摘要
目的观察比较不同培养方式下神经干细胞(neural stem cells,NSCs)的自噬发生情况。方法取出生24 h内的Sprague-Dawley(SD)大鼠海马与纹状体,并用细胞球悬浮培养和单层贴壁培养两种方法进行体外培养,在光学显微镜下观察其生长。免疫荧光标记神经干细胞特异性抗原Nestin的表达。透射电镜下观察神经干细胞自噬小体。Western blotting检测自噬相关蛋白LC3Ⅱ、Beclin-1的蛋白表达水平。结果 Nestin在悬浮培养中的阳性率为95%以上,在贴壁培养中接近100%。与贴壁培养比较,悬浮培养自噬体形成增多,自噬标记物LC3Ⅱ/LC3Ⅰ、Beclin-1蛋白表达水平显著提高(差异具有统计学意义P<0.05)。结论成功用悬浮培养和贴壁培养两种方法培养原代神经干细胞;悬浮培养可造成细胞内自噬水平增加,推测其可能的原因为神经球内细胞营养缺乏而激活自噬。
ObjectiveTo observe and compare the autophagy in neural stem cells in two different culture methods.MethodsAfter isolating hippocampus and striatum tissue from newborn SD rats24h,cells were cultured in vitro with two methods:culturing of neurospheres in floating and culturing of monolayer adherent cells.To observe the development of the neural stem cells with the light microscope.The expression of the stem cell marker Nestin was examined with immunofluorescence.We used transmission electron microscope to observe the ultrastructure of autophagosomes in neural stem cells.Western blotting was used to detect the level of autophagic related protein LC3Ⅱand Beclin1.ResultsThe positive rate of Nestin in neural stem cell spheres was more than95%,and the positive rate of Nestin in adherent culture was nearly100%.LC3Ⅱ/LC3Ⅰand Beclin1protein and autophagosomes were increased significantly in neural stem cell spheres(P<0.05).ConclusionPrimary neural stem cells are cultured in two methods successfully.Autophagy level in neural stem cell spheres increases.Presumably,the deficiency of nutrition in cells activates the autophagy.
作者
王梦影
黄国伟
程曼
董志萍
张绪梅
WANG Mengying;HUANG Guowei;CHENG Man(Department of Nutrition and Food Hygiene,School of Public Health,Tianjin Medical University,Tianjin 300070,China)
出处
《中风与神经疾病杂志》
北大核心
2017年第12期1076-1079,共4页
Journal of Apoplexy and Nervous Diseases
基金
国家自然科学基金(No.2301/2GW027)
