缺氧预适应小鼠脑匀浆提取液增强分化PC12细胞缺氧耐受性的最适浓度研究  

A study of optimal concentration of brain tissue extract of hypoxia-preconditioned mice to enhance tolerance of differentiated PC12 cells to hypoxia

作　　者：李宏[1] 高钰琪[2] 

机构地区：[1]第三军医大学高原医学系高原生理学与病理生理学教研室全军高原医学重点实验室重庆重庆教育学院生化系,重庆400067 [2]第三军医大学高原医学系高原生理学与病理生理学教研室全军高原医学重点实验室,重庆400038

出　　处：《重庆医科大学学报》2008年第5期520-525,共6页Journal of Chongqing Medical University

基　　金：国家自然科学基金重大项目(NO.30393131)

摘　　要：目的:探索缺氧预适应小鼠脑匀浆提取液(Brain tissue extract of hypoxia-preconditioned mice,BHP)对NGF诱导分化的PC12细胞缺氧耐受性的影响及保护性作用的适合浓度。方法:复制小鼠急性重复缺氧模型,制备BHP,用考马斯亮蓝法定量蛋白。在分化PC12细胞中加入BHP使其蛋白终浓度分别为1.0、10.0、100.0μg/ml(BHP组)。以等蛋白终浓度正常小鼠脑匀浆提取液(Brain tissue extract of normal mice,BN)作为对照组(BN组)。同时设置只加PBS的溶剂对照组(PBS组)。缺氧(2%o_2)培养24、48、72h后,采用回唑盐(MTT)比色法检测各组细胞活力(OD_(570nm)值)和乳酸脱氢酶(LDH)透出率、缺氧72h晚期凋亡率。结果:缺氧24h时,蛋白终浓度为10.0、100.0μg/ml的BHP组OD_(570nm)值显著高于同浓度BN组和缺氧PBS组,其LDH透出率显著低于同浓度BN组和缺氧PBS组。随缺氧时间延长,其保护作用逐渐减弱。至缺氧72h时,仅蛋白终浓度为100.0μg/ml的BHP组OD_(570nm)值显著高于同浓度BN组,其LDH透出率、晚期凋亡率均显著低于缺氧PBS组。结论:BHP对分化PC12细胞缺氧耐受性的增强作用有浓度依赖性和时间依赖性,蛋白终浓度为100.0μg/ml的BHP组保护作用稳定、持久。Objective：To investigate the effect of brain tissue extract of hypoxia-preconditioned mice （BHP） on tolerance of NGF-induced differentiated PC12 cells to hypoxia and the optimal concentration for protective effect. Methods：The mice model of acute repetitive hypoxia was reproduced and BHP was prepared. BHP was added into the cultures of 4-5 day differentiated PC12 cells and the final protein concentrations of BHP were 1.0.10.0.100.0 μg/ml（BHP groups） respectively. Brain tissue extract of normal mice（BN） at the same three protein concentrations were used as controls（BN groups） and PBS solvent control groups were also prepared in which only PBS was added（PBS groups）. Then differentiated PC12 cells were cultured in hypoxia （2%02）. After 24,48,72 hours＇hypoxia,colorimetric method（OD570nm） of tetrazolium salt MrlT staining was adopted to determine cell viability and lactate dehydrogenase（LDH） leakage assays were also conducted at the same time. Besides, apoptotic per- centages at late stage（72 hours＇ hypoxia） were detected. Resu/ts：After 24 hours＇ hypoxia,OD^o,m values of BHP groups with protein concentrations being 10.0.100.0~g/ml were significantly higher,but their LDH leakage percentages were significantly lower than those of BN groups with the same protein concentrations and of hypoxic PBS group. With hypoxia prolonging,BHP gradually lost the protective effect. At the time point of 72 hours＇ hypoxia,OD570nm values of only BHP group at 100.0μm/ml protein concentration was significantly higher than that of the corresponding BN groups, its LDH leakage percentage and apoptotic percentage was significantly lower than those of the hypoxic PBS group. Conclusion：The effects of BHP on tolerance of differentiated PC12 cells to hypoxia depend on its concentrations and on its treating time. Besides,BHP at final concentration of 100.0μ g/ml protein showed steady, prolonged protective effect.

关 键 词：缺氧 预处理 小鼠 脑匀浆提取液 分化PC12细胞 

分 类 号：R363[医药卫生—病理学]