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目的:探讨人血管平滑肌细胞(VSMC)体外培养方法及其生长的特性。方法:采用组织块贴壁结合酶消化法培养人大隐静脉VSMC并传代,以形态学观察、免疫荧光染色法、考马斯亮蓝染色法鉴定细胞;以台盼蓝染色法、四甲基偶氮唑蓝(MTT)比色法、绘制生长曲线、划痕法测定细胞成活率、增殖及迁移能力。结果:原代培养5~7 d细胞从组织块爬出,传代细胞呈“峰-谷”样生长;胞质内α-平滑肌肌动蛋白免疫荧光染色阳性;考马斯亮蓝染色可见VSMC细胞骨架呈致密束状;传代细胞成活率为97%;生长曲线呈类“S”形;细胞生长3~6 d内光密度值变化明显;无血清培养的VSMC在24 h内划痕宽度变化显著。结论:体外培养的VSMC纯度高,结构、功能良好,为收缩表型,细胞生长第3~6天增殖活力较强,24 h内迁移能力最强。
Objective: To investigate the in vitro culture methods and the growth characteristics of human vascular smooth muscle cells (VSMCs). Methods: VSMCs of human saphenous vein were cultured and adhered by tissue adherent and enzymatic digestion. Morphological observation, immunofluorescence staining and Coomassie brilliant blue staining were used to identify cells. Cell proliferation was detected by trypan blue staining, Blue (MTT) colorimetric method was used to draw the growth curve. The cell viability, proliferation and migration ability were determined by scratch method. RESULTS: Primary cultured cells grew out of the tissue blocks, and the passaged cells showed “peak-valley” growth; positive α-smooth muscle actin immunofluorescence staining in the cytoplasm; VSMC cells were stained with Coomassie brilliant blue Skeleton was densely packed, the survival rate of passaged cells was 97%, and the growth curve was in the shape of “S” shape. The optical density varied significantly within 3-6 days of cell growth; Significant changes. CONCLUSION: The VSMC cultured in vitro has high purity, good structure and good function, and has a contractive phenotype. The cells proliferated vigorously on days 3 to 6 and migrated most strongly within 24 h.