化学性缺氧致大鼠脑细胞[Ca^(2+)]_i和脑红蛋白表达变化及阿司匹林干预研究

Chemical hypoxia induced [Ca^(2+)]_i and NGB alteration and aspirin intervention

目的:观察化学性缺氧状况下大鼠脑细胞[Ca2+]i和脑红蛋白(NGB)表达的变化及阿司匹林(ASA)对其变化的干预,并初步探讨其作用机制。方法:连二亚硫酸钠致原代培养大鼠脑细胞缺氧,用荧光探针Flou-3负载和NGB荧光免疫组化技术双标,激光共聚焦显微镜动态观察。结果:[Ca2+]i和NGB表达与缺氧持续时间有关,随缺氧时间的延长,荧光强度随之增高;经ASA预防性干预,两种荧光强度增高缓慢,其增高程度明显低于单纯缺氧组;治疗性干预,用药早期两种荧光强度增高明显,其增高程度与单纯缺氧组变化相近,后期则增高缓慢,其增高程度明显低于单纯缺氧组。[Ca2+]i浓度和NGB表达呈正相关。结论:ASA对原代培养大鼠脑细胞化学性缺氧损伤具有保护作用,其作用可能与ASA抑制细胞内Ca2+超载有关;NGB的表达可能是受到Ca2+-CaM复合物的调节;ASA预防性用药的早期保护作用明显优于治疗性用药。

AIM： To observe changes of intracellular free calcium levels（[Ca^2＋]i） and neuroglobin（NGB） expression when rats′ brain cells were exposed to chemical hypoxia,and the influence of aspirin（ASA） on changes as well as to study its mechanism primarily.METHODS： Sodium dihionite was used to deprive the oxygen in culture medium,and confocal laser scanning microscope with double fluorescence labeling methods were used to measure the changes of [Ca^2＋]i and NGB expression in rats′ brain cells.Flou-3/AM and Cy-3 were used to indicate calcium concentration and NGB expression respectively.RESULTS： [Ca^2＋]i and NGB expression are related to the time of hypoxia.Compared with the normal control group,average fluorescent intensity of [Ca^2＋]i and NGB expression in the simple hypoxia group increased gradually with the time of the exposure in hypoxia.Moreover,there is the positive correlation between [Ca^2＋]i and NGB expression.[Ca^2＋]i and NGB expression in the ASA pretreated group increased slowly,the increase degree is significantly lower than that in the simple hypoxia group.[Ca^2＋]i and NGB expression in the ASA treated group were increased significantly in early stage,the increase degree is not significant diffexence from the simple hypoxia group.At 120 min,[Ca^2＋]i and NGB expression in the ASA treated group increased slowly,the increase degree is significantly lower than that in the simple hypoxia group.CONCLUSION： ASA has a protective effect on hypoxic injury of rats′ brain cells,which may be attribute to its inhibiting calcium overload,and NGB expression is regulated by Ca2＋^-CaM complex.In addition,the neuroprotective effects of ASA pretreated group were better than ASA treated group in early stage.