不同培养方式对大鼠原代神经干细胞自噬的影响

摘要

Objective To observe and compare the autophagy in neural stem cells in two different culture methods . Methods After isolating hippocampus and striatum tissue from newborn SD rats 24 h,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 immunofluo-rescence .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 LC 3Ⅱand Beclin-1.Results The positive rate of Nestin in neural stem cell spheres was more than 95%,and the positive rate of Nestin in adherent culture was nearly 100%.LC3Ⅱ/LC3Ⅰand Beclin-1 protein and autophagosomes were increased significantly in neural stem cell spheres ( P<0.05).Conclusion Primary 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 .%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.