| Aim To evaluate the promoting effects of ASCL1 gene transfection on neuronal differentiation of dental pulp stem cells and nerve regeneration in cerebral infarction area.Methods and Results Rat dental pulp mesenchymal stem cells(rDPSCs)were isolated and amplified in vitro,and the potential of multi-linage differentiation was verified by induction with osteogenic and adipogenic differentiation media.The ASCL1-lentivirus vector-transfected rDPSCs(DPSCs-ASCL1 group)were used in the experimental group,and the vector-transfected DPSCs(DPSCs-vector group)were in the control group.We observed neuronal differentiation of DPSCs after 14 d of neural induction with neuronal differentiation medium.The neuronal differentiation was testified by immunofluorescence(expression level of Nestin,a neural stem cell marker,and MAP-2,a marker of neuron,were examined)and RT-PCR(the expression of MAP-2,and DCX,a marker of neuroblast,were analized).In vitro experiments,rDPSCs could be induced by specific induction medium to undergo osteogenesis,adipogenesis and neuronal differentiation,and displayed the unique multidirectional differentiation potential of mesenchymal stem cells.Both DPSCs-ASCL1 and DPSCs-vector groups of cells could transform into nerve cells,as the Nestin expression was positive in both groups.However,the diameter of the DPSCs-ASCL1 group were much bigger than the control group(P<0.05),while the expression levels of DCX and MAP-2 in DPSCs-ASCL1 group were significantly higher than those in DPSCs-vector group(P<0.05).Immunofluorescence staining indicated that the expression level of MAP-2 in DPSCs-ASCL1 group was significantly greater than that in DPSCs-vector group(P<0.05),suggesting transcription factor ASCL1 promoted the neuronal differentiation of DPSCs.Meanwhile,we established transient middle cerebral artery occlusion model(tMCAO)of rats.24 h after modeling,gene transfection of ASCL1 was carried out by stereotactic microinjection of ASCL1-AAV(adeno-associated virus with ASCL1 gene)into the ischemic zone around the intracranial infarction area(n=5),while the control group were injected with vector virus(n=5).3 days after the injection,the 2,3,5-triphenylterazolimn chloride(TTC)staining was used to analyze the size of the infarction area in both groups.Two behavioral experiments,modified Neurological Severity Scores(mNSS)and Longa grade point standard,were utilized to evaluate the neurological deficits following MCAO.In addition,the expression levels of Nestin and MAP-2 were examined by immunofluorescence and RT-PCR test,so as to evaluate the neurogenesis of cerebral ischemia injury area after the overexpression of ASCL1 gene.In vivo experiment,TTC staining of brain tissue suggested that cerebral infarction area in ASCL1-AAV group was significantly reduced compared with that in the vector control group after tMCAO(P<0.05),and the scores of both behavior tests were also significantly decreased(P<0.05).The expression of Nestin and MAP-2 in the ASCL1-AAV group was eminently increased compared with the vector control group as examined by Immunofluorescence,and this finding was also confirmed by RT-PCR(P<0.05).Conclusion Gene transfection of transcription factor ASCL1 can promote the neuronal differentiation of rDPSCs.In vivo overexpression of ASCL1-AAV in infarction zone of tMCAO rats can facilitate the transformation of injured cells into nerve cells in the ischemic region,reduce the area of cerebral infarction,and improve neurological function.Therefore,ASCL1 plays an important role in repair of ischemic stroke. |
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