目的 观察缺氧/复氧条件下大脑星形胶质细胞水通道蛋白4(AQP4)的表达变化以及亚低温对其表达的影响,探讨脑缺血再灌注脑水肿与AQP4的关系以及亚低温对脑缺血再灌注损伤的保护机制.方法 利用新生24 h内的SD大鼠,进行原代、传代培养,将星形胶质细胞分为对照组、常温组及亚低温组.用台盼蓝染色法测定37℃及32℃时,缺氧/复氧不同时间点星形胶质细胞的存活率,作为细胞受损指标,用倒置相差显微镜对细胞进行形态学观察,应用细胞免疫化学技术检测星形胶质细胞缺氧/复氧各个时间点AQP4的表达变化及亚低温的干预效果.结果 (1)缺氧4,8h时细胞形态变化不明显,随着复氧时间的延长,可见细化逐渐明显,而亚低温干预的细胞形态及细胞存活力变化均较相应的常温组明显减轻；(2)缺氧及复氧早期AQP4的表达降低,复氧后6h随着时间延长AQP4表达明显增多,在复氧≤8h,常温组及亚低温组的表达均低于对照组(均P＜0.05或0.01),而复氧后10,12h,AQP4蛋白表达均明显高于对照组(均P＜0.05或0.01)；(3)在复氧后各时间点亚低温组AQP4的表达均明显低于常温组(均P＜0.05或0.01).结论 星形胶质细胞对缺氧的耐受能力较强,亚低温可以减轻缺氧/复氧后星形胶质细胞的损伤,通过降低AQP4的表达,可能是亚低温减轻缺血性脑水肿的作用机制之一.
Effect of mild hypothermia on the expression of aquaporin4 in astrocytes after hypoxia/reoxygenation
LIU Jun,DONG Rui-guo,LI Xiao-bin
Objective The aim the present study is to examine the expression change oi AQ, P4 on astrocytes and the effect of mild hypothermia on its expression following brain hypoxia/regoxygenation, and to investigate the relationship between cerebral ischemia--reperfusion edema and AQP4 expression, and the mechanism of protection of mild hypothermia on cerebral ischemiareperfusion injury. Methods The primary and subculture cells from the cortex of SD（less than 24 hour old）fetal rats were divided into three groups：control,normal temperature, and mild hypotherma groups. Trypan blue stai- ning was used to detect the astocytes survival rate and changes at different time points after hypoxia/ reoxygenation in 37＂C and 32~C. Cellular damage index was calculated from morphology examination of the cells under inverted phase contrast microscope. [mmunocytochemisca[ assay was carried out to meas- ure the expression change of AQP4 in astrocytes at the indicated time points after hypoxia/reoxygenation and the intervention of mind hypothermia. Results （1） The cell morphology and survival rate were not remarkable at 4 and 8 hours after hypoxia, but reoxygenation resulted in a significant damage. The lon- ger latency,the greater damage was observed in regard to the morphology and the survival rate of astro- eytes. There was lesser damage in mild hypotherma group at corresponding time points. （2）The expres- sion of AQP4 was showed a tendency to decrease at early phase but rising after reoxygenation for 6 h. As time extended it showed a tendency to increase. The expression of AQP4 in normal temperature and mild hypotherma group was significantly lower than in the control group at time points less than or equal to 8 h （P 〈 0.05 or 0.01） ,higher than in the control group atl0 and 12 hrs after reoxygenation （P 〈 0.05 or 0.01）. （3）The expression of AQP4 in mild hypothermia group were significantly lower than in common temperature group at all time points after reoxygenation （P 〈 0. 05 or 0. 01）. Conclusions Astrocytes have stronger tolerance to hypoxia. Mild hypothermia can relieve the deterioration of astrocytes after hypoxiaand reoxygenation and can restrain the expression of AQP4. Restraining AQP4 expressions might be one of the mechanisms underlying the protective effect of mild hypothermia on brain edema.