cx43基因干扰对大鼠星形胶质细胞谷氨酸基础释放的影响

《cx43基因干扰对大鼠星形胶质细胞谷氨酸基础释放的影响》由会员上传分享，免费在线阅读，更多相关内容在工程资料-天天文库。

1、Cx43基因干扰对大鼠星形胶质细胞谷氨酸基础释放的影响王凌云，田衍平,牛建钦，肖岚（400038，重庆，第三军医大学基础部组织学与胚胎学教研室，重庆市神经科学重点实验室）[摘要]目的构建能有效抑制大鼠星形胶质细胞连接蛋白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(astrocyte,AST）后，用免疫荧光染色及Westernblot检测转染干

2、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。[关键词]连接蛋白43；siRNA；星形胶质细胞；谷氨酸[中图法分类号]R394-33；R394.3

3、；R34[文献标志码]AEffectofsilencingconnexin43geneonreleaseofglutamatefromastrocytesinratsWangLingyun,TianYanping,Niujianqin,XiaoLan(TeachingandResearchSectionofHistologyandEmbryology,ThirdMilitaryMedicalUniversity,ChongqingKeyLaboratoryofNeurobiology，Chongqing40038,China)[Abstract]

4、ObjectiveToconstructthesiRNAplasmidthatcaneffectivelyinhibitexpressionofconnexin43(Cx43)anddetecttheeffectofinhibitedCx43expressiononreleaseofglutamatefromastrocytes(AST).MethodssiRNAoligonucleotidestargetingtheCx43geneinratsweresynthesized.ApGCsi.U6.neo.GFPvectorwasconstruct

5、ed.AfterplasmidsweretransfectedintoASTbyelectroblotting,effectofsilencingplasmidstransfectedintoASTonexpressionofCx43proteininASTwasdetectedbyWesternblotwithimmunofluorescencestaining.ReleaseofglutamatefromASTwasassayedwithanELISAkit.ASTwerecalculatedwithDAPIstaining.ResultsD

6、NAsequencingshowedthatthetargetsegmentsofCx43genewerecorrectlyinsertedintotheplasmidsandkeptitscorrectreadingframe.Forty-eighthoursaftertheplasmidsweretransfectedintoAST,theexpressionlevelofCx43inASTandthereleaseofglutamatefromASTweresignificantlylowerthanthoseincontrols（P<0.

7、05）withnosignificantdifferenceobservedinthenumberofAST.ConclusionThesiRNAvectorweconstructedcaneffectivelyinhibittheexpressionofCx43inASTofratsandinhibitionofCx43expressioncanreducethereleaseofglutamatefromAST.[Keywords]connexin43；siRNA；astrocyte;glutamateSupportedbytheNation

8、alNaturalScienceFoundationofChina（31171046）andtheNaturalScienceFound