星形胶质细胞条件培养液影响神经干细胞突触形成的神经营养机制探讨

Influence of astrocyte-conditioned medium on the formation of synapses in neural stem cells： the role of neurotrophin proteins

目的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）]是否参与星形胶质细胞条件培养液（ACM）影响神经干细胞（NSC）突触形成的过程。方法实验分两步，（1）PC12细胞分别经10μg／ml Aβ1-40诱导不同时间点（0、4、6、12、24h）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EHSA法检测ACM中的BDNF、NGF、NT4蛋白含量。（2）将另一部分ACM以1：3比例同DMEM／F12培养基混合，分别对胚胎大鼠皮质神经干细胞进行诱导分化，激光共聚焦扫描显微镜观察突触素和生长相关蛋白-43（GAP-43）表达，透射电镜观察成熟突触结构数目。结果在Aβ1-40作用6h时间点，PC12细胞凋亡率达高峰（P〈0．05）；星形胶质细胞与Aβ1-40诱导6h后的PC12细胞共育2d后，收集到ACM中的BDNF蛋白总量（A值=1．53±0．25）明显增高（P〈0．05），并且收集到的ACM诱导神经干细胞突触素（A值=33．39±2．71）、GAP-43（A值=49．18±6．45）表达明显升高、成熟突触结构数目（4．70±0．52个／视野）明显增多，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，ACM提高了神经干细胞突触形成，ACM中BDNF可能参与了这一过程。

Objective To discuss whether neurotrophin proteins, brain-derived neurotrophic factor （BDNF） ,neurotrophin-3 （NT-3）, and neural growth factor （NGF）, in the astrocyte-conditioned medium （ACM） are involved in the synapse formation in neural stem cells （NSCs）. Methods （ 1 ） Ceils derived from a pheochromocytoma of the rat adrenal medulla of the line PC12 were induced by amyloid-β protein （Aβ） 1-40 for 0, 4, 6, 12, and 24 h respectively. Then part of these PC12 cells underwent flow cytometry to examine the apoptotic rates. Different ceils were added into Falcon Cell Culture Insert： Group A containing astrocytes isolated from Wistar rat, Group B with PC12 cells and astrocytes, Groups C1 - C5 containing astrocytes and PC12 cells induced by Aβ1-40 for 0, 4, 6, 12, and 24 h respectively, Group D1 - 5 with PC12 cells induced by Aβ1-40 for 0, 4, 6, 12, and 24 h respectively, and Group E containing astrocytes induced by Aβ1-40 for 6 h. Flow cytometry was used to detect the apoptotic rates of different groups. Double-antibody sandwich ELISA was used to detect the levels of BDNF, NT-3, and NGF. （2） The different kinds of the astrocyte-conditioned medium as described above were mixed with DMEM/F12 medium according to the proportion of 1： 3 and then divided into 13 groups ： Group Ⅰ （ Group A ＋ NSCs）, Group Ⅱ （ Group B ＋ NSCs）, Group Ⅲ- Ⅶ （ Groups C1 - C5 ＋ NSCs）, Group Ⅶ （ NSCs without ASM）, Group Ⅸ -ⅩⅢ （Groups D1 - D5 ＋ Mscs）, and Group ⅩⅣ （ Group E ＋ NSCs）. The expression of synaptophysin and growth-associated protein-43 （ GAP-43 protein） were detected by co-focal laser scanning microscopy. The number of mature synapse was observed by transmission electron microscope （TEM）. Results Flow cytometry showed that the apoptotic rates of the PC12 cells were low 0, 2, and 4 h after Aβ1-40 induction, with the peak 6 h after induction （ P 〈 0.05 ）. The BDNF total protein level in the ACM of Group C3 was the highest （A = 1.53 ＋ 0. 25） （ P 〈 0. 05）. The expression levels of synaptophysin （A = 33.39 ＋ 2.71 ） andGAP-43 GAP =49. 18 ± 6. 45） , and the mature synapse number of NSCs （4. 70 ± 0. 52 synapse/field of vision ） of Group V were the highest in comparison with the other groups （ all P 〈 0. 05 ）. Conclusion After incubation of astrocytes with Aβ1-40-induced PC12 cells （Aβ-PC12） , the ACM induces the synapse formation in the NSCs. The BDNF in the ACM is probably involved in this process.