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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2 d后细胞(salispheres)小聚集体明显增加;接种4 d后唾液腺球直径逐渐增大,形成直径约100μm的神经球,球周围无明显刺突,存在明显细胞聚集。接种7 d后唾液腺球周围存在较多微刺,10 d后球继续增长,形状明显不同于4 d,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2 d后,唾液干细胞球开始表达LN,培养第4 d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 establish a method for isolation and culture of mouse salivary stem cells in vitro. Methods: Salivary gland stem cells were isolated from the salivary glands of C57BL / 6J mice aged 7 to 8 weeks. The salivary gland stem cells were cultured with suspension salivary spheres. The morphological changes of the cells were observed under inverted microscope. The cell growth was observed by immunocytochemistry. Protein) expression. Results: Under inverted microscope, small aggregates of salispheres increased significantly after 2 d of inoculation. After 4 d of inoculation, the diameter of salivary gland balls gradually increased to form neurospheres with a diameter of about 100 μm. There was no obvious spike around the sphere, and obvious cell aggregation . After 7 days of inoculation, there were many spikes around the salivary gland, and the sphere continued to grow after 10 days. The shape was significantly different from that of 4 days, indicating that the salivary gland stem cells had started to differentiate in an actively proliferating state. The results of growth curve showed that with the increase of days of culture, the number of salivary glands showed a significant increasing trend, meanwhile, the diameter of salivary gland balls also increased significantly. Immunofluorescence results showed that after 2 days of culture, salivary stem cells began to express LN. After 4 days of culture, the expression of laminin became more pronounced with the increase of spleen stem cells. Conclusion: Salivary gland stem cells have been obtained by this method. They have the characteristics of adult stem cells and have the potential of self-renewal and multidirectional differentiation.