褪黑激素对新生大鼠神经干细胞向神经元样细胞分化的影响

Effects of Melatonin on Differentiation of Rat Neural Stem Cell into Neuron-like Cells

【目的】探讨褪黑激素对体外培养新生大鼠神经干细胞分化的影响。【方法】从新生SD大鼠室管膜下区分离依赖碱性成纤维细胞生长因子 (bFGF)和表皮细胞生长因子 (EGF)生存的神经干细胞进行培养 ,原代及子代细胞鉴定后用于实验。实验细胞均在无生长因子而含 10mL/L胎牛血清条件下培养 ,经褪黑激素或Luzindole作用 1周后做免疫组织化学染色 ,比较Map2阳性细胞出现率。【结果】①依赖bFGF和EGF生长的神经干细胞体外呈集落样生长 ,表达抗原蛋白巢蛋白(Nestin) ,无生长因子培养基中可分化形成胶质原纤维酸性蛋白 (GFAP)、S10 0蛋白、微管相关蛋白 (Map2 )阳性细胞。②褪黑激素能够提高Map2阳性细胞生成率 (2 3 4 5 % ,2 1 82 % ) (P <0 0 1)。③褪黑激素膜受体竞争性阻断剂Luzindole可阻断高浓度 (10 0 μmol/L)褪黑激素促神经干细胞分化作用 ,但不能抑制低浓度褪黑激素 (10nmol/L)的作用。【结论】褪黑激素能促进神经干细胞向神经元样细胞分化 ,不同浓度褪黑激素可能存在不同的作用机制。

To investigate the effect of melatonin on differentiation of rat neural stem cells in vitro. Neural stem cells, which depended on bFGF and EGF for surviving, were isolated from subventricular zone of neonate rat, cultured in vitro and used for experiment after identification by immunohistochemistry All cells were cultured in medium containing 10 mL/L fetal calf serum without growth factors, processed for immunohistochemistry staining one week after treatment with melatonin or luzindol, and the formation rate of Map2 positive cells among the experimental groups was compared. ① Neural stem cells generated neural spheres in vitro and expressed Nestin protein,and differentiated into GFAP ,S100 ,Map2 positive cells without growth factors ② Melatonin increased the formation rate of Map2 positive cells (23 45%,21 82%)(P<0 01) ③ Luzindole, a competitive inhibitor of the binding of melatonin to the transmembrane receptors, blocked the effect of high concentration(100 μmol/L) of melatonin on the differentiation of neural stem cells into neuron like cells, but could not inhibit the action of melatonin in low concentration(10 nmol/L) [Conclusion] The results indicate that melatonin can accelerate differentiation of neural stem cells into neuron like cells, but there exists different effects between the low and high concentrations, which may refer to the different mechanisms of action on neural stem cells.