维生素d3抑制runt相关转录因子3依赖的结肠癌细胞增殖的研究

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1、宁夏医科大学硕士研究生学位论文英文摘要byretinoicaid.Sointhepresentwork,weexploredtherelativemechanisminvolvedincoloncancercells.【Objectives】(1)ToinvestigatewhethervitaminD3anti-cancerisassocciatedwithRunx3expressionincoloncancercelllines;(2)Toexaminethepossiblemechanismsunderlyingthein

2、ductionRunx3effectbyvitaminD3incoloncancercells.【Methods】(1)TheexpressionlevelofRunx3incoloncancercelllinesweredeterminedbywesternblot.(2)Runx3mRNAandproteinexpressiontreatmentwithvitaminD3weredeterminedbyRT-PCRandwesternblotrespectively.(3)TheRunx3-specificsiRNAvect

3、orwasdesignedandconstructedbyDNArecombinanttechnology.(4)TheRunx3-siRNAwastransfectedintoSW480cellsusinglipofectamine2000reagent.Emptyvectorwasusedascontrol.ThetransfectedcellswerescreenedwithG418for1month,singlecellcolonieswereobtainedbylimiteddilutionmethod.Semi-qu

4、antitativeRT-PCRandWesternblotwereusedtodetecttheRunx3expressioninthetransfectedcellsforconfirmationoftransfection.(5)TheeffectofvitaminD3onthegrowthoftransfectedcellsandcontrolcellsweredetectedbyMTTandflowcytometry.(6)WesternblotwasusedtodetectcyclinA,cyclinD1,cycli

5、nE,cdk2,cdk4,PCNA,Rbandp27proteinexpression.【Results】1.Runx3expressionincoloncancercellsWesternblotexaminationfoundthatRunx3expressionwasundetectableinLovoVI宁夏医科大学硕士研究生学位论文英文摘要Cellline;SW620andHT-29celllineshasalowlevelofRunx3expression;Runx3wasexpressedatahighleveli

6、nSW480cellline.2.Runx3isup-regulationbyvitaminD3intimeanddose-dependentmannerWeusedRT–PCRandwesternblottodetectRunx3mRNAandproteinlevelrespectively.Timecourse(from6hoursto72hours)experimentsshowedvitaminD3stimulationofRunx3mRNAexpressioninSW480cells.Runx3geneexpressi

7、onwasup–regulatedafter6hoursoftreatmentwithvitaminD3.Itwasenhancedat72h.VitaminD3inducedRunx3mRNAproductioninSW480cellsinadose-dependentmanner.SW480cellsweretreatedwithvitaminD3(10-10、10-9、10-8、10-7mol/Lrespectively)for48hours.VitaminD3causedincreaseofRunx3mRNAandthe

8、expressionofRunx3wasup-regulatedobviouslyontheconcentrationof10-8mol/L.Westernblotanalysiswasperformedtofurtherdetectproteinlevel.I