Caco-2细胞单层模型的建立与验证

:,,,*:,,,Tel:[phone]Email:[e-mail]2孙敏捷,盛星,胡一桥*(,210093):研究建立Caco2细胞模型的方法,并确认Caco2细胞模型的正常生长特性,以在日常培养中进行监控常规培养条件下,Caco2细胞以每1mL含8104个的接种密度接种于Millicell插入式培养皿中,培养21d后用光镜电镜细胞密度测定等方法检查细胞的形态学及生长特点,测定碱性磷酸酶活性跨膜电阻荧光素钠透过量以检验细胞单层的极化现象和致密程度Caco2细胞单层在生长10d以后均匀致密,21d后微绒毛碱性磷酸酶紧密连接等呈不对称分布,跨膜电阻达到600cm2,荧光素钠透过量为416gh-1cm-2,而同样条件下空白膜的透过量大于240gh-1cm-2本培养条件下,Caco2细胞单层形态与小肠上皮细胞类似,跨膜电阻标志物透过量均达到要求,可以作为研究小肠药物转运过程的体外模型:Caco2细胞;转运;跨膜电阻;渗透性:Q811:A:1001-2494(2006)18-1431-04EstablishmentandValidationofCaco2CellLinesforLntestinalEpithelialPermeabilitySUNMinjie,SHENGXing,HUYiqiao*(StateKeyLaboratoryofPharmaceuticalBiotechology,NanjingUniversity,Nanjing210093,China)ABSTRACT:OBJECTIVEToinvestigatetheestablishmentofCaco2cellmonolayermodel,andtoidentifyCaco2snaturalgrowthinordertomonitorthedailycultivationMETHODSCaco2cellwascultivatedinMillicellplateinsertswith8104cellsmL-1celldensityinstandardproceduresfor21dThemorphologicalandgrowthcharactersofthemonolayerwereexaminedwithopticalmicroscope,electronmicroscopeandcelldensitydeterminationThepolarizationandcompactificationofthemonolayerwasdeterminedbythealkalinephosphataseactivity,transepithelialelectricalresistance(TEER)andapicaltobasolateralofsodiumfluoresceinacrosscellmonolayersRESULTSCaco2cellmonolayerbecamewellproportionedandcompactafter10dgrowthAfter21d,microvillus,alkalinephosphataseandtightconjunctionswereasymmetricallydistributed,TEERvaluereached600cm-2,andthetransportationofsodiumfluoresceinwas416gh-1cm-2thetransportationacrossnoncellmembranewasmorethan240gh-1cm-2inthesameconditionsCONCLUSIONInthesecultivationconditions,theCaco2cellmonolayerhadbecomeananalogyofsmallintestinalepitheliumThevalueofTEERandtransportationofsodiumfluoresceinallreachedthecriterions,whichmeantthatthemonolayerbecameanvaluablemodelsystemforintestinalepithelialpermeabilityInthisresearch,Caco2cellmonolayermodelwasestablishedandevaluatedKEYWORDS:Caco2cell;transport;transepithelialelectricalresistance(TEER);permeability,(thehumancolonadenocarcinomacelllines,Caco2),[12][3],2080,Caco2,[12,4],,P(MDR)[5,7],Caco2,,,,111试剂与器材MEM/EBSSNEAA(Hyclone);(Hyclone);(1250)N2N2(Hepes)EDTA2Na2H2O1431200694118ChinPharmJ,2006September,Vol41No18(Amresco);L(Sigma);();();Millicell(,04m,12mm)Millicell-ERS()(Millipore);(UV-2450,Shimadzu);(JEM-1200EX;JEOL);()12细胞株Caco2(codeHTB037,ATCC)40~5013细胞培养Caco2T75,MEM/EBSSNEAA(pH74,10%L2mmolL-1Hepes10mmolL-1),37,5%CO224h,,4~6d90%,37[(005%)+EDTA2Na(053mmolL-1)]2~3min,1324Millicell,1mL8104,400L,600L,24h,1,121d14细胞形态学检查和单层的形成141T75MEM/