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ID:34120505
大小:3.01 MB
页数:56页
时间:2019-03-03
《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》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
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
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