Objective To analyze the effect of astrocytes conditional medium in the neuronal differentiation of bone marrow stromal cells,and discuss whether neurotrophins (brain-derived neurotrophic factor,BDNF; neural growth factor,NGF; neurotrophin-3,NT-3) was involved in the process.Methods First,PC12 cells were induced by Aβ1-40 for different time periods (0,4,6,12,24 h).The apoptosis rates of Aβ1-40-induced PC12 cells (at 0,4,6,12,24 h) were measured by flow cytometry.Aβ1-40-induced PC12 cells were then incubated with astrocytes for 2 days,and the conditioned medium was collected.Then,the total proteins of the BDNF,NGF and NT-3 in the conditioned medium were measured by ELASA in the all groups,and the BMSCs were induced to differentiate into neuronal cells by the collected cellsconditioned medium.The expression of NSE and nestin of BMSCs in all groups were detected by immunofluorescen,and neuron differentiating rates were determined.Results Flow cytometry results showed that PC12 cell apoptosis rates were lower at 0 h,2 h and 4 h,which peaked at 6 h(P ＜ 0.05).The BDNF total protein in astrocytes-conditioned medium (A=1.87±0.43) after Aβ-induced PC12 at 6h were remarkable increased,(P ＜ 0.05).The expression of NSE (A=38.63±2.72) and nestin (A=43.53±5.63) of BMSCs and neuron differentiating rates (A=44.62+3.22) induced by astrocytes-conditioned medium after Aβ1-40-induced PC12 at 6 h were significantly higher than other groups (P ＜ 0.05).There was no significant difference of the NGF total protein in astrocytes-conditioned medium among each group (P ＞ 0.05).NT-3 total protein was not detectable in all groups.Conclusion After incubation with Aβ1-40-induced PC12 cells (Aβ-PC12),astrocytes-conditioned medium (ACM) showed increased effects of inducing BMSCs to differentiate into neuron.BDNF in astrocyte-conditioned medium probably was involved in this process.%目的 星形胶质细胞(AS)与Aβ1-40诱导凋亡的PC12细胞共育,观察其共育条件培养液诱导骨髓基质细胞(BMSCs)向神经元样细胞分化的影响,探讨神经营养素家族蛋白[包括脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养素3(NT-3)]是否参与了此过程.方法 PC12细胞分别经10 μg/mL Aβ1-40诱导不同时间点(0h、4h、6h、12h、24 h)后分为两部分,一部分应用流式细胞技术检测不同时间点PC12细胞凋亡率；另一部分与星形胶质细胞共育2d,收集各组条件培养液；各组条件培养液分为两部分,一部分应用ELASA法检测各组细胞条件培养液中的BDNF、NGF、NT-3蛋白含量；将另一部分各组细胞条件培养液对BMSCs进行诱导分化,免疫荧光检测BMSCs神经元特异性烯醇化酶(NSE)、巢蛋白(nestin)蛋白表达和计数神经元样细胞分化比例.结果 在Aβ1-40作用6h时间点,PC12细胞凋亡率达高峰(P＜0.05)；与各组比较,与A31-40诱导6h后的PC12细胞共育2d后的星形胶质细胞条件培养液中(即C3组)的BDNF蛋白总量明显增高(A=1.87±0.43),具有统计学意义(P＜0.05),并且其诱导的BMSCs NSE(A=38.63±2.72)、nestin(A=43.53±5.63)表达和神经元样细胞分化比例(A=44.62±3.22)明显升高,与其他组比较,差异有统计学意义(P＜0.05).结论 星形胶质细胞与Aβ1-40诱导凋亡的PC12细胞共育后,星形胶质细胞共育条件培养液促进BMSCs向神经元样细胞分化,BDNF可能参与了这一过程.
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