NEAA,14d,,142Caco221,25%5%01molL-1PBS(pH=74)401molL-1PBS(pH=74)3,40min1%01molL-1PBS(pH=74)45min,10min,50%,70%,95%,100%11:Epon812,Epon812,6024h,(60~80nm)5%,100kV[6]143Caco2Millicell,6104,400L,1,3,15细胞极性研究Caco2,,(AP)(BL),AKPCaco28,15,21d,Hanks(pH=74)2,3720min,,AP,BL16跨膜电阻(TEER)Caco2TEER,200cm-2TEER,,1000cm-2Caco213Millicell,ERS17标志物渗漏检查Caco2,[6]Hanks21d,(2gL-1)400L,Hanks600L,05,10,15,20,25h,,490nm,48gh-1cm-2Millicell,,10min,,490nm37,5%CO2,Hanks,221细胞形态学检查和单层的形成211,Caco25,6,10,,,1212,21d,,2(a,b),,21432ChinPharmJ,2006September,Vol41No182006941181Caco2细胞生长至14d的显微镜照片Fig1OpticalmicroscopephotoofCaco2cellscultivatedfor14d(c)A,,,2(c)B,2生长于聚碳酯膜上的Caco2细胞单层亚显微结构特征a-Caco2;b-;c-(A)(B)Fig2UltrastrucctralfeaturesofCaco2cellsmonolayersgrownonmicroporousmembranesa-microvillusonapicalofCaco2monolayer;b-brushborder;c-tightjunction(A)anddismosomeamongcells(B)213Caco2,3d,4,5,604105S25,,Hidalgo[8],,,316,489%,42%33生长于Millicell中的Caco2细胞生长曲线Fig3GrowthofCaco2cellsinMillicellcellcultureplateinsertscellswereseededatadensityof60000cellswell-122细胞极性研究11,(AP/BL),2186,1558,,,15~21151Caco2细胞单层碱性磷酸酶活力比与时间关系n=3,xsTab1Relationshipbetwecnalkalinephosphataseactivityandthelengthoftimeinculturen=3,xst/dRatioofalkalinephosphataseActivity(AP/BL)82.1780.171154.0310.1012114.810.71023TEERERS,4,1,3d,,,4,7400cm2,,15600cm218d4Caco2细胞单层的跨膜电阻与生长时间关系Fig4TransepithelialelectricalresistancesofCaco2monolayersatdifferentstagesofconfluenceanddifferentiation24标志物渗漏检查,30min,22%,Caco2006%,,25h089%,51%55荧光素钠透过空白聚碳酯膜和生长21d的Caco2细胞单层的透过量时间曲线1-Millicell;2-21dCaco2Fig5Apicaltobasolateraltransportationofsodiumfluoresceinacrosscellfreemembranesandacross21dayoldcellmonolayersat371-cellfreemembrances;2-21dayoldcellmonolayers1433200694118ChinPharmJ,2006September,Vol41No183Caco250,60,[7],,60Caco27d,101,,4,,,,1400cm-2,,15600cm-2Caco221d,,,,,,(AP)(BL)1481,416gh-1cm-2,240gh-1cm-2,Caco2,21dCaco221d,[2],,REFERENCES[1]YEES.InvitropermeabilityacrossCaco2cells(colonic)canpredictinvivo(smallintestinal)absorption[J].PharmRes,1997,14(6):763766.[2]ALLENRH,ROBERTAC,PHILLIPSB.Caco2cellmonolayersasamodelfordrugtransportacrosstheintestinalmucosa[J].PharmRes,1990,7(9):902910.[3]GANLL,DHIRENRT.ApplicationsoftheCaco2modelinthedesignanddevelopmentoforallyactivedrugs:elucidationofbiochemicalandphysicalbarriersposedbytheintestinalepithelium[J].AdvDrugDelivRev,1997,23(1):7798[4]PATRICKA,RAFM.HPLCwithprogrammedwavelengthfluorescencedetectionforthesimultaneousdeterminationofmarkercompoundsofintegrityandPgpfunctionalityintheCaco2intestinalabsorptionm