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采取较小容量保存缓冲液保存急性分离的心肌细胞,进行L-型Ca2+通道特性观测时,在细胞分离6h后,L-型Ca2+通道电流峰值变异性明显增大,其原因尚不清楚。为获取更多的实验数据,且确保数据的准确与稳定性,本研究旨在观测小容量保存缓冲液保存体系pH的变化及其对心肌细胞形态、线粒体功能与L-型Ca2+通道特性的影响。将急性分离的心肌细胞分别保存于100mL大容量保存液和20mL小容量保存液中。结果显示,在10h保存过程中,100mL大容量保存液组pH保持不变;而20mL小容量保存液组,其溶液pH从7.20降至6.95,导致轻度酸中毒。20mL小容量保存液组酸中毒不仅使异常形态的长杆状心肌细胞比例增加(保存时间≥6h),而且使心肌细胞线粒体膜电位逐步降低(保存时间≥8h),线粒体NADPH自发荧光由蓝色转向绿色(保存时间≥8h),提示能量代谢受阻。由于这些变化,保存10h时,心肌细胞L-型Ca2+通道电流峰值呈显著性降低,峰值电流钳制电位从+10mV向0mV偏移。上述结果提示长时间保存急性分离心肌细胞,宜采用较大容量的缓冲液体系,或者采取心肌细胞形态与NADPH蓝色荧光两种方法相结合,选择线粒体功能良好的细胞,进行L-型Ca2+通道特性观测。
The reason why the L-type Ca2 + channel current peak variability was significantly increased 6 hours after the cells were isolated from the acutely isolated cardiomyocytes by using a smaller volume of preservation buffer to observe the characteristics of L-type Ca2 + channels was still unclear. In order to obtain more experimental data and to ensure the accuracy and stability of the data, this study aimed to observe the changes of the pH of the preservation system with small volume and its effect on the morphology of cardiomyocytes, mitochondrial function and the characteristics of L-type Ca2 + channels . The acutely isolated cardiomyocytes were stored in 100mL high-capacity preservation solution and 20mL low-volume preservation solution. The results showed that during the 10h storage, the pH of 100mL high-capacity preservation solution remained the same while that of 20mL low-volume preservation solution decreased from 7.20 to 6.95, resulting in mild acidosis. 20mL small volume preservation solution acidosis not only increased the proportion of abnormal long stem cells (preservation time ≥ 6h), but also decreased myocardial cell mitochondrial membrane potential (preservation time ≥ 8h), mitochondrial NADPH autofluorescence by the blue Turning green (save time ≥ 8h), suggesting that energy metabolism is blocked. As a result of these changes, the peak current of L-type Ca2 + channels in cardiomyocytes decreased significantly at 10h, and the peak current-clamp potential shifted from +10 mV to 0mV. The above results suggest that the preservation of acutely isolated cardiomyocytes for a long time, should adopt a larger capacity of the buffer system, or take the cardiomyocyte morphology and NADPH blue fluorescence combination of two methods, select mitochondrial well-functioning cells, L-type Ca2 + channels Characteristic observation.