The Role Of Brn-4 On The Neural Stem Cells From Hippocampus Differentiation Into Neurons

Objective: To study the role of Brn-4 on the neural stem cells from hippocampus differentiating into neurons, and to investigate the biology mechanism of neural regeneration and reparation after fimbria/fornix transection. Methods: Immunofluorescence double-labeling in experiment one: NSCs isolated and expanded from the hippocampus of embryonic rat were divided into 3 groups: the transection group, the normal group and the control group. Then the extracts from the fimbria/fornix transected and intact hippocampi of adult SD rats previously obtained were respectively added into the transection and the normal group. The neurons differentiated from NSCs were detected by immunofluorescence double-labeling of Brn-4 and MAP-2 on the 3rd, 7th, 14th and 21st day after culturing. The number of Brn-4 positive cells, the intensity of immuno- fluorescence of Brn-4 and the number, area, perimeter of Brn-4/MAP-2 double- labeling positive neurons were analyzed by image processing software respectively. Stata8.0 statistical software was adopted to analyze the results. RNA interference in experiment two: NSCs isolated and expanded from the hippocampus of embryonic rat and cultured in vitro were planted into 6-well and 24-well cell culture plate respectively with the extract from the fimbria/fornix transected hippocampi. Then the cells were divided into the silenced group and the unsilenced group from two plates respectively after siRNA sequence filtered and transfection condition optimized, and the effective fragment was added into the silenced group. The total RNA was extracted from cells of 6-well cell culture plate on the 3rd, 7th, 14th and 21st day after transfection. The relative level of Brn-4 mRNA expression was indicated by the ratio of the optical density value of Brn-4 to that of GAPDH by RT-PCR. The cells of 24-well cell culture plate were detected by immunofluorescence double-labeling of Brn-4 and MAP-2 on the 3rd, 7th, 14th and 21st day after transfection. The number of Brn-4 positive cells, the intensity of immunofluorescence of Brn-4 and the number, area, perimeter of Brn-4/MAP-2 double-labeling positive neurons were measured respectively by image processing system. The analysis of variance and group comparison were applied with Stata8.0 statistical software. Results: In experiment one, the number of Brn-4/MAP-2 double-labeling positive neurons and its body area, perimeter increased gradually after transfection. And the peak appeared on the 14th day, then the growing status degraded later. The change of the intensity of immunofluorescence of Brn-4 showed the same tendency. Among three groups, the number, body area and perimeter of double-labeling cells were the best in the transection group. Compared with the control group, the growing status was better in the normal group. The differences were significant between any two groups except the intensity of immunofluorescence of Brn-4 on the 3rd day among three groups (P<0.01). The results in the experiment two showed that the optimized transfection condition was suitable and toxico side effect of transfection reagent was indistinct. It also suggested that the efficiency of transfection was 95 percent. The relative level of Brn-4 mRNA expression of the silenced group was obviously lower than the unsilenced group on the 3rd, 7th, 14th day respectively(P<0.01), and the difference decreased on the 21st day between the two groups(P<0.05). The Brn-4/MAP-2 double-labeling positive neurons were fewer, the bodies were smaller, and the intensity of immunofluorescence of Brn-4 was thinner in the silenced group compared with the unsilenced group significantly(P<0.01). Conclusion: The expression level of Brn-4 was enhanced obviously when the neural stem cells differentiated into neurons facilitated by the extracts from the fimbria/fornix transected hippocampi. The expression of Brn-4 mRNA and protein were degraded after the siRNA aimed directly at the Brn-4 gene, and the differentiation of the neural stem cells into neurons was influenced obviously. The results suggested that Brn-4 may play an important role in the process of neural stem cells differentiating into neurons.