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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 .
Objective To establish a method for primary culture of gastric parietal cells.METHODS The inverted stomach pouch of rats was prepared by injecting digestive juice containing pronase E into gastric pouch and incubated gently with a magnetic stirrer to prepare a single The gastric gland cells were enriched by Percoll gradient centrifugation, and the fibroblasts were removed by time-lag adherent method using PBS or culture medium containing 100 ml of L-1 serum. After that, the parietal cells were inoculated into a culture plate and cultured The liquid was incubated with serum-free 1: 1 Harm’s F-12 / DMEM containing insulin 5 mg · L -1, hydrocortisone 4 μg · L -1, transferrin 5 mg · L -1, sodium selenate 5 μg · L-1, 2 g · L-1 bovine serum albumin, 25 μg · L-1 epidermal growth factor, 1.98 g · L-1 glucose were cultured continuously for more than 1wk.Results The parietal cells obtained by acridine orange acridine orange, AO) were identified more than 80% purity, the primary culture by HE staining showed that the parietal cells were scattered small group growth and good growth status.Conclusion This method is suitable for rat gastric parietal cells isolated and cultured in vitro for further Study the function of parietal cells to lay the foundation.