摘要
目的:探寻合适的初始灌流压力与定量确定酶消化终止点,以提高恒流灌注分离成年大鼠心肌细胞的产量与质量。方法:采用Langendorff装置,行主动脉插管逆向灌流,0.08%胶原酶I消化大鼠心脏,监测灌流压力的变化。结果:调节灌流流速以15 kPa初始压力恒流灌流时(n=4),灌流压力明显升高(压力峰值>25 kPa),造成左心室在松弛状态下明显扩张,使分离的心肌细胞存活率降低,且收缩功能显著降低。以10 kPa初始压力行恒流灌流时,酶消化引起的压力升高均低于18.75 kPa,左心室扩张不明显;当酶消化过程中压力降至10 kPa(n=3)或5kPa(n=4)时终止消化,心肌细胞分别呈消化不足或过度的形态,且心肌细胞存活率均低于10%,恢复正常细胞外液钙离子浓度后,大部分心肌细胞死亡。当酶消化时灌流压降至7.5 kPa时终止消化(n=15),分离即刻心肌细胞存活率为82.6%±4.8%,复钙后心肌细胞存活率为30.4%±4.5%,复钙后4 h的存活率仍为24.8%±5.4%。细胞形态完好,边缘锐利、横纹清晰,无明显博动,收缩功能正常。结论:为获得存活率较高的成年大鼠心肌细胞,宜采用低压恒流灌流分离方法,即初始灌流压力保持在10 kPa,胶原酶I消化的终止压力为7.5 kPa。
Aim： In order to get high-yield and high-quality cardiomyocytes from adult rat. We needed to clarify the optimal perfusion pressure in a constant flow condition and stopping digestion pressure. Methods： The rat was injected with heparin （1 000 IU/kg, i.p. ） 20 minutes prior to the experimental protocol. The heart was excised and the aorta was cannulated rapidly. The cannulated heart was mounted on a Langendorff perfusion apparatus with constant flow and perfusion pressure was monitored. The initial perfusion pressure was maintained at 15 kPa or 10 kPa by regulating the flow rate. The heart was digested by 0.08 % collagenase I at 37 ℃ and the enzymatic digestion was terminated immediately when the perfusion pressure was lowered to 10 kPa, 7.5 kPa or 5 kPa. Results： While the initial perfusion pressure was 15 kPa（ n=4）, enzymatic digestion induced great extent increment in perfusion pressure （maximal pressure 〉 25 kPa）. The left ventricle was enlarged significantly and the contractility of m yocytes was decreased. While the initial perfusion pressure was kept at 10 kPa, the digestion only resulted in less extent increment in perfusion pressure（maximal pressure〈 18.75 kPa）. The left ventricle was enlarged slightly. In the same initial perfusion pressure, if the digestion was stopped at 10 kPa（ n = 3）, or 5 kPa（ n = 4）, myoeytes appeared overmuch or insufficiency digestion, respectively. The viability of freshly isolated cardiomyocytes was less than 10 %. The most of myocytes died after restoration of extracellular Ca^2＋ concentration. If the digestion was stopped at 7.5 kPa（ n 15 ）. The viability of cardiomyocytes was 82.6 % ± 4.8 % in fresh isolation, 30.4 % ± 4.5 % after restoration of normal extracellular Ca^2＋ concentration, or 24.8 % ± 5.4 % after 4 h standing. The most of rod-shaped cardiomyocytes were quiescent and had visible cross striations, sharp edges, and normal contractility. Conclusion： When the initial perfusion pressure is kept at 10 kPa （low pressu
出处
《中国应用生理学杂志》
CAS
CSCD
北大核心
2005年第4期475-479,共5页
Chinese Journal of Applied Physiology
基金
国家自然科学基金资助课题(30370538)
关键词
成年大鼠
心肌细胞
分离
adult rat
cardiomyocyte
isolation
