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

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(α-SMA, myosin-II and β-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.