shRNA干擾食管癌Eca109細胞ERK2表達的實驗研究.pdf

1、新疆醫(yī)科大學碩士學位論文shRNA干擾食管癌Eca109細胞ERK2表達的實驗研究姓名：霍奇申請學位級別：碩士專業(yè)：病理學與病理生理學指導教師：盧曉梅201004新疆醫(yī)科大學醫(yī)學碩士學位論文新疆醫(yī)科大學醫(yī)學碩士學位論文—2—術(shù)有望成為治療食管癌細胞的一種新的手段，為食管癌的基因治療提供了一條新的思路。關(guān)鍵詞：關(guān)鍵詞：shRNA；Eca109；ERK2；survivin；細胞增殖ThestudyonERK2expressioninesop

2、hagealcancer109celllinebyshthairpinRNAinterferencePostgraduate:QiHuoSupervis:Prof.XiaomeiLuAbstractObjective:ShthairpinRNA(shRNA)interferencevecttargetedERK2wasconstructedusingpGeneclipU1hairpincloningsystemstoobserveERK

3、2genesilencingeffectmechanismsinhumanesophagealcancercellline（ECa109）.Methods：TheexpressionofphosphalationERK2(pERK2)，incentivedbyserumU0126(10μM50μM)inECa109weredetectedbywesternblottechnique.TwocomplementtaryoligoDNAst

4、rstargetedERK2geneweredesignedsynthesizedaccdingtotheprinciplesofsiRNA.AfterannealingoligoDNAswereedintopGeneClipTMvectbyT4DNAlingase.TherecombinantplasshRNAERK2wastransfectedintoECa109withlipofectamine2000Thetransfectio

5、nefficiencywasobservedbyfluescencemicroscopetheproliferationabilityofECa109cellwereanalyzedbyMTTat24h、48h、72h、96h.TheexpressionofERK2survivinweredetectedbywesternblotat72h96h.Thecellapoptosisthecellcyclewasanalyzedbyflow

6、cytometry（FCM）aftertransfection.Results:TheexpressionofphosphylatedERK2risedgraduallyalongwiththetimeofserumstimulationwhileitcanbedecreasedtheexpressionofphosphylatedERK2graduallywiththeincreaseofconcentrationofU0126.Ba

7、sedonenzymedigestionDNAsequencinganalysisshRNAERK2wereconstructedsuccessfully.ThetransfectionefficiencyofshRNAERK2at72hoursishigherthanthatat48h96h.TheproliferationrateofEca109cellsaftertransfectionwithshRNAERK2controlve