摘要
背景:原代培养的人正常胃黏膜上皮细胞与体内姊妹细胞相似,是进行胃生理和病理研究的理想工具,但我国尚未见简便、可靠的人正常胃黏膜上皮细胞原代培养方法的报道。目的:探讨简便的人正常胃黏膜上皮细胞原代培养方法。方法:以胶原酶Ⅱ和分散酶磁性搅拌分离人正常胃黏膜上皮细胞。以甲基噻唑基四唑(MTT)比色法检测细胞活力,以观察细胞大体生长过程;以溴脱氧尿苷(BrdU)标记法检测S期细胞数量,以观察细胞微观增殖能力。以PAS染色鉴定胃黏膜上皮细胞黏原颗粒;以细胞角蛋白(CK)-18免疫荧光染色鉴定上皮细胞。以光学显微镜和透射电子显微镜观察细胞形态结构。初步观察传代细胞的贴壁生长情况。结果:培养第2～3d,胃黏膜上皮细胞活力增幅最大,第4d达最高点,之后逐渐下降;BrdU孵育18h后,33.3%的细胞处于和曾经处于S期。接种密度高时,细胞PAS染色均呈紫红色,可见细胞核旁黏原颗粒;接种密度低时,仅成团细胞PAS染色呈阳性;培养第3d,绝大部分细胞CK-18阳性。接种第2d,胃黏膜上皮细胞成团簇状生长,增殖迅速,第4d后逐渐停止生长,第5d开始出现漂浮死亡细胞,最终完全被成纤维细胞取代;透射电子显微镜可见微绒毛、分泌颗粒、连接复合体等上皮细胞特征。传代胃黏膜上皮细胞增殖、铺展能力较原代细胞降低。结论:磁性搅拌酶分离培养可得到数量多、纯度高的人正常胃黏膜上皮细胞。
Background： Primary culture of normal human gastric mucosal epithelial cells have marked similarity with their corresponding ceils in vivo, and is an ideal tool for gastric physiological and pathological studies. However, no convenient and reliable method for primary culture of normal human gastric epithelial cells had been devised in China. Aims： To develop a convenient method for primary culture of normal human gastric mucosal epithelial cells. Methods： Gastric mucosal epithelial cells were dissociated by collagenase lI and disperse enzyme solution with a magnetic stirrer. Cell viability was estimated by methyl thiazolyl tetrazolium .（MTT） to examine the growth process in general, and bromo-deoxyuridine （BrdU） was used to estimate the cells in S-phase to observe the capability of cell proliferation. Periodic acid-Schiff （PAS） stain was used to identify the mucinogen granule of epithelial cell and cytokeratin （CK）-18 was used to identify the epithelial cells. Light microscope and transmission electron microscope were used to observe the morphological structures of cells, and the adherent growth of subculture. Results： The viability of gastric epithelial cells had a maximal increase between the 2nd and 3rd day, reached the peak on the 4th day, then declined gradually; incubating with BrdU for 18 h showed that 33.3% of the cells had entered S-phase. Almost all cells were PAS-positive when inoculated at high density and only clumped cells were PAS-positive when inoculated at low density; mos of the cells were CK-18 positive after 3 days of cultivation. On the 2nd day of inoculation, gastric mucosal epithelial ceils grew in clumps and proliferated rapidly, then gradually ceased to grow after the 4th day and began to detach and die on the 5th day. Finally they were substituted by fibroblasts. Transmission electron microscopy revealed the characteristics of epithelial cells： microvilli, secretary granules and junctional complex. The adherent growth capability of subculture was less than that of the primary culture. Conclusions： A convenient method for primary culture of normal human gastric mucosal epithelial ceils has been successfully developed, by which sufficient amount of highly purified normal human gastric mucosal epithelial cells can be obtained.
出处
《胃肠病学》
2007年第1期31-35,共5页
Chinese Journal of Gastroenterology
基金
国家自然科学基金项目(No.30270609)资助
关键词
上皮细胞
胃黏膜
细胞培养技术
Epithelial Cells
Gastric Mucosa
Cell Culture Techniques
