| Objective:Chronic cerebral ischemia(CCI)is characteristic by persistent/progressive cognitive and neurological dysfunction which is caused by chronic cerebral hypoperfusion.The mechanism of this disease is related with reduction and dysfunction of neuron.The treatment is mainly based on drug therapy;however,there is no effective drug.Mesenchymal stem cells(MSCs)have self-renewal and multiple differentiation abilities and can treat ischemic cardiomyopathy,lower extremity arterial ischemic disease,which provides a new choice for the CCI treatment.MSCs play a role in tissue repair and regeneration owing to their paracrine effects.Exosomes are the most important components of paracrine and are involved in the body's immune response,antigen presentation,metabolic response,apoptosis,tissue regeneration and angiogenesis which play a very important role in neuroprotection and neural recovery.Stem cells from exfoliated deciduous teeth(SHED)is extracted from the deciduous tooth and is a kind of stem cells derived from embryonic neural crest which homologous with central nervous tissue.It has great potential for clinical application due to the multilineage differentiation capability,and the harvest of SHED is noninvasive.It has been suggested that SHED could differentiate into functional neurons and oligodendrocytes to relieve the injury of central nervous system.In addition,it could promote the recovery of function after traumatic brain injury and spinal cord injury,and improve the ability of learning and memory in rats with Alzheimer's disease.In this study,CCI animal model in rats was established,the repaired effects on nerve injury of CCI rats by SHED transplantation in vivo were observed,and the possible molecular mechanism was explored in vitro.The present study aimed to provide a basis for the application of dental-derived stem cells on regeneration of nerve injury.Methods:1.SHED was primary isolated with enzymatic digestion.Flow cytometry was used to evaluate surface marker expression of SHED,such as CD34,CD45,CD73,CD90,and CD105.Alizarin red staining and immunofluorescence were used to evaluate the multi-lineage differentiation potential of SHED.2.CCI rat model was established and identified.Small animal ultrasound,Morris water maze,and immunohistochemical methods were used to identify CCI rat models.SHED were separately transplanted into hippocampus of CCI rats.The cognitive ability of CCI rats were measured by morris water maze,and the protein expression of BDNF,SYN,and PSD95 were examined by western blot.3.The number of neurons in the CAI hippocampus of CCI rats were measured by HE,Nissl staining.The apoptosis of neurons in the CAI hippocampus of CCI rats were measured using Tunnel staining.The protein expression levels of Caspase 3/cleaved Caspase 3 were examined by Western blot.Lentivirus was transfected with GFP,and GFP-SHED was transplanted into the hippocampus by intracellular injection.Immunofluorescence was used to detect the expression of GFP-SHED in the hippocampus of CCI rats.4.In vitro,N2a were used to establish an oxygen glucose deprivation(OGD)N2a cell model.SHED and N2a were co-cultured by transwell system.The effect of SHED on the cell viability of N2a was detected by CCK-8 assay.5.SHED-Exo was extracted by ultracentrifugation,and identified by transmission electron microscopy,nanoparticle tracking technology,and Western blot.SHED-Exo was labeled and co-cultured with N2a cells with PKH26.Immunofluorescence staining was used to detect whether SHED-Exo could be endocytosed by N2a cells.Oxygen sugar deprivation(OGD)N2a cells were treated by SHED-Exo and transfected with the over-expressing Fas gene at the same time.CCK8 was used to detect the activity of N2a cells.Western blot was used to detect the expression of Fas,Caspase 3,and cleaved Caspase 3 in N2a cells.The experimental data were statistically analyzed using SPSS 18.0 software.P<0.05 was considered statistically significant.Results:1.SHED expressed MSCs surface markers CD73,CD90 and CD105,and failed to express hemopoietic stem cells surface markers CD34 and CD45).Mineralized nodule formation could be seen after 4 weeks of osteogenic induction in vitro.Nerve cell marker?III tubulin,and neural stem cell marker Nestin were positive at 1 week induction.2.CCI model was successfully established by ligating the bilateral common carotid arteries of rats.Small animal ultrasound test showed that there was almost no blood flow through the common carotid arteries on both sides of CCI rats at the end of diastole.Morris water maze test results showed that the learning and memory abilities of CCI rats were impaired.Histological test showed a decrease in the number of neurons in the hippocampal CA1 region of CCI rats.SHED transplantation by hippocampus can alleviate the cognitive dysfunction of CCI rats(P<0.01).Western blot results showed that the expression levels of neurological function-related proteins BDNF,PSD95,and SYN in the hippocampus after SHED transplantation were significantly increased compared with CCI rats(P<0.01).The appropriate concentration of SHED transplantation by hippocampus was 2×10~5 per rat.3.HE and Nissl staining test showed a decrease in the number of neurons and the cells were unevenly distributed,the nuclear membrane was not clear,some of the nuclear membranes were shrinking,and there were coagulative necrosis and cell loss of cells compared with the control group in the hippocampal CA1 region.The number,morphology and distribution of neurons tended to be normal after SHED transplantation;Tunel staining showed that there were a large number of neurons apoptosis in the hippocampal CA1 region of CCI rats and the number of neuron apoptosis decreased significantly(P<0.01)after SHED transplantation.Western blot results showed that compared with CCI rats,the expression levels of caspase 3 and cleaved caspase 3 in hippocampus after SHED transplantation were significantly reduced(P<0.01).Immunofluorescence staining showed that there was no significant positive expression of GFP in the hippocampus of CCI rats 5 days after GFP-SHED transplantation.4.CCK-8 test showed that the survival rate of N2a cells was about 50%when N2a cells were under conditions of OGD 4 h in vitro,and the in vitro cell model was established under conditions of OGD 4 h.Compared with the model group,the survival rate of N2a cells was significantly increased after co-culture with SHED(P<0.01).5.SHED-Exo has a double-layered membrane-cup-disc structure with a particle size of30-120 nm and positive expression of CD9 and Alix.Immunofluorescence staining results showed that PKH26 labeled SHED-Exo could be endocytosed by N2a cells.CCK8 test results showed that SHED-Exo up-regulated the viability of OGD N2a cells(P<0.01),and this up-regulation effect was weakenedafter N2a cells were transfected with Fas gene(P<0.01);Western blot results showed that the expression level of Fas protein in OGD N2a cells significantly increased compared with the normal group(P<0.01),and SHED-Exo down-regulated the expression level of Fas protein in OGD N2a cells(P<0.01);Western blot results showed that the expression of Fas,caspase 3,cleaved caspase 3 were increasedsignificantly after N2a cells were transfected with the Fas gene,the inhibitory effect of SHED-Exo on apoptosis was weakened(P<0.01).Conclusions:SHED transplantation effectively reduced neuronal apoptosis in the hippocampal region of CCI rats,restored neuronal function,and alleviated cognitive dysfunction in rats;SHED improved OGD N2a cell viability;Exosomes derived from SHED could be endocytosed by the injured neurons and down-regulate the expression of Fas in neurons,then inhibited neuronal apoptosis,up-regulated neuron function,and thereby improved the cognitive dysfunction in CCI rats. |
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