Adenovirus-Mediated Transfection And Function Of Hesl In Neurogenesis In Adult Mice

Objective:As a technology vector,adenovirus(Ad)was used to explore the feasibility and effectiveness of adenovirus-mediated Hesl gene upregulation 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=108,were randomly divided into four groups:1.shame-operated group(n=24);2.stereotactic injection of PBS group(n=24);3.Stereotactic injection of Ad-EGFP group(n=24);4.Stereotactic injection of Ad-Hesl group(n=24).All mice were fed for 1 week after purchase.At 3 days after dealed with different factors,immunofluorescence and western blot method was used to observe the expression of Hesl protein,all of the rest mice were subjected to moderate fluid percussion injury(FPI).Then motor function was tested with modified neurologic severity scores(mNSS).At Id,3d after FPI,each group was randomly selected 3 mice to execute and the brain sections were stained by BrdU,the numbers of BrdUu cells were recorded under the fluorescence microscopy.At 5 day after FPI,3 mice of each group were randomly selected and the staining of DCX was carried out to observe the immature neurons,another 6 mice were used for hippocampus protein extraction and western blotting.Results:1.3 days after the transfection,EGFP was highly expressed in the dentate subgranular zone(SGZ)of the hippocampus.The results of western blotting showed that Hesl protein relative expression of transfection group was significantly increased.2.24-72 hour after FPI,the mNSS score of control group mice was significantly higher than Hesl transfection group mice(P<0.05).On 6 day,transfection group and control group was no difference(P>0.05).3.Immunofluorescence results showed that:3 day after FPI,compared with the control groups,the number of neural precursor cells in SGZ(BrdU+)was significantly reduced in the Hes1 transfection group.4.Immunofluorescence results showed that the number of immature neurons(DCX+)in SGZ was significantly reduced in the Hesl transfection group 5 days after FPI;western blot also showed that DCX protein expression of Hes1 transfection group were significantly lower than other groups(P<0.05).Conclusion:1.Ad-Hesl transfection in vivo effectively increased Hesl expression in the mouse hippocampus tissue,moreover,the Hesl protein mainly expressed in the dentate subgranular zone(SGZ)of the hippocampus.2.Compared with the control groups,traumatic injury significantly reduced the number of BrdU+and DCXI cells at different days,which suggest that the increase of Hesl expression may not only inhibit the proliferation of neural stem cells,but also inhibit the new born cells to differentiate and mature.3.Hes1 gene is one of an important transcriptional repressor gene in the Notch signaling syetem and the Hesl protein could combined with the targeted zone of DNA.Sustained Hes1 expression leaded to inhibition of both cell proliferation and differentiation.In addition,a large number of Hesl proteins accumulated within the cells may also affect the functional status and impede the proliferation and differentiation of the cells,for that sense,single-gene seemed to be insufficient for the accommodation of the cell fate,multiple genes expressed at correct time and space can play a good regulation,therefore,the research should be focused on to explore the impact on the neurogenesis through the regulation of polygene in the future.