FLT3靶向短发夹状干扰RNA体外转录合成及作用.doc

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1、FLT3靶向短发夹状干扰RNA体外转录合成及作用作者：卢洁，盛光耀，邹湘，方营旗，赵晓明，徐学聚，白松婷，许培荣，王建人【摘要】FMS样酪氨酸激酶3(FLT3)是一种酪氨酸激酶受体，在大多数急性髓系白血病(AML)患者中持续激活，与患者预后差密切相关。为探讨3种FLT3靶向短发夹状干扰RNA(shRNA)对急性髓系白血病细胞株THP1的沉默效应，设计和体外转录合成3个FLT3靶向shRNA(shRNA1、shRNA2、shRNA3),体外转染THP1细胞;以RTPCR法检测FLT3mRNA水平表达，用流式细胞术、免疫荧光测定法检测FLT3蛋白的表达。结果显示：shRNA1

2、、shRNA3可显著下调FLT3mRNA的表达，其中shRNA1的抑制作用较强。25nmol/LshRNA1转染48小时对FLT3mRNA的抑制率是(72.95±2.07)%，作用可达72小时。5nmol/L及以上浓度的shRNA1对FLT3mRNA表达有下调作用，作用存在量效关系。15nmol/LshRNA1的抑制率是(67.53±0.66)%。FLT3蛋白位于细胞膜上，shRNA1对其有较强的抑制作用，转染72小时蛋白抑制率达(79.67±0.66)%。结论：FLT3shRNA1具有较好的FLT3基因靶向抑制作用，可作为进一步研究该基因作用机制及靶向治疗可能性的工具。【

3、关键词】急性髓系白血病InVitroTranscriptionSynthesisandEffectsofFLT3TargetedShortHairpinRNAAbstractFMSliketyrosinekinase3(FLT3)isareceptoroftyrosinekinasethatisconstitutivelyactivatedinmostofacutemyeloidleukemiapatientsandseemstogiveanadverseprognosis.InordertoexplorethesilencingeffectofFLT3targetedsho

4、rthairpinRNA(FLT3shRNA)onacuteleukaemiacelllineTHP1,threeFLT3shRNAs(shRNA1,shRNA2,shRNA3)weredesignedandsynthesizedbytranscriptionsysteminvitroandthentransfectedintoTHP1cells.FLT3mRNAwasanalyzedbysemiquantitativeRTPCR,FLT3proteinwasdetectedbyFlowcytometryandimmunofluorescence.Theresult

5、sindicatedthatFLT3expressionwasdownregulatedbyshRNA1andshRNA3,andshRNA1showedstrongerinhibitoryeffect.At48hoursfollowingtransfection,theinhibitoryrateof25nmol/LshRNA1was72.95±2.07%,lasting72hours.The5nmol/LandmoreconcentrationofFLT3shRNA1coulddownregulateFLT3mRNAlevel,whichdisplayedaquantity

6、effectrelation;theinhibitoryrateof15nmol/LshRNA1was67.53±0.66%.FLT3proteinwaslocatedonTHP1cellmembrance,itsexpressionwasdownregulatedobviouslybyshRNA1，at72hoursfollowingtransfectiontheinhibitoryrateofshRNA1was779.67±0.66%.shRNA1showedthebestinhibitoryeffectonFLT3protein,theoptimaltimeofwhi

7、chwas72hourswithaninhibitoryrateof79.67%.ItisconcludedthatFLT3shRNA1showsadesireableFLT3targetedinhibitoryeffect,whichcanbeusedforfurtherinvestigationofFLT3mechanismorFLT3targetingtreatment.Keywordsacutemyeloidleukaemia；FLT3；FLT3shRNA;RNAinterfe