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1、·612·ChineseJournalofReparativeandReconstructiveSurgery,May2009,Vol.23,No.5上皮细胞条件培养液对BMSCs分化的影响杨朋魏人前谭波李秀群王佳周昆鹏左潇李顺解慧琪【摘 要】目的探讨上皮细胞条件培养液（epithelialcellconditionedmedium，ECCM）体外诱导犬BMSCs向上皮细胞分化的可行性。 方法成年健康雄性比格犬1只，体重10kg。于犬髂后上棘采用骨髓穿刺法取骨髓5mL，分离培养BMSCs。取犬口腔黏膜下组织，剪成约4mm×4mm大小，制备ECCM。取第2代BMSCs以2×104个/孔细胞
2、密度接种，实验组于ECCM中培养，对照组于低糖DMEM完全培养基中培养。观察两组细胞形态特征，MTT法绘制生长曲线，诱导培养21d免疫组织化学染色鉴定上皮细胞特异性标志物——细胞角蛋白19（cytokeratin19，CK-19）和细胞角蛋白AE1/AE3，透射电镜观察细胞超微结构。 结果倒置相差显微镜下见两组细胞均呈长梭形，均匀分布，折光性较强，增殖迅速。两组细胞生长曲线均呈“S”形，实验组生长曲线较对照组右移，提示ECCM对细胞增殖有抑制作用。实验组CK-19和细胞角蛋白AE1/AE3免疫组织化学染色均呈阳性反应，对照组呈阴性。透射电镜下实验组细胞胞浆内可见紧密连接。 结论ECCM体
3、外有诱导同种BMSCs向上皮细胞分化的作用。【关键词】组织工程 BMSCs上皮细胞诱导分化犬中图分类号：R318Q813文献标志码：AEFFECTSOFEPITHELIALCELLCONDITIONEDMEDIUMONDIFFERENTIATIONOFBMSCs/YANGPeng,WEIRenqian,TANBo,LIXiuqun,WANGJia,ZHOUKunpeng,ZUOXiao,LIShun,XIEHuiqi.DivisionofStemCellandTissueEngineering,NationalKeyLaboratoryofBiotherapy,WestChinaHos
4、pital,SichuanUniversity,ChengduSichuan,610041,P.R.China.Correspondingauthor:XIEHuiqi,E-mail:xiehuiqi@163.com【Abstract】 ObjectiveToinvestigatethefeasibilityofinducingcanineBMSCstodifferentiateintoepithelialcellsinvitrowithepithelialcellconditionedmedium(ECCM).MethodsFivemLBMSCswereobtainedfromili
5、acspineofahealthyadultmalecaninewithweighing10kg,andthenisolatedandcultured.Theoralmucosawasharvestedandcutinto4mm×4mmafterthesubmucosatissuewaseliminated;ECCMwasprepared.BMSCsofthe2ndpassagewereculturedanddividedintotwogroups,culturedinECCMasexperimentalgroupandinL-DMEMascontrolgroup.Thecellmorp
6、hologicalcharacteristicswereobservedandthecellgrowthcurvesoftwogroupsweredrawnbythecontinualcellcounting.Thecellswereidentifiedbyimmunohistochemicalstainingthroughdetectingcytokeratin19(CK-19)andanti-cytokeratinAE1/AE3onthe21stdayofinduction.Theultra-structurecharacteristicswereobservedundertrans
7、missionelectronmicroscope.ResultsThecellsoftwogroupsshowedlong-fusiforminshapeanddistributeduniformlyunderinvertedphasecontrastmicroscope.ThecellgrowthcurvesoftwogroupspresentedStype.Thecellgrowthcurveoftheexperimental
