所获的细胞波形蛋白(Vimentin)表达阳性/a-平滑肌肌动蛋白(a-SMA)表达阴性,证实培养的细胞为AFs。两种方法获得的细胞在细胞增殖期、对数生长期和平台期的时间大致相同,二者的生长、增殖比较,差异无统计学意义(P>0.05)。结论贴块法和消化法均能成功培养AFs,所获得的细胞性状稳定、存活率高,可在体外稳定地传代培养,在血管重构研究领域具有应用价值。

Objective To compare the differences of two methods in adventitial fibroblasts culture and summarize the experience. Methods Aortic vascular adventitia were separated from 6 specific pathogen free wistar rats, and cut into small pieces. Half of adventitial fibroblasts were isolated and cultured using tissue explants adherent method, others by digestion method. Phase contrast microscope was used to observe the cell morphology, immunohistochemistry was used to identify the expression of vimentin and a-smooth muscle actin, and cell counts to determine rate of cell growth and proliferation. The differences between two groups were compared. Results Massive adventitial fibroblasts were obtained successfully by both tissue explant adherent method and digestion method. All of them were confirmed to be Vimentin （＋）/a-SMA （-） cells by immunohistochemistry, which represented adventitial fibroblasts. The rate of cell growth and proliferation were comparable between two groups and showed no statistically significant difference （P 〉 0.05）. Conclusion Adventitial fibroblasts can be obtained and cultured successfully both by tissue explants adherent method and digestion method. Stability and high survival rate of adventitial fibroblasts gained from both two methods make it valuable in the future study of vascular remodeling.