心肌细胞缺氧引起微管结构破坏导致线粒体通透性转换孔开放的实验研究

目的观察缺氧引起的心肌细胞微管破坏能否导致线粒体通透性转换孔(MPTP)开放及其呼吸功能的变化情况。方法将原代培养的SD大鼠乳鼠同批心肌细胞随机分为对照组、缺氧组、常氧 解聚剂组、缺氧 稳定剂组。对照组常氧培养。常氧 解聚剂组加入8μmol/L秋水仙素室温平衡30 min后,与对照组一起行常氧培养。缺氧 稳定剂组加入10μmol/L紫杉醇室温平衡30 min后,和缺氧组一起行缺氧处理。检测对照组及其他组细胞处理0．5、1．0、3．0、6．0、12．0h后微管免疫荧光、MPTP的开放及线粒体内膜电位变化,噻唑蓝法测定线粒体的呼吸功能。结果处理0．5h,缺氧组和常氧 解聚剂组微管免疫荧光强度分别为(76．1±3．9)%、(74．8±5．O)%,微管发生明显破坏,MPTP开放,线粒体内膜电位损耗和细胞呼吸功能下降,且随着处理时间的延长,以上现象愈发显著。处理0．5h,缺氧 稳定剂组微管免疫荧光强度为(92．8±4．0)%,与对照组(100．0±0．0)%相近(P＞0．05)；随着处理时间的延长,该组各检测指标的改变程度均明显轻于缺氧组(P＜0．05或0．01)。结论缺氧可引起心肌细胞微管明显破坏,导致MPTP开放,进而影响线粒体的呼吸功能。微管解聚剂能较好地模拟缺氧引起的微管破坏；微管稳定剂则能有效地减轻缺氧引起的微管破坏,改善线粒体通透性转换及其呼吸功能。

Study on the influence of hypoxia induced microtubule damage on the opening of mitochondrial permeable transition pore of cardiac myocytes in rat

OBJECTIVE: To investigate the influence of hypoxia induced microtubule damage on the opening of mitochondrial permeable transition pore (MPTP)of cardiac myocytes and on the decrease of respiratory function in rat. METHODS: Primary cultured myocardial cells from 30 neonatal rats were randomized as normoxic group (A), hypoxia group (B), normoxia with microtubule destabilizing agent group (C, with treatment of 8 micromol/L colchicines for 30 minutes before normoxia), and hypoxia with microtubule stabilizing agent group (D, with treatment of 10 micromol/L taxol for 30 minutes before hypoxia). beta-tubulin immunofluorescence ,the opening of mitochondria permeability transition pore, and the mitochondrial inner membrane potential were detected at 0.5, 1, 3, 6 and 12 post-treatment hours (PTH), and the mitochondrial respiratory function was determined by MTT method. The changes in these indices were also determined in A group at the corresponding time-points. RESULTS: Obvious damage of polymerized microtubule, opening of MPTP, mitochondrial inner membrane potential loss and decrease of myocardial respiratory activity were observed in both group B and C at 0.5 PTH, and they became more and more serious afterwards. However, the changes in the above indices in D group were much better than those in B group (P < 0.05 or 0.01), and no difference was found between D (92.8 +/- 4.0)% and C [(100.0 +/- 0.0) %, P > 0.05] groups. CONCLUSION: Hypoxia played a role in the myocardial microtubule damage as well as in the opening of MPTP. Moreover, hypoxia could also impair the mitochondrial respiratory function. Microtubule destabilizing agent could reproduce well the process of hypoxia induced microtubule damage, while the stablizing agent exerted protective effect by improving the transition of mitochondrial permeability and the mitochondria respiratory function.