IntJOphthalmol，Vo1．10，No．2，Feb．2010www．UO．caTel：029-8224517283085628Email：IJO．2000@163．com组织工程人角膜内皮的体外重建及其形态结构樊廷俊，赵君，王晶，丛日山，杨秀霞，史伟云，王宜强·实验论著·基金项目：中国国家高技术研究发展863计划资助项目(No．2006AA02A132)；中国山东省眼科学重点实验室一省部共建国家重点实验室培育基地开放基金课题资助项目(No．2006K05)作者单位：(266003)中国山东省青岛市，中国海洋大学角膜组织工程重点实验室；(266071)中国山东省青岛市，山东省医学科学院山东省眼科研究所作者简介：樊廷俊，男，教授，理学博士，中国海洋大学海洋生命学院副院长，国家生命科学与技术人才培养基地主任，国家细胞生物学教学团队带头人，山东省高等学校教学名师，研究方向：动物细胞工程与细胞分化，主要成果：在国际上建立了首个非转染的人角膜内皮细胞系，在体外重建出结构和功能与在体角膜内皮类似的组织工程人角膜内皮等。通讯作者：樊廷俊．tjfan@oue．edu．an收稿日期：2010—0l一08修回日期：2010—0l一28|nvitroreconstructionoftissue—engineeredhumancorneaIendotheliumandcharacter-ologyandstructureTing．JunFan，JunZhao，JingWang，Ri-ShanCong，Xiu．XiaYang，Wei-YunShi，Yi-QiangWangFoundationitems：NationalHighTechnologyResearchandDevelopmentProgram(“863”Program)ofChina(No．2XI6AA02A132)；OpeningProgramfromTheStateKeyLaboratoryCultivationBase，ShandongProvincialKeyLaboratoryofOphthalmology(No．2006K05)KeyLaboratoryforCornealTissueEngineering，OceanUniversityofChina，Qingdao266003，ShandongProvince，China；ShandongEyeInstitute，ShandongAcademyofMedicalSciences，Qingdao266071，ShandongProvince，ChinaCorresp0ndenceto：Ting—JunFan．KeyLaboratoryforCornealTissueEngineering，OceanUniversityofChina，Qingdao266003，ShandongProvince，China．tjfan@oue．edu．anReceived：2010一O1．08Accepted：2010—01—28Abstract·AIM：TOreconstructtissue—engineeredhumancornealendothelia(TE—HCE)invitroandcharacterizetheminmorphologyandstructure．·METHODS：MonoclonalHCEcells(mcHCEcells)wereclonedfr0muntransfectedHCEcellliRebylimiteddilution，andtheirkaryotypeswereanalyzedbyroutinemethodsofchromosomalpreparingandkaryosyste—matics．Modifieddenudedamnioticmembranes(mdAMs)werepreparedfrOmamnioticmembranebyinvertedtrypsindenudationandcoatedwithextracellularmatrixproteins．TE—HCEswereinvitrorec0nstructedbyusingmcHCEcellsatIogarithmicphaseasseedcellsandmdAMstIledonwellbottomsofa24一wellcuItureplateassca仟oldcarriers．whichwereculturedin200mL／LfetaIboviReserum(FBS)一containingDMEM／F12mediumat37℃ina50mL／LC02incubator．Themorphologyofseedcells，formationofcelljunctions，integralityofendothelialmonolayeranditsintegratedstatustomdAMwereinvestigatedbyAlizarinredstaining，freeze—sectionshematoxylin—eosin(HE)staining，inve~edmicroscopyandscanningelectronmicroscopy．TheultrastructureofseedcellsonmdAMandformationofcelliunctionswereexaminedbytransmissionelectronmicroscopy．Theexpres—sionpaternsofdifierentcelliunctionproteinsofTE—HCEseedercellsweredetectedbyimmunofluorescenttechniques．·RESULTS：SevenmcHCEcellstraiaswithnormaIkaryotype(2n=46)werescreenedoutfrOmtheuntrans—fectedHCEcellliRe．About30hoursafterrec0nstructiOninitiation．mcHCEseedercellsformedanintegratedmono—IayeronmdAMwithacelldensityashighas3413／mm．Mostofseedcellswereinpolygonalmorphology，integralendotheIialmonolayerwasrecOnstructedwithvariouscell-cellandcell_mdAMiunctions．AndtheultrastructureofseedcellswassimilartothatofHCEceIlsvivo．withalotofmitochondriascatteredin...
