在多药耐药机制中twist与nf-κb、akt、fhit表达差异性与相关性分析

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1、关键词：Twist、NF-κB、Akt、FHIT、5-Fu、TSA、Taxol、多药耐药性User第4页StudyofvariabilityandcorrelationbetweentheexpressionofTwistandNF-κB、AKT、FHITInmultidrugresistanceofmechanismAbstract:bjective:TofurtherexploretherelationshipofexpressionofTwistwithNF-κB、AKT、FHITinInmultidrugresistanceofmechanism，byobservingthec

2、hemosensitivityoftrichostatinA(TSA)、5-fluorouracil(5-Fu)andpaclitaxel(Taxol)inHEK-293-twistcellllines，andinvestigatingtheexpressionlevelchangesofTwist、NF-κB、AkTandFHITinHEK-293-TwistandHEK-293-vector.Methods:（1）CellsScarificationexperiment：tomarkoff5well-distributedtransverselinesatthebackof6-po

3、replate，intervalbeing0.5~1cm，througheveryhole，5andtoputculturemediumcontaining5×10cellsintothe6-poreplate，aftertheday,andtoperpendicularlytotransverselinesscuffingwithtransferpettoronthecells，then，baptizingthe6-poreplatewithPBSthreetimes,andaddingserum-freeculturemediainittocontinuecultivation,a

4、ndtotakephotographsafter7hoursand24hours.(2)toobservethedifferentialdrugsensitivityofTSA、5-Fu、TaxolinHEK-293-TwistandHEK-293-vectorcelllines，CellCountingKit-8(CCK-8)skillwasapply.wedividedHEK-293-TwistandHEK-293-vectorcellintocelllinesfourgroupssuchastaxolgroup、TSAgroup、5-Fugroupandcontrolgroup，

5、everygrouphaving3double-Pored.Allcelllineswereculturedfor2days，afterthat，themediumcontainingtaxol，TSAand5-FutakesUser第5页theplaceofculturemediaofthetestgroupsrespectively.After48hours，toaddedCCK-8intoeverygroups,incubatingfor4hoursawayfromlight，andtodetectOD.valueat450nmwithMicroplateReader.（3）RT

6、–PCRWasusedtodetecttheexpressionsofgeneofTwistandFHITinHEK-293-TwistandHEK-293-vectorcelllines.establishedasfollowingsteps：First,byusingtotalRNAstandardextractionkit,TotalRNAintendedofHEK-293-TwistandHEK-293-vectorcelllinesweredrawn,andTwistandFHITgeneswerebothamplifiedusingRT-PCR.Afterthat，coll

7、ectingimage，ultimum，usingQuantityOnesoftwaretoanalyzethegrayvalue（4）TheproteinexpressionsofTwist、NF-κB、AkTandFHITinHEK-293-TwistandHEK-293-vectorcelllineswasdetectedusingWesternBlot.RIPAwasUsedtoextractcellsprotein，Coomassie