Ca2+-tolerant adult canine myocytes: preparation and response to anoxia/acidosis.
Ca2+-tolerant adult canine myocytes: preparation and response to anoxia/acidosis.
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Ca2 耐受的成年犬肌细胞：准备和对缺氧/酸中毒的反应。
DOI：
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发表时间：
1982
影响因子：
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通讯作者：
W. Weglicki
中科院分区：
文献类型：
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作者：
A. M. Spanier;W. Weglicki
一个程序描述了大量的钙耐受成年犬心室肌细胞的生产。在存在毫摩尔水平的Ca 2+的情况下，使用含有胶原酶、透明质酸酶和胰蛋白酶的320 mosM酶缓冲液在旋转瓶中实现温和的组织解离。该技术允许完全去除红细胞，温和地去除紧密粘附的非肌细胞（少于3%的污染），并选择性地从未受损的肌细胞中去除受损的肌细胞。总肌细胞产量平均为4-6 x 10（6）/g湿重; 61.0 +/- 6.5%的细胞具有杆状形态，并且基于台盼蓝排除法是“活的”。只有3.9 +/- 1.1%的杆状细胞在用2 mM Ca 2+激发时自发搏动。基于台盼蓝摄取和肌酸激酶释放，在37 ℃下暴露于2 mM Ca 2+导致最小的活力损失（2.1 +/- 1.3%/h）。模拟蜂窝（即，通过对犬肌细胞悬浮液分别施加缺氧、酸中毒和低葡萄糖的扰动，进行了体外细胞（膜）损伤;数据表明，这些肌细胞独立地受到这些扰动的影响，并且与其他人使用分离的大鼠肌细胞系统获得的结果非常一致。
A procedure is described for the production of large numbers of Ca2+-tolerant adult canine ventricular myocytes. Gentle tissue dissociation is achieved in Spinner flasks using 320 mosM enzyme buffer containing collagenase hyaluronidase, and trypsin in the presence of millimolar levels of Ca2+. The technique allows for the complete removal of erythrocytes, the gentle removal of closely adhering nonmuscle cells (less than 3% contamination), and the selective removal of damaged from nondamaged myocytes. Total myocyte yields averaged 4-6 x 10(6)/g wet wt; 61.0 +/- 6.5% of the cells have rod-shaped morphology and are "viable" based on trypan blue exclusion. Only 3.9 +/- 1.1% of the rod-shaped cells beat spontaneously when challenged with 2 mM Ca2+. Exposure to 2 mM Ca2+ at 37 degrees C results in minimal loss of viability (2.1 +/- 1.3%/h) based on both trypan blue uptake and creatine kinase release. Simulation of cellular (i.e., membrane) injury in vitro is performed by the separate application of the perturbations of anoxia, acidosis, and low glucose to the canine myocyte suspensions; the data suggest that these myocytes are affected independently by these perturbations and are in good agreement with the results obtained by others using the isolated rat myocyte system.