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血管平滑肌细胞(vascular smooth muscle cells,VSMCs)迁移参与血管发育和多种疾病的发生、发展过程,然而其迁移机制尚不清楚。本课题建立了一种适合研究VSMCs迁移的方法——玻片倒扣法,基于该方法进一步研究VSMCs在不同细胞外基质上的迁移特性及机制。原代培养大鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs),采用免疫细胞化学方法鉴定其纯度。将细胞接种于小玻片上,待生长至单层汇合后,将小玻片倒扣于包被有不同细胞外基质的大玻片上,培养24h后,固定、拍照、统计软件测量迁移出的细胞数量和迁移距离。进一步采用细胞划痕实验和免疫细胞化学技术,研究细胞外基质对PASMCs迁移的作用及机制。结果显示:(1)原代培养PASMCs纯度高达(97±3)%;(2)玻片倒扣24h后,层粘连蛋白(laminin)组、基质胶(Matrigel)组比对照(phosphate buffered saline,PBS)组、多聚右旋赖氨酸(poly-D-lysinehydrobromide,PDL)组迁出的细胞数量更多,迁移距离更远,差异具有明显统计学意义;(3)细胞划痕8、12、24h后,细胞在层粘连蛋白、基质胶上的面积愈合率比在PBS上的高,差异具有统计学意义;(4)免疫细胞化学结果显示迁移能力较强的PASMCs中肌动蛋白在与细胞运动方向相一致的边缘聚集现象越明显;vinculin(粘附斑的标记物)染色显示,种植于层粘连蛋白和基质胶上的PASMCs中,vinculin的分布较PBS和PDL上的更少。上述结果表明:玻片倒扣法是一种适合研究VSMCs迁移的方法;层粘连蛋白和基质胶可能通过抑制粘附斑的形成和调节骨架蛋白,促进VSMCs迁移。
Migration of vascular smooth muscle cells (VSMCs) is involved in the development of blood vessels and the development of various diseases. However, its migration mechanism is still unclear. This project established a suitable method for studying the migration of VSMCs - slide-down method, based on the method to further study VSMCs migration characteristics and mechanisms of different extracellular matrix. Primary cultured rat pulmonary arterial smooth muscle cells (PASMCs), the use of immunocytochemistry to identify its purity. The cells were seeded on glass slides and allowed to grow until confluent with the monolayer. The glass slides were snapped onto glass slides coated with different extracellular matrix. After 24 hours of incubation, the cells were fixed, photographed, and counted by statistical software The number and migration distance. The cell scratch assay and immunocytochemistry were further used to study the effect of extracellular matrix on the migration of PASMCs. The results showed that: (1) Purity of primary cultured PASMCs was as high as (97 ± 3)%; (2) After lamina for 24 hours, the expression of laminin, Matrigel and phosphate buffered saline PBS group and PDL group had more migrated cells and longer migration distance with statistically significant difference. (3) Cell scratches 8 and 12 , The healing rate of cells on laminin and matrigel was higher than that on PBS in 24h, and the difference was statistically significant; (4) The results of immunocytochemistry showed that actin in PASMCs with strong migration capacity was significantly different The more prominent edge aggregation was observed in the direction of cell motility. The vinculin staining showed that the distribution of vinculin in PASMCs on laminin and matrigel was less than that in PBS and PDL. The above results indicate that the slide-down method is a suitable method to study the migration of VSMCs. Laminin and matrigel may promote the migration of VSMCs by inhibiting the formation of adhesion spots and regulating the skeleton proteins.