新生小鼠肠边缘群细胞的体外分离

背景: 目前分离干细胞的方法主要基于其细胞标志，近年来发展一种非基于细胞标志的干细胞分离方法，即利用荧光激活细胞分类法将组织中干细胞和成熟细胞分离。目的：分离新生小鼠小肠黏膜来源的侧群细胞，探讨利用荧光活化细胞分选系统构建鼠肠干细胞群的可行性。方法：取新生小鼠小肠全长，制作小肠黏膜的类器官片段并制备成单细胞悬液。使用Hoechst33342和碘化丙啶染色后用流式细胞仪分选侧群细胞。提取细胞总RNA和蛋白，RT-PCR及Western-blot方法分别检测其中MSI-1 mRNA及MSI-1蛋白的表达水平。结果与结论：新生小鼠来源的小肠黏膜单细胞悬液中包含一个特定的细胞群体即侧群细胞，染色液中加入维拉帕米后，侧群细胞被阻断后消失。侧群细胞中显示有MSI-1 mRNA及蛋白的表达。提示新生小鼠的小肠黏膜侧群细胞富集小肠黏膜干细胞，荧光活化细胞分选系统可用于构建鼠肠干细胞群。

In vitro isolation of intestinal side population cells from newborn mice

BACKGROUND: Current methods of stem cell separation are mainly based on their cell markers. A method for stem cells separation which is not based on cell markers developed in recent years, that is fluorescence activated cell sorting method, has been applied for stem cells and mature cells separation.OBJECTIVE: To isolate side population cells from newborn mice small intestinal mucosa, and to investigate the feasibility of constructing the murine intestinal stem cell population by fluorescence activated cell sorting. METHODS: Small intestine mucosa organoids of mice were isolated and dissociated into single cells. The side population cells were stained with Hoechst 33342 and propidium iodide, then sorted using fluorescence activated cell sorting. Total RNA and protein were purified from sorted fractions to detect Musashi-1 expressions by RT-PCR and Western-blotting.RESULTS AND CONCLUSION: Single cell suspension from mouse small intestine mucosa contained a viable population of cells, which showed the side population phenotype and were sensitive to verapamil. These cells were enriched for Musashi-1 mRNA and MSI-1 protein expression. Results demonstrated that the side population fraction separated from mice intestinal mucosa is enriched for intestinal stem cells, the murine intestinal stem cell population can be successfully constructed with fluorescence activated cell sorting.