摘要
目的 完成胃壁细胞分离及纯化 ,建立原代培养胃壁细胞的方法 .方法 制备大鼠翻转的胃囊用含链霉蛋白酶E的消化液注入胃囊内孵育 ,并用磁力搅拌器轻轻搅拌而制备单个的胃底腺细胞 ,Percoll梯度离心富集胃壁细胞 ,用含10 0 m L· L- 1 血清的 PBS或培养液 ,时差贴壁去除成纤维细胞 ,然后 ,将壁细胞接种于培养板中 ,培养液用无血清的 1∶ 1Harm's F- 12 / DMEM培养 ,内含胰岛素 5 mg· L- 1 ,氢化可地松 4μg· L- 1 ,转铁蛋白 5 mg· L- 1 ,硒酸钠 5μg· L- 1 ,牛血清白蛋白 2 g· L- 1 ,表皮生长因子 2 5 μg· L- 1 ,葡萄糖 1.98g· L- 1持续培养可达 1wk以上 .结果 获得的壁细胞经吖啶橙 (acridine orange,AO)鉴定纯度达 80 %以上 ,原代培养经HE染色可见壁细胞呈分散小组式生长 ,且生长状态良好 .结论 此方法适宜于大鼠胃壁细胞的分离及体外培养 ,为体外进一步研究壁细胞的功能打下基础 .
AIM To establish a new model of isolation andprimary culture in vitro for further study. METHODS The stomachs of rats were reverted to form sacs with the mucosa facing outward and serosa facing inward. Pronase E solution was injected into the sac lumina. The stomachs were incubated in 950 mL·L -1 O 2 and 50 mL·L -1 CO 2 at 37℃for 60 min, then stirred at room temperature and the dispersed cells were collected to be loaded on a density gradient (40%~60% per cell) centrifugation. For removal of fibroblasts, the cells were suspended in PSS containing 100 mL·L -1 fetal bovine serum FBS and preattached in flasks coated with rat tail glue at 37℃ for 30 min. The rich parietal cells were maintained in 1∶1 Ham's F 12/DMEM contained insulin 5 mg·L -1 , hydrocortaison 4 μg·L -1 , tranferrin 5 mg·L -1 , sodium selenite 5 μg·L -1 , BSA 2 g·L -1 , epidermal growth factor (EGF) 25 μg·L -1 , D glucose 1.98 g·L -1 . They were cultured continuously in serum free media and survived for over one week. RESULTS Identified by acridine orange (AO), the purity of isolated and cultured rat gastric parietal cells was over 80% and they grew very well. CONCLUSION Our methods and condition are suitable for the isolation and primary culture of rat gastric parietal cells.
出处
《第四军医大学学报》
北大核心
2002年第9期769-771,共3页
Journal of the Fourth Military Medical University
基金
国家自然科学基金资助项目 (3 9770 3 88)
关键词
胃壁细胞
细胞分离
细胞培养
大鼠
gastric parietal cell
cell isolation
cell culture
