資源描述：
《cx43基因干擾對大鼠星形膠質(zhì)細(xì)胞谷氨酸基礎(chǔ)釋放的影響》由會員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在工程資料-天天文庫。
1、Cx43基因干擾對大鼠星形膠質(zhì)細(xì)胞谷氨酸基礎(chǔ)釋放的影響王凌云，田衍平,牛建欽，肖嵐（400038，重慶，第三軍醫(yī)大學(xué)基礎(chǔ)部組織學(xué)與胚胎學(xué)教研室，重慶市神經(jīng)科學(xué)重點(diǎn)實(shí)驗(yàn)室）[摘要]目的構(gòu)建能有效抑制大鼠星形膠質(zhì)細(xì)胞連接蛋白43（connexin43，Cx43）基因表達(dá)的siRNA載體，并檢測抑制Cx43表達(dá)對星形膠質(zhì)細(xì)胞谷氨酸釋放的影響。方法合成針對大鼠Cx43基因的siRNA寡核苷酸，構(gòu)建pGCsi.U6.neo.GFP質(zhì)粒載體。質(zhì)粒通過電轉(zhuǎn)方法轉(zhuǎn)染原代星形膠質(zhì)細(xì)胞(astrocyte,AST）后，用免疫熒光染色及Westernblot檢測轉(zhuǎn)染干
2、擾質(zhì)粒對大鼠ASTsCx43蛋白表達(dá)的影響。用谷氨酸ELISA定量試劑盒檢測釋放至培養(yǎng)基中谷氨酸的濃度,并用DAPI染色進(jìn)行細(xì)胞計(jì)數(shù)。結(jié)果測序結(jié)果顯示，Cx43基因的目的片段正確插入載體，并保持正確的讀碼框。干擾質(zhì)粒轉(zhuǎn)染ASTs48h后與對照組相比，Cx43蛋白表達(dá)明顯下降(P<0.05)，細(xì)胞存活未受影響，而谷氨酸釋放明顯減少(P<0.05)。結(jié)論成功構(gòu)建了抑制大鼠Cx43基因表達(dá)的siRNA載體，抑制Cx43表達(dá)可降低ASTs谷氨酸的基礎(chǔ)釋放。[關(guān)鍵詞]連接蛋白43；siRNA；星形膠質(zhì)細(xì)胞；谷氨酸[中圖法分類號]R394-33；R394.3
3、；R34[文獻(xiàn)標(biāo)志碼]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
