Objective To set up the methods of rat pulmonary artery smooth muscle cells ( PASMCs) isolation,culture and immunological identification in vitro. Methods The PASMCs cultured in vitro with type I collagenase. Be-fore the PASMCs cultured,male SD rat pulmonary trunk separated were subjected to outer membrane peel and endothelial cells remove by enzymatic digestion in a sterile environment. We observed the status and characteristics of PASMCs with inverte phase contrast microscope,determined the cell viability with trypan blue staining,and indentified theα-smooth muscle actin (α-SM actin) with immuncytochemistry staining. Results The cultured PASMCs subjected to enzymatic digestion and isolation presented shuttle shape at d 3,typical peak - valley -like growth at d6,and 90‰ confluence at d9. The morphological observation and immuncytochemistry staining identification showed that:the cells cultured were PASMCs;cell survival rate up to 98. 5‰;the primary cultures can be passaged at d8~ d10 and cells can be used for cell experiments at 3th generation to 10th for stable morphology and fast growth. Conclutions The method type I collage-nase digestion is easy operation and reliable,the primary cultured PASMCs presented fast growt and short cell cycle can be used for pulmonary arterial hypertension and pulmonary vascular remodeling study.%目的：建立体外胶原酶消化法分离培养大鼠肺动脉平滑肌细胞( PASMCs)及免疫鉴定的方法。方法采用I型胶原酶对PASMCs进行体外培养。在无菌环境下,分离提取雄性Sprague Dawley( SD)大鼠肺动脉主干,剥离外膜、剔除内皮细胞,经酶消化法,体外培养PASMCs。倒置相差显微镜观察PASMCs生长状态及特点；台盼兰测定细胞活力；免疫细胞染色法进行平滑肌α-肌动蛋白(α-SM actin)鉴定。结果酶消化法分离培养的PASMCs 3 d后,可见细胞贴壁呈梭形生长,6 d后生长迅速呈典型的峰-谷状生长,9 d后达90‰融合。通过形态学观察及免疫荧光染色法鉴定表明：培养的细胞为PASMCs；细胞存活率为98.5‰；原代培养8~10 d后即可传代,3-10代细胞生长迅速、细胞形态不易发生改变,可用于进行细胞实验。结论采用I型胶原酶消化法简单易行、可靠、PASMCs生长迅速、传代周期短的特点；尤为重要的是,培养的原代PASMCs可用于肺动脉高压和肺血管重构的研究工作。
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