| 王爱平,李严兵,谢巍,曹宇辉,彭田红,周小兵,龚邵新.酶消化法分离培养原代肺动脉平滑肌细胞及免疫组化鉴定.[J].中南医学科学杂志.,2015,43(1):78-81. |
| 酶消化法分离培养原代肺动脉平滑肌细胞及免疫组化鉴定 |
| Methods of Culture of Rat Pulmonary Artery Smooth Muscle Cells and Identification |
| 投稿时间：2014-06-07 |
| DOI： |
| 中文关键词: 酶消化法 大鼠肺动脉平滑肌细胞 免疫细胞化学 SD大鼠 |
| 英文关键词:Enzyme Digestion PASMCs SD-rat immunocytochemistry |
| 基金项目:湖南省教育厅科研项目（13C837），湖南省科技厅项目（2014FJ3016） |
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| 中文摘要: |
| 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SpragueDawley（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-SMactin）鉴定。结果酶消化法分离培养的PASMCs3d后，可见细胞贴壁呈梭形生长，6d后生长迅速呈典型的峰-谷状生长，9d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8～10d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。 |
| 英文摘要: |
| 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.Before 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 the α- 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 collagenase 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. |
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