人大隐静脉平滑肌细胞体外培养及其生长特性分析

In vitro growth characteristics of human great saphenous vein smooth muscle cells in culture

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5～7d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3～6d内光密度值变化明显；无血清培养的VSMC在24h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，24h内迁移能力最强。

Objective： To explore in vitro culture method and growth characteristics of human great saphenous vein smooth muscle cells （VSMCs）. Methods： VSMCs were primarily cultured and passaged by digestive and explant attached method, and then the cells were subcultured. The cultured cells were identified by morphological observation, immunofluorescence staining and Coomassie brilliant blue staining. The survival percentage, proliferation ability and migration ability of cells were determined by trypan blue staining, MTT assay, drawing growth curve and wound healing assay. Results： he primary cultured VSMCs passage cells maintained ＂hill-valley＂revealed by immunofluorescence, and the cytoskeleton structure was observed as blue dense bundles with Coomassie brilliant blue staining. Cell survival percentage was 97%. The growth curve of VSMC resembled ＂S＂ in shape, a significant change of optical density was detected after culture for 3-6 d, and the wound width of the serum-free cultured VSMCs changed markedly within the first 24 h. Conclusion： In vitro cultured VSMCs are characterized by contractile phenotype and high purity with good structure and function. Proliferation activity is higher after growth for 3-6 d and migration ability is strongest within the first 24 h of culture.