星形胶质细胞与Aβ1-40诱导凋亡的PC12细胞共育条件培养液对神经干细胞分化的影响机制

Effect of conditioned medium from coculture of astrocytes and β-amyloid-induced apoptotic PC12 cells on the differentiation of neural stem cells and the possible mechanism

目的将星形胶质细胞与β-淀粉样蛋白（Aβ1-40）诱导凋亡的PC12细胞共育，观察星形胶质细胞条件培养液（ACM）对胚胎大鼠皮层神经干细胞（NSCs）体外定向分化为神经元的比例影响及机制，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）]是否参与此过程。方法PC12细胞分别经10μg/mL Aβ1-40诱导不同时间（0、4、6、12、24h）后分为两部分，第一部分应用流式细胞技术检测不同时间点PC12细胞凋亡率；第二部分分别与星形胶质细胞共育2d，将收集的ACM分为两部分，一部分应用ELISA法检测ACM中BDNF、NGF、NT-3蛋白含量，另一部分以1：3比例同DMEM／F12培养基混合，对NSCs进行体外诱导分化，应用激光共聚焦显微镜、NSE免疫荧光技术鉴定和计数神经元分化比例。结果在Aβ1-40作用6h时间点，PC12细胞凋亡率达高峰，与其共育的ACM中BDNF蛋白总量明显增高，诱导的NSCs神经元分化比例明显升高，与其他组比较，差异均有统计学意义（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，ACM提高了NSCs向神经元的分化比例，ACM中BDNF可能参与了这一过程。

Objective To observe the effect of the conditioned medium obtained from the coculture of astrocytes and β-amyloid （Aβ1-40）-induced apoptotic PC12 cells on the neuronal differentiation efficiency of neural stem cells （NSCs）, and investigate the possible involvement of the neurotrophins proteins including brain-derived neurotrophic factor （BDNF）, nerve growth factor （NGF） and neurotrophin-3 （NT-3） in the induced differentiation of the NSCs. Methods PC12 cells were induced by Aβ1-40 for different time lengths （0, 4, 6, 12, and 24 h）, and after detection of the apoptotic rates using flow cytometry, the cells were cocultured with astrocytes for 2 days. The astrocyte-conditioned medium （ACM）, after analysis of the neurotrophins proteins using enzyme-linked immunosorbent assay （ELISA）, was mixed with DMEM/F12 medium at the proportion of 1：3 to induce the differentiation of the NSCs. The cell differentiation was identified by laser confocal microscopy, NSE immunofluorescent labeling, and neuronal counting. Results Flow cytometry showed that induction by Aβ1-40 for 6 h resulted in the highest apoptosis rate of PC 12 cells （P〈0.05）. BDNF content in the ACM derived from the coculture of astrocytes and the PC12 cells induced for 6 h was significantly increased, and the NSCs induced in this ACM showed the highest neuronal differentiation rates, showing significant difference from those induced by other ACMs （P〈0.05）. Conclusion The ACM derived from the coculture of Aβ1-40-induced PC12 cells and astrocytes can increase the neuronal differentiation rates of NSCs in vitro, and BDNF in the ACM may play a role in this process.