氯化钴诱发缺氧对体外星形胶质细胞水通道蛋白4表达的影响  

The effects of CoCl2-induced in vitro hypoxia on aquaporin-4 expression in cultured astrocytes

作　　者：鲁宏[1] 胡惠[1] 何占平[1] 

机构地区：[1]中南大学湘雅医学院附属海口医院(海口市人民医院)放射科,海口570208

出　　处：《中华神经科杂志》2011年第2期117-121,共5页Chinese Journal of Neurology

基　　金：国家自然科学基金资助项目（30960399）;海南省科技计划资助项目（081007）

摘　　要：目的 研究缺氧对星形胶质细胞水通道蛋白4（AQP4）表达水平的影响.方法 体外培养星形胶质细胞,用氯化钴建立星形胶质细胞缺氧模型,采用随机数字表法分为对照组和缺氧组,每组再分为15、30 min,1、2、4、6和12 h时间点亚组,共14组,每组6孔.观察星形胶质细胞形态;采用原位杂交、荧光定量、免疫细胞化学、Western blot方法检测AQP4基因及蛋白表达;用Ex-△△Ct表示AQP4基因表达量、D表示AQP4蛋白表达量.结果 星形胶质细胞的AQP4基因和蛋白变化具有一致性（r=0.85,P＜0.01）.对照组星形胶质细胞呈弱阳性表达.缺氧组星形胶质细胞内AQP4表达从15 min开始随时间的延长而增强,以1～4 h内最明显（mRNA表达缺氧组分别为0.26±0.04、0.31±0.02、0.36±0.04,对照组分别为0.06±0.01、0.09±0.01、0.08±0.01;t=16.51、18.20、15.26,均P＜0.01）,6 h后缓慢增强,与对照组对应时间点相比差异均有统计学意义.随缺氧时间延长,细胞肿胀逐渐加重,以1～4 h时间点最显著,电镜下见细胞核增大、内质网和线粒体肿胀,并随时间推移而加重.12 h见少数细胞破裂.结论 缺氧所致星形胶质细胞的病理变化是细胞水肿,水肿程度与缺氧时间和AQP4高表达呈正相关,AQP4可能是星形胶质细胞水肿的重要分子基础.Objective To investigate the expression of aquaporin-4 （AQP4） in cultured astrocytes after in vitro hypoxia induced by CoCl2. Methods After primary culture and subculture, the astrocytes were placed in a controlled atmosphere culture chamber. Both control group and hypoxia groups were established.These groups were further divided into seven sub-groups according to the different time intervals： 15, 30minutes and 1,2, 4, 6, 12 hours, respectively （6 apertures for each group）. The shape of the astrocytes in each group was observed with light microscopy and transmission electron microscopy （ TEM ）. All groups were examined using in situ hybridization, real time fluorescence quantitative reverse transcriptase polymerase chain reaction, immunocytochemistry and Western blot. The data was analyzed statistically with SPSS 13.0 software. Results There was significant consistency between the AQP4 mRNA and protein （ r =0. 85, P 〈0. 01 ）. There was slight positive expression of AQP4 in a few astrocytes of the control groups. In the hypoxia groups, the expression of AQP4 increased within 15 minutes; the increase was most prominent between 1 and 4 hours（ mRNA in hypoxia groups： 0. 26 ± 0. 04, 0. 31 ± 0. 02, 0. 36 ± 0. 04; control groups：0. 06 ±0. 01,0. 09 ±0. 01,0. 08 ±0. 01 ）after hypoxia and became less between 6 and 12 hours; There was significant difference in the AQP4 expression between the hypoxia groups and control groups among different time points （t = 16. 51, 18.20, 15.26,all P〈0. 01 ）. The corresponding pathological changes were cellular edema, which was most prominent between 1 and 4 hours. Under TEM, increase in size of the nucleolus and swelling of endoplasmic reticulum and mitochondria; these changes became more marked with time.Disruption of a few astrocytes was detected in the hypoxia groups at 12 hours. Conclusions The pathological change of astrocytes is cellular edema following hypoxia. There is a positive relationship between the presence and degree of cellular edema

关 键 词：星形细胞 细胞 培养的 细胞低氧 水通道蛋白质4 

分 类 号：R285.5[医药卫生—中药学]