【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.更多还原

【Abstract】 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 fetal-bovine-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 (CK14), cytokeratin 13 (CK13) and vimentin. RESULTS: Fibroblasts were not seen in all the cultures. Population doubling time (PDT) in K-SFM was (51.5±11.5) 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<0.01). 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 serum-free keratinocyte medium are feasible and credible methods. Serum may induce the differentiation of the sells.更多还原