成人食管上皮细胞的体外培养及生物学特性

CULTURE AND BIOLOGICAL PROPERTIES OF ADULT ESOPHAGEAL EPITHELIAL CELLS IN VITRO

目的培养成人正常食管上皮细胞,建立能够体外长期培养的食管上皮细胞系,为上皮细胞的体外研究提供实验材料。方法取食管癌患者正常食管上皮,用0.25%Dispase酶和0.25%胰蛋白酶/0.02%EDTA消化获取成人食管上皮细胞,使用无血清角化细胞培养液培养,通过细胞形态学观察和角蛋白、上皮膜抗原(EMA)免疫组织化学染色鉴定细胞。结果原代培养8d后,细胞汇合成片呈铺路石样生长,细胞角蛋白、上皮膜抗原表达阳性,可连续传代。结论为体外分离培养成人正常食管上皮细胞建立了方便可行的方法。

Objective To culture normal adult esophageal epithelial cells and establish a normal esophageal epithelial cell line to provide experimental materials for further studies in vitro. Methods Normal esophageal epithelium samples were obtained in sterile phosphate buffered saline supplemented with antibiotics from the normal part of the esophagus of a patient with esophageal carcinoma. Tissue was cleaned of all connective tissues and muscles and rinsed in phosphate buffered saline 5-6 times. The mucosal layer was minced into small pieces and dissociated into a single cell suspension by 0.25% Dispase and 0.25% trypsin/ 0.02% EDTA. Cells were grown in keratinocyte serum-free media. The cultured cells were identified through their morphological characteristics and immunohistochemical staining. The proliferative capacity of the cultured cells was also examined. Resets The cultured cells showed microscopic features of epithelial cells and were positive in keratin and epithelial membrane antigen staining. Eight days after primary culture, the cells displayed a cobblestone morphology reaching 80%-90% confluency and were passaged successfully with 0.25% trypsin/0.02% EDTA. The cell cycle analysis showed about 77.60% of the cultured cells was in G0/G1 phase and the others in S/G2/M phase. Conclusion The culture methods and techniques used in the experiments are convenient and suitable for the primary culture and subculture of normal adult esophageal epithelial cells.