微管解聚与心肌细胞缺氧性损害的实验研究

目的观察缺氧引起的心肌细胞微管解聚是否能导致细胞线粒体通透性转换孔开放,降低细胞活性,引起细胞缺氧性损害.方法将原代培养的乳鼠心肌细胞随机分为对照(常氧)组(A组)、缺氧组(B组)、常氧 微管解聚剂组(C组)、缺氧 微管稳定剂组(D组).检测各组0.5、1、3、6、12 h微管免疫荧光,了解微管结构变化,以免疫印迹(Western blot)法半定量分析聚合态微管蛋白变化,用酯化钙黄绿素和氯化钴共孵育的方法检测线粒体通透性转换孔开放,用MTT法测定细胞活性.结果 B组和C组聚合态微管在缺氧0.5 h后即发生明显破坏,同时能观察到线粒体通透性转换孔开放和细胞活性下降,且随着缺氧时间的延长,以上现象愈发显著.而同一时相点D组聚合态微管被破坏程度、线粒体通透性转换孔开放情况和细胞活性下降均明显轻于B组.结论缺氧可引起心肌细胞聚合态微管明显破坏,导致细胞线粒体通透性转换孔开放,进而影响细胞活性,引起细胞缺氧性损害.微管解聚剂能较好地模拟缺氧引起的微管破坏作用,导致细胞活性下降,而微管稳定剂能有效减轻缺氧引起的微管破坏,保护细胞活性,明显减轻心肌细胞的缺氧性损害.

Effects of microtubule disassembly on hypoxic injury of cultured cardiomyocytes

Objective To investigate whether microtubule disassembly plays an important role in the pathogenesis of the opening of mitochondria permeability transition pore (MPTP) in hypoxic cardiomyocytes and the decrease of its activity, resulting in its hypoxic injury. Methods Neonatal rat cardiomyocytes in primary culture were randomized as normoxia group (A), hypoxic group (B), normoxia treated with microtubule destabilizing agent (Colchicine) group (C), hypoxia treated with microtubule stabilizing agent (Taxol) group (D). At 0.5, 1, 3, 6, 12 h after treatment, polymeric tubulin was detected by immunofluorescence and Western blotting, mitochondria permeability transition pore (MPTP) open by coloading with calcein AM and cobalt chloride, and the activity of cells by measuring the mitochondrial-dependent reduction of MTT to formazan. Results Early microtubule disassembly, MPTP open and activity decrease of cardiomyocytes in both groups B and C were observed at 0.5 h after treatment. These phenomena all became more and more significant with the prolongation of treatment. However, microtubule disassembly, MPTP open and activity decrease of cardiomyocytes of group D were significantly lower than those of group B. Conclusion Microtubule disassembly happened at 0.5 h after hypoxic treatment. Microtubule stabling agent Taxol and destabilizing agent Colchicine can regulate microtubule integrity efficiently. The microtubule damage plays an important role in the hypoxic injury of cardiomyocytes.