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时间:2018-08-31
《人正常食管黏膜上皮细胞的纯化分离和传代培养.doc》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
1、人正常食管黏膜上皮细胞的纯化分离和传代培养作者:张茹,龚均,王晖,王利,雷娟,冉力伟【关键词】细胞培养Isolationandsubcultureofhumanesophagealsquamousepithelialcells 【Abstract】AIM:Toinvestigatetheoptimalmethodforthecultureofhighlypurifiedhumannormalesophagealepithelialcellsandtoyieldsignificantcellnumbers.METHODS:Cellswereisol
2、atedfromtissuesbydispasedigestionandtrypsinizationandthenprimarilyculturedandsubculturedinserumfreekeratinocytemedium(KSFM)orfetalbovineserum(FBS)supplementarymedium(KSFMwith100ml/LFBS).Thebiologicalcharacteristicsofthecellswereobservedthroughcellattachmentsandgrowthkineticcur
3、vesandwereconfirmedimmunohistochemicallyusingantihumanmonoclonalantibodyofthecytokeratin14(CK14),cytokeratin13(CK13)andvimentin.RESULTS:Fibroblastswerenotseeninallthecultures.Populationdoublingtime(PDT)inKSFMwas(51.5±11.5)h(n=3),butitcouldnotbecalculatedinKSFMwith100ml/LFBS.Th
4、eadherentcellssignificantlyincreasedinKSFM(P<0.01).ThepercentagesofCK14positivecellsinKSFMweresignificantlyhigheratanycomparableperiods(P<0.05),butdecreasedwiththeprolongationofthegrowthtimeinthese2kindsofmedia.Contrarily,thepercentagesofCK13positivecellswerelowerandincr
5、eased.Vimentinwasnegativeinallthecultures.CONCLUSION:Tocultureandproliferatehumannormalesophagealsquamousepithelialcellsproductivelyinvitro,dispasedigestionandserumfreekeratinocytemediumarefeasibleandcrediblemethods.Serummayinducethedifferentiationofthesells. 【Keywords】esopha
6、gus;epithelialcells;cellculture 【摘要】目的:探讨新的食管黏膜细胞的培养方法,以便能够获得大量细胞.方法:采用中性蛋白酶(Dispase)和胰酶先后消化从食管黏膜上分离上皮细胞,比较细胞在无血清培养基(KSFM)和含100ml/L胎牛血清的培养基中的细胞生长曲线和贴壁性;采用细胞角蛋白14、13(CK14,CK13)和波形丝蛋白抗体三者共同鉴定细胞.结果:细胞倍增时间在KSFM中为(51.5±11.5)h(n=3),而在含1005ml/L血清培养基中不能计算;在KSFM中,贴壁细胞明显为高(P<0.01);C
7、K14阳性细胞百分率KSFM组在不同时段均高,但两组内均随生长时间增加而减少;CK13阳性细胞则恰与其相反;两组中均未见波形丝蛋白阳性表达.结论:Dispase消化分离细胞,KSFM作为培养基是食管鳞状上皮可靠的新培养方法. 【关键词】食管;上皮细胞;细胞培养 0引言 目前食管黏膜的传代培养,就报道的极少的几种方法还存在着很多问题,比如大量的成纤维细胞污染[1],不能传代扩增或传代次数较少[2],细胞数量较少.我们通过改善原代取材时消化方法,进行含血清及不含血清培养基比较研究,探讨出能获得大量细胞的新的正常食管黏膜细胞的培养方法. 1材料和
8、方法 1.1材料 人角朊细胞培养液(KSFM,Gibco),胰酶(Sigma),DMEM(Hyclone),Dispa
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