人参皂苷Rg3对体外培养小鼠神经干细胞分化的影响

摘要

背景:神经干细胞来源非常有限,而且在自然分化过程中绝大多数分化为神经胶质细胞,分化为神经元的比例相对较少,因此采取有效措施诱导其向合适的子细胞方向分化是非常重要的科研课题.以往研究证明人参皂苷类成分,如Rb1和Rg1,对神经干细胞的定向分化有一定影响,但Rg3是否对神经干细胞的分化起作用鲜有报道.目的:研究人参皂苷Rg3对小鼠神经干细胞分化为神经元和星形胶质细胞的影响.方法:分离孕14 d的C57BL/6小鼠的胎鼠大脑皮质神经干细胞并传代培养,用神经干细胞特异性免疫抗体Nestin和Sox2检测神经干细胞纯度.用分化培养基联合人参皂苷Rg3(空白对照、50 nmol/L和250 nmol/L)诱导神经干细胞分化3 d,然后用神经元特异性免疫抗体Tuj1和星形胶质细胞特异性免疫抗体GFAP检测神经干细胞分化为神经元和星形胶质细胞的情况,计算Tuj1+/DAPI和GFAP+/DAPI的比例;通过实时荧光定量PCR技术检测各组细胞Tuj1和GFAP mRNA的表达水平.结果与结论:①经细胞免疫荧光实验检测传代培养的小鼠胎鼠细胞几乎全为 Nestin 和 Sox2双阳性细胞,表明成功分离培养出纯度较高的神经干细胞,可以用于后期实验;②诱导分化3 d 后,与空白对照组和250 nmol/L人参皂苷Rg3组相比,50 nmol/L人参皂苷Rg3组对神经干细胞分化为神经元具有明显促进作用(P < 0.01),而空白对照组和250 nmol/L人参皂苷Rg3组之间差异无显著性意义(P > 0.05);③诱导分化3 d后,与空白对照组相比,50 nmol/L人参皂苷Rg3组和250 nmol/L人参皂苷Rg3组对神经干细胞分化为星形胶质细胞具有促进作用(P < 0.05),而50 nmol/L人参皂苷Rg3组和250 nmol/L人参皂苷Rg3组之间差异不明显(P > 0.05);④real time PCR结果总体与细胞免疫荧光结果相似;⑤结果表明,人参皂苷Rg3在50 nmol/L浓度时能明显促进神经干细胞向神经元和星形胶质细胞方向分化;但在250 nmol/L浓度时主要促进其向星形胶质细胞方向分化,对神经元分化没有显著影响.%BACKGROUND: Application of neural stem cells (NSCs) is of great current interest in neuroscience, but NSCs origin is very limited. And they always differentiate into a large percentage of glial cells and small percentage of neurons in natural differentiation process, so researchers should take effective measures to promote NSCs differentiation into certain offsprings. Previous studies have shown that ginseng saponin ingredients, such as Rb1 and Rg1, have certain influence on NSCs differentiation, but it is unclear whether Rg3 plays a role on NSCs differentiation. OBJECTIVE:To preliminarily investigate the effect of ginsenoside Rg3 on mouse NSCs differentiation into neurons and astrocytes in vitro. METHODS: The fetal cortices of embryonic 14 days (E14) C57BL/6 mice were isolated for culturing primary NSCs. Then passaged NSCs were identified by their purity with NSCs specific antibodies, Nestin and Sox2, by immunofluorescence staining. NSCs were induced for 3 days in the differentiation medium containing ginsenoside Rg3 of different concentrations (blank control, 50 and 250 nmol/L). After that, immunofluorescence staining was used to identify differentiated neurons with neuronal specific antibody, Tuj1, and differentiated astrocytes with astrocyte specific antibody, GFAP. Then, we calculated and statistically analyzed Tuj1+/DAPI and GFAP+/DAPI percentages in the three different groups. Besides, real-time PCR assay was used to test Tuj1 and GFAP mRNA expression in the three groups after 3 days of differentiation. RESULTS AND CONCLUSION: Primary and passaged NSCs were successfully cultured and almost of cells were positive for both Nestin and Sox2, so these high-purity NSCs could be used in the following experiments. Immunofluorescence staining and statistical analysis results showed that compared with the blank control and 250 nmol/L groups, 50 nmol/L group had an obviously increased neuronal percentage after 3 days differentiation (P < 0.01), while the blank control and 250 nmol/L groups had no significant difference (P > 0.05); compared with the blank control group, 50 and 250 nmol/L groups had significantly increased astrocyte percentages (P < 0.05), whereas there was no obvious difference between 50 and 250 nmol/L groups (P > 0.05). The results of real-time PCR assay were similar with the above immunofluorescence results. In conclusion, 50 nmol/L ginsenoside Rg3 can enhance mouse NSCs differentiation into neurons and astrocytes, while 250 nmol/L ginsenoside Rg3 can only promote mouse NSCs differentiation into astrocytes.