RNA干扰介导新生大鼠星形胶质细胞水通道蛋白-4基因沉默

RNA interference mediates silencing of aquaporin-4 gene in cultured neonatal rat astrocytes

目的研究RNA干扰(RNAinterference,RNAi)表达载体对原代培养星形胶质细胞水通道蛋白-4(AQP4)基因的抑制作用。方法构建特异性抑制大鼠AQP4基因表达的AQP4RNAi表达载体和对照载体,用电转染法转染原代培养的新生大鼠星形胶质细胞。将原代培养的星形胶质细胞随机分为未转染组、转染对照载体组和转染AQP4RNAi表达载体组。在细胞转染AQP4RNAi表达载体和对照载体后1~20d,分别采用倒置显微镜和细胞记数观察细胞形态学改变和测定细胞生长率。采用Westernblot和实时荧光定量PCR分别测定AQP4蛋白和mRNA表达水平;用放射性[3H]标记的甲基D-葡萄糖测定细胞体积。结果①转染AQP4RNAi表达载体和对照载体后,星形胶质细胞形态学和生长率均无统计学意义(P>0.05)。②在培养的星形胶质细胞转染AQP4RNAi表达载体后经Westernblot和Real-TimePCR分析显示,AQP4蛋白和mRNA表达水平与其对应的转染对照载体和未转染组细胞比较,呈进行性降低(P<0·05),从而确定了AQP4RNAi对AQP4基因的沉默作用。而且在转染后48h达到最大抑制作用,AQP4蛋白和mRNA抑制率分别约为85·7%和77·5%,同时该最大抑制作用持续至转染后14d。而转染对照载体组与未转染组比较,AQP4蛋白和mRNA表达水平差异均无统计学意义(P>0·05)。③星形胶质细胞在250mmol·L-1低渗溶液中分别培养10,20,30和40min,随着培养时间延长,细胞体积呈进行性增加。而且在相同作用时间点,AQP4基因沉默星形胶质细胞较转染对照载体组细胞,细胞体积增加缓慢(P<0·05),同时转染对照载体组较未转染组细胞体积差异无统计学意义(P>0·05),提示AQP4基因沉默导致星形胶质细胞在低渗液中水渗透性明显减低。结论RNAi表达载体能有效抑制星形胶质细胞AQP4基因的表达,而且AQP4可能是星形胶质细胞水转运的主要调控因素。

Objective To investigate the inhibitory effects of RNA interference （RNAi） expression vector on the expression of aquaporin-4 （AQP4） gene in astrocyte primary cultures. Methods RNAi expression vector, which specifically suppressed AQP4 gene expression（ AQP4 RNAi） and a control vector served as a non-silencing control were constructed, and the neonatal rat astrocytes in primary cultures were nucleofected with the constructed vector. The astrocytes in primary cultures were randomly assigned to non-transfected group, transfected with control vector group, and transfected with AQP4 RNAi expression vector group. The changes of astrocytic morphology were observed under light microscopy and cell growth rate was judged by cell counting at 1～20 day after transfection. The expression levels of AQP4 protein and mRNA were detected by Western blot and real-time PCR respectively; cellular volume was determined using [ 3H ^-3-O-methyl-D-glucose. Results ①There was no difference in astrocytic appearance and cell growth rate between AQP4 RNAi expression vector transfected and control vector-treated cells （ P 〉 0. 05 ）. ② Real-time PCR experiment and Western blotting analysis showed that a progressive and parallel reduction of the expression of AQP4 mRNA and protein in cultured astrocytes after transfection with AQP4 RNAi expression vector compared to that of control vectortreated and untransfected wild type astrocytes（ P 〈0. 05 ）, and reached maximal inhibition, around 85.7% and 77. 5% in AQP4 protein and mRNA respectively at 48 hours, and maintained 14 days after transfection. But AQP4 mRNA and protein expression levels were unaffected in control vector-treated and untransfected cells （P 〉 0. 05 ）. ③After exposure to hypotonic medium （250 mmol·L^-1） for 10,20,30 and 40minutes, the astrocytes showed a time-dependent increase in cellular volume, and the cellular volume was significantly decreased in astrocytes with AQP4 gene silencing than that of control vector-treated cells at the same hypotonic medium（ P 〈 0. 05 ）. Also, there was no difference in cellular volume between control vector-treated and untransfected astrocytes, the results suggested that cellular water permeability in hypotonic medium were decreased significantly in astrocytes with AQP4 gene silencing. Conclusions The RNAi expression vector can effectively inhibit the expression of AQP4 gene in cultured rat astrocytes, and AQP4 may be the major factor responsible for the water transport of cultured astrocytes.