缺氧预适应小鼠脑匀浆去蛋白液中抗缺氧有效成分的初步研究  

Deproteinized brain tissue extracts from hypoxia-preconditioned mice protect differentiated PC12 cells

作　　者：李宏[1,2] 高钰琪[1] 

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400038 [2]重庆教育学院生命科学与化学系,重庆400067

出　　处：《第三军医大学学报》2008年第11期1028-1032,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金重大项目(30393131)~~

摘　　要：目的探讨缺氧预适应小鼠脑匀浆去蛋白液(deproteinized brain tissue extracts of hypoxia-preconditioned mice,DHP)对NGF诱导分化的PC12细胞缺氧耐受性的影响及其与腺苷的关系。方法复制缺氧预适应小鼠模型,高氯酸法制备DHP。HPLC法检测DHP中腺苷含量及正常小鼠脑匀浆去蛋白液(deproteinized brain tissue extracts of normal mice,DN)中腺苷含量。在已分化PC12细胞中加入DHP,以DN作对照,以1.0、10.0、100.0μmol/L的腺苷为阳性对照,分别于缺氧(2%O2)培养24、48、72h后检测MTT值、LDH透出率以及72h晚期凋亡率;在DHP、10.0μmol/L腺苷阳性对照组中分别加入腺苷A1、A2A受体阻断剂DPCPX、SCH58261(5.0μmol/L),以DN作对照,缺氧24、48、72h后分别检测各组MTT值、LDH透出率。结果DHP中腺苷含量显著高于DN;缺氧24h时,DHP组MTT值显著高于DN组,其LDH透出率显著低于DN组。随缺氧时间延长,DHP保护作用逐渐减弱,至72h时,DHP组MTT值、LDH透出率、晚期凋亡率与DN组均无显著差异;A2A受体阻断剂SCH58261可阻断DHP的保护作用。结论DHP在缺氧初期对分化PC12细胞的保护作用与缺氧预适应中腺苷含量增加有关,保护作用可能通过A2A受体实现。Objective To investigate the effects of deproteinized brain tissue extracts from hypoxia-precon-ditioned mice （DHP） on the tolerance of differentiated PC12 cells （DPC12） to hypoxia and its relationship with adenosine （ADO）. Methods The mouse model of acute repetitive hypoxia was reproduced and deproteinized brain tissue extracts were prepared by means of perchloric acid. High performance liquid chromatogram （HPLC） was used to determine ADO contents in the extracts. DHP was added into the culture of 4-day DPC12 cells, and deproteinized brain tissue extracts of normal mice （DN） were used as control. Besides, ADO at final concentrations of 1.0, 10.0, 100.0 mol/L was used as positive controls. Then DPC12 cells were cultured in hypoxia （2%O2）. After 24-, 48-, 72-hour hypoxia exposure, colorimetric method of tetrazolium salt MTr as-says （A570） and lactate dehydrogenase （LDH） release assays were conducted. Furthermore, apoptotic percent-ages at late stage （72-hour hypoxia） were detected with Hoechst 33258 stained fluorescence microscopy; Then, adenosine A1 , A2A receptor antagonists, DPCPX and SCH 58261, were respectively added into DHP groups and 10.0 μmol/L adenosine positive control groups, with DN group as control; MTF, LDH assays were performed after 24-, 48-, 72-hour hypoxia. Results ADO contents in DHP significantly increased; MTr value of DHP group was significantly higher than that of DN group, and LDH release percentage was significantly lower than that of DN group after 24-hour hypoxia. As the period of hypoxia prolonged, DHP gradually lost its protective effect. After 72-hour hypoxia, MTT value, LDH release percentage and apoptotic percentage at late stage of DHP group showed no significant differences from those of DN group. SCH 58261 but not DPCPX blocked the protective effect of DHP. Conclusion The protective effects of DHP on DPC12 cells at early stage were related to ADO increase in DHP and the effects might be mediated via A2A receptor.

关 键 词：缺氧 预适应 小鼠 脑匀浆去蛋白液 分化PCI2细胞 

分 类 号：R322.81[医药卫生—人体解剖和组织胚胎学] R363[医药卫生—基础医学]