微管解聚剂在缺氧早期大鼠心肌细胞线粒体损害中的作用

Effect of the microtubule depolymerization on mitochondria damage in rat myocadiocytes early after hypoxia

目的了解微管解聚剂在缺氧早期大鼠心肌细胞线粒体损害中的作用。方法常规方法分离培养Wistar乳鼠心肌细胞,分为正常组、微管解聚组(培养液中加入4μmol/L秋水仙碱)、缺氧组、缺氧解聚组(4μmol/L秋水仙碱联合缺氧处理)。分别采用激光共聚焦显微镜检测4组细胞线粒体分布情况,透射电镜观察线粒体形态变化,生物氧耗呼吸仪检测呼吸调节比(RCR),高效液相色谱仪检测腺苷三磷酸(ATP)含量。缺氧组及缺氧解聚组所设时相点为缺氧后10、20、30、60 min。结果正常组线粒体呈粒状、排列规则,微管解聚组较之发生轻微改变。缺氧20、30、60 min,缺氧解聚组线粒体分布的无规律性及形态结构损害均较缺氧组严重;细胞RCR分别为1.58±0.37、1.51±0.32、1.12±0.11,明显低于缺氧组3.85±0.56、2.98±0.44、1.79±0.73(P<0.01);ATP含量分别为(419±83)、(326±73)、(295±58)ng/mg,亦明显低于缺氧组(475±68)、(397±59)、(336±67)ng/m0g(P<0.01)。结论微管解聚剂可加重缺氧引起的心肌细胞线粒体分布紊乱和形态结构损害,以及线粒体呼吸功能和能量代谢障碍,它在线粒体缺氧损害机制中具有重要作用。

Objective To investigate the effect of microtubule depolymerization on mitochondria damage in rat myocardiocytes early after hypoxia. Methods Myoeardiocytes from Wistar rats were isolated according to routine procedure, and they were randomly divided into control group, depolymerization group （with treatment of 4 μmol/L colchicines in the culture medium） , hypoxia group, hypoxia and depolymerization group（ with treatment of 4μmol/L colchicines in the culture medium combined with low oxygen tension）. The changes in distribution of the mitochondria were examined with laser confocal microscopy , the morphology and the structure of mitochondria was observed by transmission electron microscope, the respiration control ratio（RCR） was determined by respirometer, and the content of adenosine triphosphate （ATP） in endochylema was detected with liquid chromatograph at 20, 30, 60 post - hypoxia minutes （ PHM ）. Re- sults In control group, the mitochondria was in granular form, with regular arrangement, while mild changes were observed in depolymerization group. At 20,30, and 60 PHM, the disarrangement in distribution and morphologic damage were aggravated in hypoxia depolymerization group, and the RCR （1.58 ± 0.37, 1.51±0.32,1. 12 ± 0. 11, respectively ） were evidently lower than those in hypoxia group（3.85 ± 0.56, 2.98±0.44,1.79±0.73,respectively , P 〈0.01）.The content of ATP showed the same tendency at the same time-points （419 ± 83,326 ± 73,295 ± 58 ng/mg） compared with hypoxia depolymerization group [ （475 ±68,397 ±59,336 ±67 ng/mg） in hypoxia group ]. Conclusion The disarrangement in distribution of mitochondria , as well as the damage in mitochondrial structure, respiratory function and energy metabolism, can be aggravated by microtubule depolymerization after hypoxia, which indicates that microtubule depolymerization plays an important role in the mitochondria damage.