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人正常食管黏膜上皮细胞的纯化分离和传代培养 |
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作者：张茹，龚均，王晖，王利，雷娟，冉力伟
【关键词】 细胞培养
Isolation and subculture of human esophageal squamous epithelial cells
【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 serumfree keratinocyte medium (KSFM) or fetalbovineserum (FBS)supplementary medium (KSFM 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 antihuman 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 KSFM was (51.5±11.5) h (n=3), but it could not be calculated in KSFM with 100 ml/L FBS. The adherent cells significantly increased in KSFM （P<0.01）. The percentages of CK14positive cells in KSFM 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 CK13positive 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.
【Keywords】 esophagus; epithelial cells; cell culture
【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100 ml/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100 ml/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）；CK14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[1] [2] [3] [4] 下一页
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