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Cx43基因干扰对大鼠星形胶质细胞谷氨酸基础释放的影响

, PP. 701-704

Keywords: 连接蛋白,siRNA,星形胶质细胞,谷氨酸

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Abstract:

目的构建能有效抑制大鼠星形胶质细胞(astrocytes,ASTs)连接蛋白43(connexin43,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Westernblot检测转染干扰质粒对大鼠ASTsCx43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ASTs48h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。

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