摘要目的 寻找一种方法能够有效去除胃黏液,把胃黏膜制备成单细胞悬液以进行胃黏膜流式细胞术等单细胞的蛋白分析研究,为即将在胃组织开展细胞组学做好准备.方法 应用酶反应去除胃黏液和分离黏膜层后,再使用机械法制备成单细胞悬液,并应用细胞免疫组织化学流式细胞术和激光共聚焦显微镜技术初步检测胃黏膜细胞主要周期蛋白cyclin D、E、A、B1的表达.结果 0.1%的胃蛋白酶和1.2～2.4 U/L的中性蛋白酶分别能够较好地去除胃黏液,获得黏膜层,从而制备成胃黏膜单细胞悬液,从流式中首次系统地显示出其细胞主要周期蛋白的时相性表达规律:cyclin D3、B1明显表达,cyclin D2较弱,cyclin D1、A、E表达不明显,并得到激光扫描共聚焦显微镜的验证.结论 利用胃蛋白酶,中性蛋白酶作用后能够去除胃黏液,较容易获得胃黏膜单细胞悬液;并提示体内的细胞周期蛋白的表达规律与体外培养的细胞系的表达规律不同.

abstractsObjective To explore a method which can remove the gastric mucus in order to prepare mucous membrane single cell suspension for the research of cytomics. Methods Enzymology was used to remove the mucus gel and to separate mucous layer from the normal fresh gastric tissue.The mucous layer was broken to prepare single cell suspension with machine method. Expression of major cyelins in mucous layer cells was examined by cytoimmunochemistry, flow cytometry (FCM) and confocal microscopy. Results The 0.1% pepsin could dissolve the mucus gel and 1.2-2.4 U/L dispase could separate the mucous layer completely. The single mucous cell suspension was prepared successfully. FCM results from mucous single cell suspension revealed that expression of cyclin D3, B1 was obvious, that of cyclin D2 was weak and that of cyclin D1, A, E was the least. Similar results were found with confocal microscopy. Conclusions Single cell suspension from mucous layer can be easily prepared by pepsin and dispase. Cyclins schedule expression in vivo is different from cyclins schedule expression in vitro.