梯度密度离心法分离肝癌细胞及其转移性细胞株的建立【摘要】【目的】建立新的肝细胞癌细胞系，为肝癌实验研究提供肝癌；细胞株；核型；比较基因组杂交Abstract:【Objective】Toestablishhepatocellularcarcinoma(HCC)celllinesandprovidemorepurelivercarcinomacellsforHCCstudies.【Methods】ThecarcinomatissueswereobtainedfromtheprimarylesionandmetastaticlesioninportalveinofHCC.ThecarcinomacellswereisolatedbyPercolldensitygradientcentrifugeandcultivated.Thecellcloneswerethenobtainedbymicro-environmentaldigestionandcelllineswereestablished.Thegrowthcurves,Gbandchromosomekaryotype,comparativegenomichybridization(CGH)oftheestablishedcellswerecharacterized.Thecellswereimplantedinnudemicefortumorigenesis.【Results】20casesofHCCcellswereisolatedandcultivated.Twocelllines(designatedasH2MandH4M)weresuccessfullyestablishedfrom2casesofemboliofportalvein.Thekaryotypeshowedthatbothcelllinesareahypertriploid(71～78chromosomes).Amarkerchromosomecontaining1qand6p［t(1;6)］wasfoundinH2Mcellsandahugemarkerchromosomecontainingalonghomogeneouslystainingregion(hsr)inH4Mcells.ThemaingeneticalterationsanalyzedbyCGHwereahighcopynumberamplificationof1q,3q,5p,6p,7qand8qandlossof4q,13q,16q,17p19pinH2Mcells,whereas,amplificationof6p,7p,11p,11q13andlossof8p,9,13q,16qinH4Mcells.ImplantationofH2Mcellsinnudemiceforamonthproducestypicalhumanhepatocellularcarcinoma,butnotumorforH4Mcells.【Conclusions】Percolldensitygradientcentrifugeandmicro-environmentaldigestionisaneffectivemethodforcloningofprimarycarcinomacellandcelllineestablishment.TwoestablishedHCCcelllineswillprovideculturecellsforHCCstudies.ThegeneticalterationsdetectedinbothcelllinesalsoprovidecluesandcellmodelsforfurtherscreeningofoncogenesandtumorsuppressorgenesinHCC.Keywords：hepatocellularcarcinoma;HCCcelllines;karyotype;comparativegenomichybridization肝细胞癌是常见癌症之一。尽管已有多个肝癌细胞系供实验室研究用，但这些细胞多由国外研究人员建立，而且极少从门脉转移灶建立[1]。此外，已建立的细胞系缺乏细胞遗传学改变的资料，不能为肝癌癌基因、抑癌基因的筛选及肝癌转移机制研究提供线索。本文描述2例门脉转移性肝癌细胞系的建立过程并检测其分子遗传学改变，旨在为HCC的深入研究提供实验工具。1材料和方法标本来源20例肝癌组织取自中山大学附属第一医院肝胆外科手术标本，每份癌组织均取原发灶和门脉癌栓，经病理检查证实和进行细胞培养。细胞培养原发瘤及门脉癌栓组织用含5倍浓双抗PBS冲洗后剪成约1mm3大小，用%的胶原酶在37℃下消化30