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

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) Cells derived from a pheochromocytoma of the rat adrenal medulla of the line PC12 were induced by amyloid-beta protein (Abeta)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 cells 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 Abeta(1-40) for 0, 4, 6, 12, and 24 h respectively, Group D1-5 with PC12 cells induced by Abeta(1-40) for 0, 4, 6, 12, and 24 h respectively, and Group E containing astrocytes induced by Abeta(1-40) 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 VIII (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 Abeta(1-40) 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) and GAP-43 (A = 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 Abeta(1-40)-induced PC12 cells(Abeta-PC12), the ACM induces the synapse formation in the NSCs. The BDNF in the ACM is probably involved in this process.