人血管平滑肌细胞的原代培养及其钙化模型的建立

目的利用β-甘油磷酸盐处理人血管平滑肌细胞（human vascular smooth muscle cells,HVSMCs）制备体外血管钙化模型。方法用组织贴壁法从人胚胎脐带动脉中分离原代人主动脉平滑肌细胞,原代细胞通过抗体α-sm-actin染色鉴定,将传4～6代的细胞分为钙化组和对照组,对照组用正常DMEM培养基培养,钙化组加入10mmol/Lβ-甘油磷酸盐诱导细胞钙化,连续培养10d。茜素红S染色及碱性磷酸酶法鉴定。结果：分离的原代细胞经S-P染色鉴定为阳性,呈淡黄色。钙化组细胞诱导钙化后,细胞增殖缓慢,并形成囊泡结构,茜素红S染色形成红色的钙化结节,钙化组碱性磷酸酶活性较对照组在不同时间点（4,6,8,10d）都有所增加（P〈0.01）。结论：β-甘油磷酸盐体外能够诱导HVSMC钙化,此方法诱导的钙化模型是一种良好的研究血管疾病方面的体外模型。

Primary culture of human vascular smooth muscle cells and their calcification mode

Objective To establish a calcification mode in vitro of human vascular smooth muscle cells (HVSMCs) induced by β-GP. Methods Primary HVSMCs were obtained from human embryo by plant method and confirmed by stain with α-sm-actin antibody. The cells after 4-6 passages were divided into two groups.The control group was incubated with normal DMEM medium while the calcification group was incubated with the medium containing 10 mmol/L β-glycerophosphate for 10 days.Calcification was confirmed by alizarin red S stain and alkaline phosphatase(ALP) assays. Results The primary cells observed by S-P stain were positive and the cells after being stained were pale yellow. After being induced with β-GP, the cells of calcification group began concentric growth and formed vesicles. Alizarin red S staining showed that the reaction of calcifying nodules was red,ALP activity was higher than that of controls at various time points(4 d,6 d,8 d and 10 d,P＜0.01 ).Conclusion The HVSMCs could be induced into calcification in vitro by β-GP, and this model contribates to further studies of vascular diseases.