缺血期亚低温保存再灌注后心肌细胞收缩功能的研究

Study of the effects of mild hypothermia on improvement of cardiomyocyte contractility after ischemia-reperfusion in rats

目的探讨缺血期亚低温对常温再灌注后心肌细胞的保护作用及其可能机制。方法本实验在中山大学心肺脑复苏研究所完成。取50只SD新生1—2d乳鼠心脏，建立心肌细胞体外模型，利用氧糖剥夺（oxygen and glncose deprivation，OGD）和氧糖恢复（oxygen and glucoserestoration，OGR）模拟缺血一再灌注过程。将细胞随机（随机数字法）分入常温对照组、32℃ OGD-37℃OGR组（低温组）、37℃ OGD—OGR组（常温组），并在OGR0，0．5，1．0，1．5，2．0h分别测定心肌细胞收缩频率和平均收缩速率；在OGR0，2h分别收集细胞进行透射电镜观察超微结构以及线粒体变化，同时采取差速离心法制备心肌细胞线粒体匀浆液，使用Clark氧电极测定上述两个检测时点的线粒体呼吸控制率（respiratory control rate，RCR）。采用SPSSl3．0统计软件分析结果。结果低温组和常温组在OGD1h后都出现收缩频率和收缩速率下降，但是常温组下降更明显（P=0．000）。随着OGR时间延长，两组的收缩参数都呈上升趋势，但低温组在1h后收缩功能接近正常，而常温组在观测时点始终低于对照组和低温组（P=0．000）。心肌细胞超微结果提示，常温组的线粒体肿胀明显，基质密度降低。线粒体RCR测定提示，低温组和常温组在OGR0h都明显降低，但后者损伤效应显著延长，持续到OGR2h。结论单纯缺血期亚低温可以减轻细胞线粒体的损伤，并有助于心肌细胞早期恢复收缩功能。

Objective To study the effects of mild hypothermia on cardiomyocyte contractility improvement after ischemia-reperfusion injury and on the preservation of well-functioning mitochondrial respiratory capability. Methods A total of 50 newborn SD rats 1 -2 days after delivery were sacrificed and their hearts taken to preserved in 4 ℃ cold D-hanks buffer solution with 0. 12% pancreatic proteinase and collagenase and then processed with 37 ℃ water bath to collect the cardiomyocytes cultured in DMEM medium with 10% FBS for 5 days. The cardiomyocytes of rats were subjected to ischemia/reperfusion, in vitro, by oxygen and glucose deprivation （OGD） /oxygen and glucose restoration （OGR）. The cardiomyocytes of rats after ischemia/reperfusion were divided into three groups： control group, hypothemia group and normothermia group. Contractile frequency and velosity were determined before OGD and 0 h, 0. 5 h, 1 h, 1.5 h and 2 h after OGR. Ultrastructure changes of cardiomyocytes and mitochondrion wereobserved under transmission electron microscope （TEM） 0 h and 2 h after OGR as well as assessment of respiratory rate and respiratory control rate （RC1R） with Clark oxygen electrode in each group. All data were analyzed with statistical software of SPSS 13.0. Results Contractile function of cardiomyocytes in hypothermia group and normothermia group declined to nadir at 0 h after OGR （ P = 0. 000 ） and the contractile function of cardiomyocytes in hypothermia group was improved one hour later, compared with the normothermia group （P = 0. 000 ）. Obvious swelling of mitochondrion was observed under TEM in normothermia group with little alteration after OGR. The RCR assessments indicated respiratory function in normothermia group was impaired after OGR （P = 0. 000） and this may be responsible for contractility dysfunction. Conclusions Mild hypothemia used after ischaemia can optimize the contractility of cardiomyocytes after a normothermia OGR, and the well-functioning respiratory capability of mitocbondrion may be preserved in this process.