The Study Of The Role For Hes1 In Adult Neurogenesis After Hippocampus Injury

Objective:As a technology carrier, RNAi was used to explore the feasibility and effectiveness of RNAi-mediated Hes1 gene downregulation in vivo, further to demonstrate its role for adult neurogenesis and repair of neurological function after TBI.Through this study, we preliminarily detect the possible mechanism that Hesl gene regulated adult neurogenesis, to find the approach that can promote adult neurogenesis and to provide a new idea for the clinical treatment of TBI.Methods:C57BL/6 mice.25-30g body weight, total n=130, were randomly divided into three groups:â‘ stereotactic injection with PEI dissolved in 5% glucose and negative control siRNA (n=50)(NC group);â‘¡shame-operated group(n=15);â‘¢Stereotactic injection of PEI and Hesl-siRNA mixture dissolved in 5% glucose(n=65) and made moderate fluid percussion brain injury. All mice were fed for 1 week after purchase, then put into the Morris water maze to adapt swimming 4 times per day, and randomly entered into the tank to swim each time from a different point, training for 5 days. On 2 day after the training, we injected siRNA into mice hippocampus. After dealed with different factors, all mice were subjected to moderate FPI(2.1-2.2 ATM). Then motor function was tested with modified neurologic severity scores (mNSS). On 24h after RNAi, the total mRNA of hippocampus was extracted for real time RT-PCR test to select the optimal siRNA sequence. On 3 day after siRNA transfection, each group were randomly selected five mice to slice. Slices were used for HE, Hes1 and BrdU staining. On 1 day,2 day,3 day,14 day after siRNA transfection,6 mice of each group were randomly selected to extract Hesl protein that was used for Western Blot. On 3 day after trauma, the others were trained again for 4 days and 7 days after the second training to detect, in order to observe the changes in mice behavior. After water maze training was over, the double staining (BrdU+NeuN) were carried out to observe the cell types in SGZ. All slices were observed by fluorescence microscopy and confocal microscope and record the number of positive cells.Results:1, The results of real time RT-PCR showed that Hesl mRNA relative expression of transfection group were significantly decreased, in which inhibition efficiency of Mus-Hesl-siRNA-7 was 70% or more, was the optimal.2,24-72 hour after FPI, the mNSS score of control group mice was significantly higher than transfection group mice (P<0.05). On 6 day, transfection group and control group was no difference (P>0.05).3, Morris water maze after traumatic brain injury, the results of transfection group was significantly better than two control groups (P<0.05); 7 day after the second training, navigation and spatial search was detected, the difference between transfected and control group still existed (P<0.05).4, Immunohistochemistry results showed that:24h after transfection, fluorescent protein successfully expressed in the hippocampus; 3 day after RNAi, compared with NC group, the Hesl expression of transfection group significantly reduced and the number of neural precursor cells in SGZ (BrdU+) did not change; but mature neurons (BrdU+/NeuN+) increased significantly on 14 day.5. Western blot showed that Hesl protein expression of transfection group were significantly lower than NC group'on 3 day after RNAi (P<0.05).Conclusion:1, Hesl siRNA transfection in vivo effectively knocked down Hesl expression in the mouse hippocampus tissue.2, Moderate hippocampal injury model did not obviously affect motor function in mice. The damage of the hippocampus mainly appeared the decline of spatial learning and memory ability.3, Early trauma, hippocampus neurons may occur apoptosis and the relevant neural circuit was damaged, making long-term cognitive function decline in mice; Hesl gene was likely to promote the proliferation of neural stem cells, make new nerve cells into mature neurons and integrate into existing neural network, to repair cognitive function.4, Compared with the control group, traumatic injury did not change the number of BrdU positive cells, which suggest that the decrease of Hesl expression may inhibit the proliferation of neural stem cells, but promote the new nerve cells to differentiate and mature.5, The results of Western blot showed that after TBI, the Hes1 protein relative expression of transfection group was significantly reduced compared with NC group, indicating that transfection of siRNA successfully inhibited Hesl gene expression.