RNA干扰CyclinD1基因表达对瘢痕疙瘩成纤维细胞增殖及G1期调控的影响_英.pdf

ResearchPapers 研究报告 生物化学与生物物理进展 ProgressinBiochemistryandBiophysics 2008,35(2):159~169 InfluenceofRNAInterferenceMediatedCyclinD1 GeneSilencingonTheProliferationandG1Phase RegulatorsofFibroblastsDerivedFromKeloid 1) 1)* 1) 2) YUDong-Mei,HAOLi-Jun,WANGDong-Yan,WANGHui-Dong 1) (PlasticandAestheticSurgery,SecondAffiliatedHospital,HarbinMedicalUniversity,Harbin150086,China; 2)FourthAffiliatedHospital,HarbinMedicalUniversity,Harbin150007,China) Abstract Inordertoinvestigatetheeffectofsequence-specificsmallinterferingRNAonsuppressingcyclinD1expressionand proliferationandcellcycleandexpressionofG1phaseregulatorsoffibroblastsderivedfromkeloid,theplasmidexpressionvectorof TM siRNAtargetedagainstcyclinD1wasconstructedandtransfectedintofibroblastswithLipofectamine 2000.Thechangesof cyclinD1expressionweredetectedbyfluorescentquantitativePCR(FQ-PCR),semi-quantitativeRT-PCR.Theeffectofsequence- specificsmallinterferingRNAinsuppressingtheproliferationoffibroblastswasdetectedbyMTTassay.Flowcytometrywereusedfor evaluationofcellcycle.TheexpressionofcyclinD1,CDK4,pRbandP16wasdetectedbyimmunohistochemicalmethod.Theresults showedthat:(1)Thesequence-specificsiRNAeffectivelysuppressedcyclinD1expressionatbothmRNAlevelswithinhibitionrate of63.68%and92.83% (0.01).(2)Significantlyinhibitedtheproliferationoffibroblasts,andchangedcellcycleinpercentageof P G0/G1phasecellswasincreasedcomparedwiththecontrolsgroupsinfibroblasts(0.05).(3)72haftertransfection,theexpression P ofcyclinD1,CDK4andpRbdecreas