| Objective:The aims of this study were to to explore the effect of transplantation of oligodendrocyte precursor cell,which induced from mouse embryo fibroblasts-derived pluripotent stem cell,on rats suffered with contusive spinal cord injury.Methods:Rat spinal cord injury(SCI)model was established by Allen’s way to induce spinal cord contusive injury(SCC).Basso,Beattie and Bresnahan(BBB)score and MRI scan were used to assure the success of rat SCC model.Immunofluorescence staining was performed for identifying oligodendrocyte precursor cells(OPCs)nduced from mouse embryo fibroblasts-derived pluripotent stem cell(ipsC).All rats were randomly divided into sham group,SCC group,OPC group and Medium group according to the animal models and treataments.BBB score and pain score were used to assess the effects of different treatments on SCC rat motor and sensory functional rehabilitation.Results:(1)The results showed that BBB score of rats in SCC group were significantly lower than that in Sham group(P<0.05).MRI scan also showed there were significant edema and the formation of cavities in SCC group at T10 level,compared with that in sham group,which suggested the successful establishment of SCC model.(2)When GFP-OPCs were cultured in basal-OPC-medium,almost all of the cells displayed the typical morphology of OPCs,which further confirmed the successful building of OPCs.(3)With the exception of day 1 and 3,post-transplantation,the BBB score demonstrated that rats in the OPC group exhibited a significant motor improvement compared with those in the Medium group.(4)At 1 and 3 days post-transplantation,there were no significant differences between the average paw withdrawal threshold for rats in the Medium and OPC groups.However,on 7,14,21 and 28 days following the transplantation,the rats in OPC group showed a significant attenuation of mechanical allodynia(i,e.an increase in paw-withdrawal threshold)compared with that in Medium group.Conclusion:The SCC rat models were successfully established,confirmed by BBB score and MRI scan.Immunofluorescence staining identified that mouse embryo fibroblasts-derived pluripotent stem cell could differentiate into OPCs.OPC transplantation may promote motor and sensory functional recovery of rats with SCC.Objective:MicroRNAs assay was used to analyze the differentially expressed microRNAs of spinal cord in sham,SCC,OPC and Medium groups.Bioinformatics analysis was performed to predict the possible target genes,functions and pathways of microRNAs for further discussion of the molecular regulatory mechanism involved in SCC rats transplanted with OPCs.Methods:Spinal cord of rats,at 7 days after operation in Sham,SCC,OPC and Medium groups,were harvested to perform microRNA chip assay.Gene ontology and KEGG pathway were used to annotate the functions and pathways of target genes of differentially expressed microRNAs among the groups.Results:(1)There were 217 microRNAs(62 up-regulated and 155 down-regulated)expressed differentially in the spinal cord of rats from the OPC group compared with that from the Sham group,according to the following criteria:Fold change>2 and P<0.05.While,there were 45 differentially expressed microRNAs(40 up-regulated and 5 down-regulated)in the spinal cord of rats from the OPC group compared with that in the Medium group,according to the following criteria:Fold change>2 and P<0.05.3 common up-regulated microRNAs were found between SCC and Sham groups,OPC and Medium groups.3 common microRNAs were found in upregulated microRNAs between Sham and Sham groups,down-regulated microRNAs between OPC and Medium groups.There were 24 common microRNAs found in down-regulated microRNAs between Sham and Sham groups,up-regulated microRNAs between OPC and Medium groups.There were no common down-regulated microRNAs between SCC and Sham groups,OPC and Medium groups.(2)There were 33 specially GOs,enriched by targeted genes of up-regulated microRNAs between SCC and Sham groups.99 GOs were specially enriched by targeted gene of down-regulated microRNAs between SCC and Sham groups.323 GOs were specially enriched by targeted gene of up-regulated microRNAs between OPC and Medium groups.105 GOs were specially enriched by targeted gene of down-regulated microRNA between OPC and Medium groups.(3)There were specially enriched 3 pathways,targeted by gene of up-regulated microRNA between SCC and Sham groups.9 pathways were specially enriched by targeted gene of down-regulated microRNAs between SCC and Sham groups.10 pathways were specially enriched by targeted gene of up-regulated microRNAs between OPC and Medium groups.2 pathways were specially enriched by targeted gene of downregulated microRNAs between OPC and Medium groups.Conclusion:There were 217 differentially expressed microRNAs(62 upregulated and 155 downregulated)in the spinal cord of rats from the OPC group,compared with that in the Sham group.While,there were 45 differentially expressed microRNAs(40 up-regulated and 5 down-regulated)in the spinal cord of rats from the OPC group compared with that in the Medium group.The GO and pathway of targeted gene of microRNAs were both common and special between SCC and Sham,OPC and Medium groups.Objective:The aims of this study were to identify the top 10 up-regulated expression microRNAs and 5 down-regulated expression microRNAs differerntially expressed between OPC group and medium group,and to investigate the role of miR-363 on primary spinal cord neuron and the possible mechanism for the regulation of DCLK1 regulation.Methods:Q-PCR was used to verify the top 10 up-regulated expression microRNAs and 5 down-regulated expression microRNAs differerntially expressed between OPC group and medium group.The role of miR-363 was investigated through transfection of miR-363 mimics and miR-363 inhibitor.And also,dual luciferase reporter assay was used to verify the regulation between miR-363 and DCLK1.QPCR was used to confirm the differentially expression of DCLK1 mRNA between the miR-363 mimics group and the miR-363 inhibitor group.Results:(1)QPCR analysis further confirmed that the most highly up-regulated(miR-375-3p and miR-1-3p)and down-regulated(miR-363,miR-449a-5p,miR-3074)spinal cord miRs were differentially expressed between OPC and medium groups.(2)In vitro,interference of miR-363 expression could increase the number of spinal cord neuron,magnify the area of spinal cord neuron body,and enhance neurite length of spinal cord neuron.While over-expression of miR-363 led to the opposite results.(3)The results of dual luciferase reporter assay confirmed the regulation between miR-363 and DCLK1.(4)Compared with Normal group,the expression of DCLK1 mRNA in primary spinal cord neuron was decreased in miR-363 mimics group.While,the expression of DCLK1 mRNA in primary spinal cord neuron was increased in miR-363 inhibitor group.Conclusion:MiR-375-3p,ro-miR-1-3p,ro-miR-363,ro-miR-449a-5p and ro-miR-3074 were differentially expressed between OPC and medium groups.In vitro,interference of miR-363expression could increase the number of spinal cord neurons,magnify the area of spinal cord neurons body,and enhance neurite length of spinal cord neurons.While over-expression of miR-363 led to the opposite results.Moreover,miR-363 could regulate the expression of DCLK1.These results suggested that miR-363 could modulate the growth and proliferation of primary spinal cord neuron through regulating DCLK1 expression. |
PDF Full Text Request