成年大鼠心肌细胞分离培养及兴奋-收缩耦联表征

Isolation and culture of adult rat cardiomyocytes and characteristics of excitation-contraction coupling

目的比较两种细胞分离液分离成年大鼠心肌细胞,进一步表征成年大鼠心室肌细胞兴奋-收缩耦联。方法 Langendorff装置进行主动脉逆流灌流,分别用两种细胞分离液分离成年大鼠心肌细胞,无血清培养并进行腺病毒感染。显微镜下观察单个心肌细胞的形态学特点,荧光显微镜下检测病毒感染。采用IonOptix仪器检测心肌细胞肌节收缩-舒张指标以及心肌细胞钙离子摄入-排出指标。结果两种分离液均可获得70%横纹清晰的长杆状心肌细胞,培养可存活7 d以上。腺病毒感染48 h,绿色荧光蛋白持续表达7 d以上。分离液一获得的心肌细胞不能很好地随电场刺激产生收缩,分离液二获得的细胞可用于检测兴奋-收缩耦联特性,心肌细胞肌节缩短分数为11.61%±2.15%,舒张时间为(0.177±0.031)s,钙瞬变幅度为30.79%±9.74%,钙瞬变衰减时间为(0.300±0.074)s。结论两种分离液均可用于分离和培养成年大鼠心肌细胞,并用于腺病毒转染等长时程研究。分离液二更适用于检测成年大鼠心肌细胞的兴奋-收缩耦联特性。

Objective To compare two separation medium of isolation of adult rat cardiomyocytes , and to observe the characteristics of excitation-contraction coupling of cardiomyocytes .Methods The isolated adult rat heart was hanged on to the Langendorff apparatus for aortic counter-current perfusion and collagenase digestion using two different separation medium.The single cardiomyocytes were cultured and infected with adenovirus . The morphological features of cardiomyocytes were observed with microscope and fluorescent microscope . The shortening-re-lengthening features of sarcomere and the intake-discharge features of calcium were simultaneously recorded by IonOptix equipment .Results 70%rod-shaped with clear-striation adult rat cardiomyocytes could be obtained with the stated two separation medium and cultured in serum-free medium for more than 7 days.GFP could express more than 7 days when the cardiomyocytes were infected with adenovirus .Cardiomyocytes obtained by the first separation medium could not contract with the electrical stimulation, while cardiomyoctyes obtained by the second separation medium could be used for the detection of excitation -contraction coupling .The shortening fraction of sarcomere was 11.61%±2.15% and the relaxing time was （ 0.177±0.031） s.The amplitude of calcium transient was 30.79%±9.74 % and the decaying time of calcium transient was （0.300 ±0.074） s.Conclusion With the stated two separation medium , adult rat cardiomyocytes can be well isolated , cultured and infected with adenovirus .The second separation medium can be used for the detection of excitation-contraction coupling characteristics .