低渗液对星形胶质细胞水通道蛋白-4表达的影响

目的　探讨体外培养星形胶质细胞在低渗状态下水通道蛋白 4 (AQP4 )表达的变化特点及其所起的作用。方法　取生后 2d的Wistar大鼠大脑皮质进行星形胶质细胞纯培养 ,随机分为对照组和低渗组 ,每组分为 3、6、12、2 4h共 4个时间点 ,每个时间点细胞孔数为 6。对照组予以正常培养液常规培养 ,低渗组分别予以不同程度的低渗液作用于细胞 ,以建立星形胶质细胞对低渗液反应的实验模型。利用倒置显微镜和透射电镜观察低渗液对星形胶质细胞的形态学影响 ,并通过免疫细胞化学、逆转录 聚合酶链反应 (RT PCR)、乳酸脱氢酶活性的测定及图像分析等方法研究星形胶质细胞对低渗液的反应及AQP4的表达变化。结果　对照组在正常渗透压培养液培养 3、6、12、2 4h后 ,AQP4表达无明显差异 (均P >0 .0 5 )。在相同的作用时间条件下 ,各低渗组AQP4mRNA和蛋白表达水平均明显高于对照组 (均P <0 .0 5 )。而在同一低渗状态下的不同培养时间点和不同低渗状态下的同一培养时间点 ,AQP4表达随作用时间的延长和低渗程度的增加而增强 ,AQP4mRNA和蛋白的表达呈明显正相关 (r >0 .836 9,P <0 .0 5 ) ;同时星形胶质细胞表现出特征性的细胞水肿形态学变化 ,细胞存活能力明显下降 ,其严重程度与低渗液的作用时间和渗透压有关。结论　低渗液

Effect of hypotonic medium on expression of aquaporin-4 in astrocytes

Objective To investigate the expression of aquaporin-4 (AQP4) and its role in cultured rat astrocytes after exposure to hypotonic medium. Methods Rat cerebral cortical astrocytes from 2 days newborn Wistar rats were undergone the pure culture. The cells were divided into control group and hypotonic groups randomly, in which group was examined at interval times of 3, 6, 12 and 24 h, respectively (n=6), and its model of cell edema was established by exposed to hypotonic medium. The cells of the control group were cultured in normal culture medium. Inverted microscope and transmission electron microscope were used to observe the cells changes. The reactivity and rules of AQP4mRNA and protein expression of astrocyte to hypotonic medium were studied by RT-PCR, immunocytochemistry, the activity of LDH and imaging analysis. Results There was no significant change in the control group. After astrocytes cultured for 3, 6, 12, 24 h with hypotonic medium (268, 254, 240 mmol/L), the astrocytes showed typical features of cell edema, especially in lower osmolality group. The activity of LDH showed a significant change than those in the control group after astrocytes exposed to hypotonic medium( P <0.05). RT-PCR and immunocytochemistry analysis confirmed that the expression of AQP4mRNA and protein was remarkably increased( P <0.05), up-graduated expression of AQP4 was correlated with hypotonic medium and time dependent manner . The AQP4mRNA expression showed a positive relationship with the AQP4 protein expression during the astrocytes edema formation( r >0.8369, P <0.05). Conclusion Hyponotic medium could induce cell edema and expression of AQP4mRNA and protein increased, and vibility of astrocytes inhibited. The AQP4 expression showed a positive relationship with the osmolality. The results suggest that AQP4 participated in the brain edema formation.