Investigation for morphology and function of astrocyte end-food membrane

本研究采用Westernblot方法检测大鼠原代星形胶质细胞水通道蛋白4（AQP 4）、5（AQP 5）、9（AQP 9）和Kir4.1的表达。在3至4次传代后，用十二烷基硫酸钠（SDS）和二硫苏糖醇溶解细胞。裂解物用SDS-PAGE分离并转移到聚偏二氟乙烯膜上。用一抗和二抗（辣根过氧化物酶标记的IgG）孵育细胞膜，产生化学发光，用X光片检测，发现星形胶质细胞特异性胶质细胞酸性蛋白（GFAP）在约50 KDa处有强表达。经蛋白质数据库SWISS-PROT分析，GFAP的分子大小为49.9KDa，经鉴定为GFAP。AQP 4在30 KDa和60 KDa左右呈双带。根据SWISS-PROT，AQP 4产生两种不同分子大小的蛋白质，因为AQP 4 mRNA中的两个起始密码子分别为M1和M23，产生的蛋白质分子量分别为34.5KDa和32.5KDa，AQP 5和AQP 9在60 KDa处各显示一条很弱的条带。经SWISS-PROT分析，AQP 5和AQP 9的分子量分别为28.4KDa和31.4KDa。Kir 4. 1在50 KDa左右有一条阳性条带，表明其为AQP 5和AQP 9的二聚体。SWISS-PROT分析表明Kir4.1的分子量为42.4KDa。由于我们的发现与SWISS-PROT数据之间的差异很小，因此可以在培养的星形胶质细胞中检测到Kir4.1。下一步计划是对细胞组分中水通道蛋白表达进行定量分析，并对脑水肿下星形胶质细胞中水通道蛋白的定位进行形态学分析。

The aim of this study was to demonstrate the expression of aquaporin4 (AQP4), aquaporin5 (AQP5), aquaporin9 (AQP9) and Kir4.1 in rat primary astrocytes using Western blot.Trypsinized new-born rat brain was harvested into culture flasks. After 3 to 4 passages, the cell was solubilized with sodium dodecyl sulfate (SDS) and dithiothreitol. The lysate was separated with SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was incubated with primary antibodies and the secondary antibody (Horseradish peroxidase labeled IgG), then generated chemiluminescence was detected with X-ray film.Glial fibrillary acidic protein (GFAP) specific for astrocytes showed was strongly expressed at about 50KDa. According to the protein database SWISS-PROT, the molecular size of GFAP is 49.9KDa, and then we identified the band as GFAP. AQP4 showed dual band at about 30KDa and 60KDa. According to SWISS-PROT, AQP4 is produced two different molecular size of protein, because two initiation codons in AQP4 mRNA called M1 and M23, then produced protein were 34.5KDa and 32.5KDa.AQP5 and AQP9 showed a very weak band at each 60KDa. According to SWISS-PROT, molecular size of AQP5 and AQP9 are 28.4KDa and 31.4KDa, respectively. As a result, this may be dimer of AQP5 and AQP9 in our experiments.Kir4.1 showed a positive band at about 50KDa. SWISS-PROT showed that the molecular size of Kir4.1 was 42.4KDa. It was possible to detect Kir4.1 in cultured astrocytes because this discrepancy between our finding and SWISS-PROT data was very little.The next plans are to make a quantitative analysis of aquaporin expression in cell fraction and a morphological analysis of localization of aquaporin in astrocytes under brain edema.