大鼠胸主动脉血管平滑肌细胞原代培养方法的改进  

An improvement in primary culture of rat vascular smooth muscle cells and its identification

作　　者：孟立平[1,2,3] 蒋承建[1,2] 赵飞[1,2,3] 郭艳[4] 池菊芳[1,2] 郭航远[1,2,3] 

机构地区：[1]绍兴市人民医院 [2]浙江大学绍兴医院心内科,浙江绍兴312000 [3]温州医科大学第一临床医学院,浙江温州325035 [4]浙江中医药大学研究生院,浙江杭州310000

出　　处：《温州医学院学报》2015年第8期593-596,共4页Journal of Wenzhou Medical College

基　　金：浙江省自然科学基金资助项目(LY14H020002);浙江省卫生高层次创新人才培养工程资助项目(CXRC201201);浙江省公益技术研究社会发展项目(2012C33040);绍兴市科技局科研基金资助项目(2013B70072)

摘　　要：目的:探索提高大鼠血管平滑肌细胞(VSMCs)原代培养效率和纯度的方法。方法:在原有组织贴块法培育大鼠原代VSMCs的基础上进行改良,分离出胸主动脉后进一步分离中膜,DMEM高糖培养基中加入10μg/L血小板源性生长因子B(PDGF-B)。用细胞形态学及细胞免疫荧光对VSMCs进行鉴定。结果:6 d左右组织块周围有细胞爬出,12 d左右可以传代。镜下见细胞呈梭形,典型"峰谷"状排列生长;免疫荧光鉴定可见大量平滑肌肌动蛋白(SMA),比传统方法获得的细胞纯度高。结论:加入PDGF-B可以缩短大鼠VSMCs原代培养时间,提高效率;剥离中膜可以提高大鼠VSMCs原代细胞的纯度。Objective：To explore a new effective way on primary culture of rat vascular smooth muscle cells（VSMCs）,which could shorten the cultivate time and enhance the VSMCs＇ purity.Methods：The primary culture of rat VSMCs was conducted by a new way reformed from the traditional modified tissue explant technique,in which the medial layer was isolated and 10 μg/L PDGF-B was added to the high glucose DMEM.The VSMCs were identified by cell morphology and immunocytochemistry.Results：About six day later,VSMCs could be found around some tissue blocks,and passage of the VSMCs can be done about 12 days later.Under the phase contrast microscope,the VSMCs looks like spindle and the specific ＂hill and valley＂ phenomenon could be seen.Immunocytochemistry showed that there was a large number of smooth muscle actin（SMA） and the VSMCs＇ purity was much higher than the cells got by the old way.Conclusion：Adding PDGF-B could shorten the cultivate time of primary VSMCs and isolation of the medial layer could improve the VSMCs＇ purity.

关 键 词：原代细胞 血管平滑肌细胞 组织贴块法 血小板源性生长因子B 

分 类 号：R331[医药卫生—人体生理学]