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(K-SFM)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K-SFM中为(51.5±11.5)h(n=3),而在含100ml/L血清培养基中不能计算;在K-SFM中,贴壁细胞明显为高(P<0.01);CK14阳性细胞百分率K-SFM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,K-SFM作为培养基是食管鳞状上皮可靠的新培养方法.
AIM： To investigate the optimal method for the culture of highly purified human normal esophageal epithelial cells and to yield significant cell numbers. METHODS： Cells were isolated from tissues by dispase digestion andtrypsinization and then primarily cultured and subcultured in serum-free keratinocyte medium （K-SFM） or fetalbovine-serum （FBS）-supplementary medium （K-SFM with 100 ml/L FBS）. The biological characteristics of the cells were observed through cell attachments and growth kinetic curves and were confirmed immunohistochemically using anti-human monoclonal antibody of the cytokeratin 14 （CKI4）, cytokeratin 13 （CKI3） and vimentin. RESULTS： Fibroblasts were not seen in all the cultures. Population doubling time （PDT） in K-SFM was （51.5 ± ll.5） h （n = 3）, but it could not be calculated in K-SFM with 100 ml/L FBS. The adherent cells significantly increased in K-SFM （P〈0.0l）. The percentages of CK14-positive cells in K- SFM were significantly higher at any comparable periods （ P 〈0.05）, but decreased with the prolongation of the growth time in these 2 kinds of media. Contrarily, the percentages of CK13-positive cells were lower and increased. Vimentin was negative in all the cultures. CONCLUSION： To culture and proliferate human normal esophageal squamous epithelial cells productively in vitro, dispase digestion and serumfree keratinocyte medium are feasible and credible methods. Serum may induce the differentiation of the sells.
出处
《第四军医大学学报》
CAS
北大核心
2005年第16期1468-1471,共4页
Journal of the Fourth Military Medical University
基金
卫生部临床学科重点课题(20012130)
关键词
食管
上皮细胞
细胞培养
esophagus
epithelial cells
cell culture
