Efficient Enrichment of Hepatic Cancer Stem-Like Cells from a Primary Rat HCC Model via a Density Gradient Centrifugation-Centered Method 英文参考文献.docVIP

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Efficient Enrichment of Hepatic Cancer Stem-Like Cells from a Primary Rat HCC Model via a Density Gradient Centrifugation-Centered Method 英文参考文献.doc

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Efficient Enrichment of Hepatic Cancer Stem-Like Cells from a Primary Rat HCC Model via a Density Gradient Centrifugation-Centered Method 英文参考文献

EfficientEnrichmentofHepaticCancerStem-LikeCells fromaPrimaryRatHCCModelviaaDensityGradient Centrifugation-CenteredMethod Wei-huiLiu1,2.,XingWang2.,NanYou2.,Kai-shanTao2.,TaoWang1,2,Li-junTang1*,Ke-fengDou2* 1PLACenterofGeneralSurgery,GeneralHospitalofChengduArmyRegion,Chengdu,SichuanProvince,China,2DepartmentofHepatobiliarySurgery,XijingHospital, FourthMilitaryMedicalUniversity,Xi’an,ShaanxiProvince,China Abstract Background:Becausefewdefinitivemarkersareavailableforhepaticcancerstemcells(HCSCs),basedonphysicalrather thanimmunochemicalproperties,weappliedanovelmethodtoenrichHCSCs. Methodology: After hepatic tumorcells (HTCs) werefirst isolated fromdiethylinitrosamine-induced F344 rat HCC model using percoll discontinuous gradient centrifugation (PDGC) and purified via differential trypsinization and differential attachment(DTDA),theywereseparatedintofourfractionsusingpercollcontinuousgradientcentrifugation(PCGC)and sequentiallydesignatedasfractionsI–IV(FI–IV).Morphologicalcharacteristics,mRNAandproteinlevelsofstemcellmarkers, proliferative abilities, induced differentiation, in vitro migratory capacities, in vitro chemo-resistant capacities, and invivo malignantcapacitiesweredeterminedforthecellsofeachfraction. Findings:Asthedensityofcellsincreased,22.18%,11.62%,4.73%and61.47%ofprimaryculturedHTCsweresegregatedin FI–FIV,respectively.ThecellsfromFIII(densitybetween1.041and1.062g/ml)displayedahighernuclear-cytoplasmicratio and fewer organelles and expressed higher levels of stem cell markers (AFP, EpCAM and CD133) than cells from other fractions(P,0.01).Additionally,invitro,thecellsfromFIIIshowedagreatercapacitytoself-renew,differentiateintomature HTCs, transit across membranes, close scratches, and carry resistance to chemotherapy than did cells from any other fraction;invivo,injectionofonly16104cellsfromFIIIcouldgeneratetumorsnotonlyinsubcutaneoustissuebutalsointhe liversofnudemice. Conclusions:Throughournovelmethod,HCSC-likecellsweresuccessfullyenrichedinFIII.Th