改良的两步复合酶消化法制备大鼠胸主动脉血管平滑肌细胞  

Preparation of rat thoracic aortic vascular smooth muscle cells by modified enzyme double digestion

作　　者：周玥华 刘星[1,2] 张艳军[1,2] 汪辛[1,2] 陈渝萍[1,2] ZHOU Yue-hua LIU Xing ZHANG Yan-jun WANG Xin CHEN Yu-ping(Key Lab for Advanced Technologies of Materials, Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China School of Materials Science And Engineering, Southwest Jiaotong University, Chengdu 610031)

机构地区：[1]西南交通大学材料先进技术教育部重点实验室,成都610031 [2]西南交通大学材料科学与工程学院,成都610031

出　　处：《中国比较医学杂志》2017年第7期17-23,共7页Chinese Journal of Comparative Medicine

基　　金：国家自然科学基金(编号:81371675;81330031)

摘　　要：目的本文报道一种基于复合酶消化法进行改良的大鼠胸主动脉血管平滑肌细胞的分离方法。方法采用胶原酶、胰肽酶复合酶对胸主动脉进行初步消化去除内外膜、再对中膜进行二次消化释放血管平滑肌细胞。结果细胞分离后贴壁生长,多具梭形形态,呈现典型的"峰-谷"生长状态,1周可进行传代;多种收缩型血管平滑肌细胞的分子标记基因均在所分离培养的细胞中高表达;超过95%的细胞为alpha smooth muscle actin(α-SMA)和myosin-II显著阳性。结论本分离方法简捷易行、重复性好,所得细胞活性佳、纯度高,可确保短时间内获得大量收缩型血管平滑肌细胞。Objective This paper reports a modified method for the isolation of rat thoracic aortic vascular smooth muscle cells based on combined enzyme double digestion. Methods An enzyme mixture containing collagenase II, soybean trypsin inhibitor and elastase was prepared and used to remove the aortic tunica intima and tunica adventitia, and then the tunica media was subjected to second digestion using the same enzyme mixture and to isolate vascular smooth muscle cells. Results The isolated VSMCs were cultured in vitro and the growing cells had an elongated spindle-shape, reached confluence within a week, and displaying a typical ＂ hill-and-valley＂ pattern. After 1 week, the cells were passaged. Contractile smooth muscle markers （ct-SMA, myosin-II and 13-tubulin） were highly expressed in the isolated cells. More than 90% of the cells significantly expressed alpha smooth muscle actin and myosin-II. Conclusions Themethod established in this study has advantages of simple and easy to operate, and good reproducibility, with a high activity and purity of the separated cells. It can ensure to obtain a large amount of contraction-type vascular smooth muscle cells within a short time.

关 键 词：血管平滑肌细胞 复合酶 两步消化法 

分 类 号：R575.2[医药卫生—消化系统]