小鼠肠干细胞群的构建

Construction of Murine Intestinal Stem Cell Population

目的探索利用干细胞体外培养技术构建小鼠肠干细胞群。方法取孕17 d昆明系小白鼠的胚胎鼠肠组织,剪碎后采用酶消化法分离肠干细胞群;将通过离心获得的细胞溶于选择性培养液并种于一次性培养瓶中;利用角蛋白18亚型抗体行免疫组织化学染色鉴定细胞。结果细胞培养48 h后,开始贴壁生长。镜下可见,上皮样细胞呈集落状生长,其中,由典型上皮构成的克隆约占30%,由扁平上皮细胞构成的克隆约占20%,混合生长的克隆约占50%。培养出的肠干细胞群在第3、4代达到生长分化的顶峰;而后干细胞逐渐减少,成熟的肠上皮细胞逐渐增多,直至最终凋亡。培养出的肠干细胞群角蛋白18亚型抗体染色阳性。结论在现有技术条件下,体外构建肠干细胞群是完全可以实现的。

Objective To culture the murine intestinal stem cell population and identify the cells by immunohistochemistry methods.Methods The intestinal stem cell population was isolated by enzyme digestion from the embryonic intestine of the 17-day Kunming pregnant mice,centriguged and dissolved in the selective culture.The cells were implanted in the culture bottle and identified by immunohistochemistry methods with anti-cytokeratin peptide 18.Results The cells were found to adhere 48 h later.Under the microscopy,colonies were observed.About 30% of colonies were found to be lined with a homogeneous population of large,undifferentiated epithelial-like cells,and about 20% of the colonies were lined with a layer of flattened epithelial cells.The remaining colonies(about 50%) appeared to be lined with a more heterogeneous epithelium containing both large and flattened cells.The cells from passages 3 and 4 were the most active.Then,the number of the stem cells was gradually decreased and the mature intestinal epithelial cells gradually increased.In the end the apoptosis of the cells was found.The intestinal stem cell population was positive for the anti-cytokeratin peptide 18 staining after several passages.Conclusion The murine intestinal stem cell population can be successfully constructed with available condition.