Preparation of functional smooth muscle cells from the rabbit aorta.
Preparation of functional smooth muscle cells from the rabbit aorta.
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DOI：
10.1084/jem.148.5.1400
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发表时间：
1978-11-01
期刊：
影响因子：
--
通讯作者：
Jamieson JD
中科院分区：
文献类型：
--
作者：
Ives HE;Schultz GS;Galardy RE;Jamieson JD
一个程序分离成单一的兔子主动脉，功能平滑肌细胞描述。去除外膜和内膜后，培养基切片与纯化的胶原酶、弹性酶和大豆胰蛋白酶抑制剂一起在用Hepes、氨基酸和0.2 mM的[Ca2+]修饰的Krebs-Ringer缓冲液中孵育。酶切和机械剪切后，根据回收的DNA，分散细胞的产量约为25%。95%以上的细胞排除台盼蓝，约80-90%的细胞粘附在组织培养皿上。通过相差显微镜，大多数细胞呈细长状，大小约为10微米× 30微米。其余的要么是球形的，要么是高度圆齿化和收缩的。电镜观察显示，游离后的细胞95%以上为平滑肌，但与原位细胞相比，大多数细胞发生了一定程度的结构变化。根据制剂的不同，这些细胞中有5%到50%对激动剂有反应而收缩。当受到血管紧张素II去甲肾上腺素或氨甲酰胆碱刺激时，细胞缩短10-15%，并出现大量外翻。细胞在激动剂洗脱后松弛，随后可被重新刺激。每种激动剂的特异性抑制剂阻断了收缩反应。分散培养1-5天的细胞比新鲜制备的细胞收缩更多，表明在培养中恢复了激素结合和/或收缩功能。这种制备提供了一个系统，其中可以研究单个血管平滑肌细胞的生理。
A procedure for dissociating the rabbit aorta into single, functional smooth muscle cells is described. After removal of adventitia and intima, slices of media were incubated with purified collagenase, elastase, and soybean trypsin inhibitor in a Krebs-Ringer buffer modified with Hepes, amino acids, and a [Ca2+] of 0.2 mM. After enzymatic digestion and mechanical shear, the yield of dispersed cells was approximately 25% based on DNA recovered. Greater than 95% of the cells excluded trypan blue and approximately 80-90% adhered to tissue culture dishes. By phase contrast microscopy, most of the cells were elongate and approximately 10 micron X 30 micron in size. The remainder were either spherical or highly crenated and contracted. Electron microscopy of the cells showed that immediately after dissociation greater than 95% could be identified as smooth muscle, though most had undergone some degree of structural change compared to cells in situ. Depending on the preparation, from 5 to 50% of these cells contracted in response to agonists. Cells shortened by 10-15% and developed numerous evaginations when stimulated by angiotensin II norepinephrine, or carbamylcholine. Cells relaxed after washout of agonists and could subsequently be restimulated. Specific inhibitors of each of the agonists blocked the contractile response. Dispersed cells cultured for 1-5 days contracted in even higher numbers than the freshly prepared cells, suggesting restoration of hormone binding and/or contractile function in culture. This preparation provides a system in which the physiology of individual vascular smooth muscle cells may be studied.