小鼠唾液腺干细胞体外分离培养方法研究  

A Method for Isolation of Mouse Salivary Gland Stem Cells

作　　者：李林蔚[1,2] 李国庆[1] 胡劲松[1] 谢红艳[1] 罗莎莎[1] 王燕[1] 易岚[1] 

机构地区：[1]南华大学药学与生物科学学院,湖南衡阳421000 [2]南华大学附属第二医院检验科,湖南衡阳421000

出　　处：《现代生物医学进展》2015年第32期6230-6233,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(81400117);中国博士后基金项目(2014M562115);湖南省自然科学基金项目(2015JJ4042);南华大学归国人员科研启动基金项目(2014XQD46)

摘　　要：目的：建立小鼠唾液腺干细胞体外分离培养方法。方法：从7～8周龄的C57BL／6J小鼠唾液腺分离唾液腺干细胞，采用悬浮唾液球法培养唾液腺干细胞，应用倒置显微镜动态观察细胞形态；细胞计数观察细胞生长状况；免疫荧光检测LN（层连蛋白）的表达。结果：倒置显微镜下，接种2d后细胞（salispheres）小聚集体明显增加；接种4d后唾液腺球直径逐渐增大，形成直径约100斗m的神经球，球周围无明显刺突，存在明显细胞聚集。接种7d后唾液腺球周围存在较多微刺，10d后球继续增长，形状明显不同于4d，表明处于活跃的增殖状态，唾液腺干细胞已经开始分化。生长曲线结果显示随着培养天数的增加，唾液腺球的数目呈明显递增趋势，同时，唾液腺球的直径也明显增大。免疫荧光结果显示，培养2d后，唾液干细胞球开始表达LN，培养第4d后，随着唾液干细胞球的增加，层连蛋白表达更明显。结论：应用该方法获得了唾液腺干细胞，具有成体干细胞的特征，具有自我更新和多向分化的潜能．Objective： To develop an efficient method for isolation and culture of mouse salivary gland stem cells. Methods： Salivary gland stem cells were isolated and cultured from 7-8 week old C57BL/6J mice salivary gland. Inverted microscope was used to observe the cells＇ morphology; Growth curve was determined by cell counting; the expression of laminin was analyzed by Immunofluorescence. Results： After two days in culture, small aggregates of cells （salispheres） will be apparent in the cultures. Salispheres will continue to grow in size over a period of four days in culture. The diameter is about 100 μm and there are no obvious spikes around the salispheres. After seven days in culture, obvious spikes occurred. The morphology of the salispheres after ten days was different from those in four days; the salispheres have a trend to differentiation. Growth curve show the number of the salispheres was increased in a time-dependent manner. At the same time, the diameter of the salispheres also increased significantly. These results demonstrated that the salivary gland stem cells had high proliferation ability. Immunofluorescence results showed that salivary stem cells began to express LN after 2 days in culture, and the level of LN was increased significantly after 4 days in culture. Conclusion： The results of this study demonstrated that the mouse salivary gland stem cells from the mice salivary gland through this improved method possessed the biological characteristics of adult stem cells. These cells had the ability to undergo both selfrenewal and multi-directional differentiation.

关 键 词：小鼠唾液腺干细胞 细胞分离培养 悬浮唾液球 层连蛋白 

分 类 号：Q95-3[生物学—动物学] Q593