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ID:32838543
大小:6.57 MB
页数:47页
时间:2019-02-16
《satb1-shrna下调bc12表达诱导a549细胞凋亡》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
1、重庆医科大学硕士学位论文SATB1--shRNA下调Bc12表达诱导A549细胞凋亡姓名:蒋金申请学位级别:硕士专业:病理学与病理生理学指导教师:曹友德201205重庆医科大学硕士研究生学位论文SATBl-shRNA下调BcI2表达诱导A549细胞凋亡摘要目的:应用RNAi干扰技术干扰肺癌A549细胞株中特异性AT序列结合蛋白一1(SpecialAT—richsequence—bindingprotein—I,SATBl)基因的表达,探讨其对A549宝m胞凋亡的影响。方法:设计合成针对SATBl基因的shRNA表达质粒(SATBl一shRNA)。采用阳离子脂质体法将SATBl一shRN
2、A转染入肺癌A549兰m胞。分别采用RT—PCR、Western—Blot法检钡,USATBl一shRNA转染A549细胞中SATBl、Bc12、BAX及Caspase一3的表达。FCM检钡USATBl一shRNA转染的A549细胞凋亡率。结果:经凝胶电泳、DNA测序鉴定分析,成功构建了SATBl一shRNA。RT—PCR和Western—Blot结果显示SATBl一shRNA转染成功后可显著下调A549宝m胞中SATBlmRNA矛FI蛋白的表达水平(P<0.05)。RT—PCR矛lWestern—Blot结果显示Bcl2表达水平下降(P(O.05),SATBl一shRNA转染组BAX
3、、Caspase一3表达水平上调(P<0.05)。FCM检测结果示SATB1一shRNA转染组细胞凋亡率显著增加(P4、wn—regulationofSpecialAT-richsequence—bindingprotein一1(SATB1)inducedbyRNAinterference(RNAi)oncellapoptosisofhumanhmgcancercelllineA549andtoexploreitspossiblemechanism.Methods:SATBl一targetedshRNAexpressionplasmid(SATBl一shRNA)wasconstructed.SATBl一shRNAwastransfectedintoA549cellsbyLipofectAMINE20005、.3.ThenlRNAsandproteinsexpressionofSATB1,Bcl2,BAXandCaspase一3wereexaminedbyRT—PCRandWestern—Blot,respectively.TheapoptosisrateofA549cellswasdetectedbyFCM.SATB1一shRNAwasconstructedandtransfectedintoA549cellssuccessfully.Results:SATB1一shRNAwerevalidatedbygelelectrophoresisandDNAsequenceanalysis.Th6、emRNAandproteinexpressionofSATB1wasdecreasedmarkedlyaftertransfectionwithSATB1一shRNAl,P7、andCaspase一3wasincreasedaftertransfectionwithf,P
4、wn—regulationofSpecialAT-richsequence—bindingprotein一1(SATB1)inducedbyRNAinterference(RNAi)oncellapoptosisofhumanhmgcancercelllineA549andtoexploreitspossiblemechanism.Methods:SATBl一targetedshRNAexpressionplasmid(SATBl一shRNA)wasconstructed.SATBl一shRNAwastransfectedintoA549cellsbyLipofectAMINE2000
5、.3.ThenlRNAsandproteinsexpressionofSATB1,Bcl2,BAXandCaspase一3wereexaminedbyRT—PCRandWestern—Blot,respectively.TheapoptosisrateofA549cellswasdetectedbyFCM.SATB1一shRNAwasconstructedandtransfectedintoA549cellssuccessfully.Results:SATB1一shRNAwerevalidatedbygelelectrophoresisandDNAsequenceanalysis.Th
6、emRNAandproteinexpressionofSATB1wasdecreasedmarkedlyaftertransfectionwithSATB1一shRNAl,P7、andCaspase一3wasincreasedaftertransfectionwithf,P
7、andCaspase一3wasincreasedaftertransfectionwithf,P
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