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目的构建能有效抑制大鼠星形胶质细胞(astrocytes,ASTs)连接蛋白43(connexin 43,Cx43)基因表达的siRNA载体,并检测抑制Cx43表达对星形胶质细胞谷氨酸释放的影响。方法合成针对大鼠Cx43基因的siRNA寡核苷酸,构建pGCsi.U6.neo.GFP质粒载体。质粒通过电转方法转染原代星形胶质细胞后,用免疫荧光染色及Western blot检测转染干扰质粒对大鼠ASTs Cx43蛋白表达的影响。用谷氨酸ELISA定量试剂盒检测释放至培养基中谷氨酸的浓度,并用DAPI染色进行细胞计数。结果测序结果显示,Cx43基因的目的片段正确插入载体,并保持正确的读码框。干扰质粒转染ASTs 48 h后与对照组相比,Cx43蛋白表达明显下降(P<0.05),细胞存活未受影响,而谷氨酸释放明显减少(P<0.05)。结论成功构建了抑制大鼠Cx43基因表达的siRNA载体,抑制Cx43表达可降低ASTs谷氨酸的基础释放。
Objective To construct an siRNA vector that can effectively inhibit the gene expression of connexin 43 (Cx43) in rat astrocytes and detect the effect of inhibiting Cx43 expression on glutamate release from astrocytes. Methods siRNA oligonucleotides targeting rat Cx43 gene were synthesized to construct pGCsi.U6.neo.GFP plasmid vector. Plasmids were transfected into primary astrocytes by electroporation. The expression of Cx43 protein in ASTs was detected by immunofluorescence staining and Western blot. The concentration of glutamate released into the medium was measured with a glutamate ELISA kit and the cells were counted with DAPI staining. Results The sequencing results showed that the target fragment of Cx43 gene was correctly inserted into the vector and maintained the correct reading frame. Compared with the control group, the expression of Cx43 protein was significantly decreased (P <0.05), but the cell survival was not affected, but glutamate release was significantly reduced (P <0.05). Conclusion The siRNA vector that inhibits the expression of Cx43 gene in rat is successfully constructed. Inhibition of Cx43 expression can reduce the basic release of glutamate in ASTs.