水通道蛋白-4 mRNA沉默可抑制离体缺氧星形胶质细胞水通道蛋白-4的表达

AQP4 expression in hypoxic cultured astrocytes can be inhibited by AQP4 mRNA silence in vitro

目的：研究水通道蛋白-4（AQP4） mRNA沉默对体外缺氧星形胶质细胞AQP4表达的影响.方法： 用氯化钴诱导体外星形胶质细胞缺氧,建立AQP4 mRNA沉默缺氧星形胶质细胞模型.随机分为正常组、对照组、缺氧组和干扰组,观察星形胶质细胞形态,免疫细胞化学、荧光定量PCR、免疫印迹法检测AQP4 mRNA 及蛋白表达.结果： 星形胶质细胞的AQP4 mRNA 和蛋白表达一致.正常组有少数弱阳性表达细胞.对照组与缺氧组成相同的改变：从15min始AQP4 表达增强,以1～2h显著,4h后缓慢增强.干扰组与对照组对比,在15min～4h时间段AQP4表达明显受抑制,尤其在1、2h最突出,与对照组对应时相点相比差异有统计学意义,4h后AQP4表达缓慢回升,12h差异无统计学意义.对照组的星形胶质细胞水肿逐渐加重,以1、2h变化最明显.干扰组在30min～4h细胞水肿明显减轻,尤以1、2h变化明显,4h后细胞水肿继续加剧.结论： 星形胶质细胞水肿与AQP4表达高度一致,AQP4 mRNA沉默可以明显抑制早期缺氧（小于4h）星形胶质细胞的AQP4表达,并减轻细胞水肿.推测AQP4是产生细胞水肿的＂最后共同通道＂.

Objective： To investigate the aquaporin-4 （AQP4） expression of cultured astrocytes in vitro during hypoxia after AQP4 mRNA silencing. Methods： Cultured astrocytes were divided into 4 groups randomly： normal, control, hypoxia （induced by cobalt chloride）, and AQP4 siRNA groups （using AQP4 mRNA interference technology）. Each group was further divided into 7 subgroups based on the observation time point （15 min, 30 min, 1 h, 2 h, 4 h, 6 h and 12 h） and each subgroup contained 6 apertures of astrocytes. The shape of the astrocytes in each subgroup was observed under a light microscope and a transmission electron microscope. AQP4 expression of each subgroup was measured by real time fluorescence quantitative reverse transcript polymerase chain reaction, immunocytochemistry and Western-blotting. Results： There was a significant positive correlation between the expression of AQP4 mRNA and protein. A few astrocytes in the normal group showed slight positive expression of AQP4. There was significant consistency between the control and hypoxia groups. The expression of AQP4 increased rapidly between 15 min and 4 h after hypoxia, and then increased slowly during 4-12 h. However, in the AQP4 siRNA silenced group, AQP4 expression decreased significantly within 4 h, compared with that in the control group. AQP4 expression increased slowly during 4-12 h without significant difference between those two groups at 12 h Intracellular edema along with swelling of the mitochondria, increased nucleolus size, increased endoplasmic reticulum cisternae were observed in the hypoxia group within 4 h In the AQP4 siRNA group, intracellular edema decreased dramatically between 30 min and 4 h, especially at 1-2 h However, intracellular edema was increased after 4 h, even in the AQP4 siRNA group. Conclusion： Cellular edema in astrocytes increases with AQP4 expression. AQP4 expression in astrocytes as well as cellular edema can be sharply inhibited by RNA interference technology within 4 h after hypoxia, which implies that the AQP4 may be the ＂final common channel＂ of celluar edema.