热休克蛋白70对过氧化氢所致乳鼠心肌细胞核仁损伤的保护作用

为探讨氧化应激时体外培养的新生Wistar大鼠心肌细胞核仁损伤以及热休克蛋白70对损伤核仁的保护作用。用0.5mmol/L过氧化氢处理原代培养的心肌细胞0,30,60min，采用甲苯胺兰染色核仁及电镜技术观察核仁结构的改变；并通过热休克预处理及反义技术诱导或阻断热休克蛋白70的表达，观察其对核仁损伤的保护作用。结果发现，光镜下过氧化氢损伤组心肌细胞核仁染色颗粒数增多，电镜下核仁结构松散，核仁组份分离。热休克预处理导致心肌细胞中热休克蛋白70表达明显增加，并使过氧化氢缺致心肌细胞核仁损伤明显减轻，免疫组织化学显示过氧化氢可引起热休克蛋白70从胞浆到胞核，再到核仁的移位，热休克蛋白70反义寡核苷酸很大程度上能阻断热休克预处理对心肌细胞核仁损伤的保护作用。结果提示，过氧化氢可导致体外培养的新生大鼠心肌细胞核仁结构损伤，热休克蛋白70高表达及其向核仁的移位对上述损伤具有明显保护作用。

The Protective Effect of Heat Shock Protein 70 on The Nucleolar Impairment of Cultured Neonatal Cardiomyocyte Induced by Hydrogen Peroxide

Aim To investigate the nucleolar changes and the effect of heat shock protein 70 (HSP70) during the oxidative stress using an injury model of neonatal rat cardiomyocyte induced by hydrogen peroxide (H 2O 2). Methods The nucleolar impairment induced by 0.5 mmol/L H 2O 2 was observed with toluidine blue staining and electronic microscopy. The protective effect of HSP70 on nucleolar damage was investigated with heat shock pretreatment and antisense oligonucleotides of HSP70. Results H 2O 2 (0.5 mmol/L) significantly increased numbers of stained nucleolar granules, and resulted in obvious segregation of nucleolar components as indicated by staining with toluidine blue and electronic microscopy. Western blot analysis demonstrated that heat shock pretreatment induced expression of HSP70. Heat shock pretreatment significantly relieved the nucleolar impairment induced by H 2O 2. Moreover, it was showed by immunocytochemistry that H 2O 2 mediated HSP70 translocation from cytoplasm to nucleus and nucleolus in cardiomyocyte. Additional investigation showed that HSP70 antisense oligonucleotide can remarkably block the protective effect of HSP70 against the nucleolar injury induced by H 2O 2. Conclusions The nucleolar impairment of primary cultured cardiomyocyte can be induced by H 2O 2 and be protected by HSP70, which is over expressed and translocated to the nucleolus in the process.