Experimental Research Of Exosomes Derived From MiR-133b Modified MSCs Promoting Neurological Recovery Following Spinal Cord Injury In Rats And Possible Mechanism

Part I:The changes of miR-1 33b expression in spinal cord tissue after spinal cord injury in ratsObjective:The expression of miR-133b in spinal cord tissue after spinal cord injury in rats.Method:48 adult male SD rats were randomly divided into 8 experimental groups:The normal group,12 hours after spinal cord injury group,spinal cord injury group after 24 hours,48 hours after spinal cord injury group,spinal cord injury group after 72 hours,96 hours after spinal cord injury group,spinal cord injury group after 5 days and 1 weeks after spinal cord injury group.Each experimental group of 6 rats.The aneurysm clipping T1 0 in rats spinal cord for 10s,so as to establish the experimental animal model of spinal cord injury.After the establishment of the model of 12 hours,24 hours,48 hours,72 hours,96 hours,5 days and 1 weeks respectively,the rats were sacrificed.Remove the T1 0 rat spinal cord tissue specimens,Using fluorescence quantitative PCR method,check the changes of miR-133b expression in spinal cord tissue,and statistical analysis.Results:PCR results showed that:The expression of miR-1 33b in both SCI group and sham operation group.MiR-1 33b in the SCI group the expression level decreased at 1 2h after injury,compared with the sham operation,the expression of miR-1 33b in 24h were significantly lower(p<0.05),and over time decreased.Conclusion:1.mir-1 33b was expressed in normal rats spinal cord and in spinal cord after spinal cord injury;2.The expression of miR-1 33b level decreased one week after spinal cord injury in rats,At 24 hours after spinal cord injury miR-1 33b expression have significant difference.Part Ⅱ:The expression of miR-1 33b in exosomes derived from bone marrow mesenchymal stem cellsObjective:To study the expression of miR-133b in exosomes derived from bone marrow mesenchymal stem cells(MSCs).Method:1.Construction miR-133b expression plasmid and its control plasmid:Extraction and cultured primary rat MSCs,MiR-133b plasmid and control plasmid were transfected into MSCs and the establishment of stable expression of miR-133b MSCs and control group MSCs.2.Exosome isolation and identification derived from MSCs:The separation of MSCs derived exosomes by centrifuge,Western blot was used to identify the exosome protein markers(CD 9,CD 63 anfd CD81).Extraction RNA of exosomes and identification of miR-133b expression by real-time PCR method.Results:PCR results showed that:miR-133b express in transfected MSCs,Compared with the control group the expression of miR-133b has significant difference(p<0.01).The expression of miR-1 33b was significantly in exosomes,Compared with the control group there were significant differences(p<0.01).Western blot results showed that:The centrifugation suspension contains a lot of exosomes,Exosome marker proteins CD9,CD63 and CD81 were expressed.Conclusion:1.The suspension derived from MSCs contains exosomes;2.MiR-133b express in exosomes derived from MSCs after transfection.Part III:Experimental research of exosomes derived from miR-1 33b modified MSCs promoting neurologicalrecovery following spinal cord injury in rats Objective:Establish spinal cord injury(SCI)model of rat,In study the function of miR-1 33b on nerve recovery after spinal cord injury in rats.Method:30 adult male SD rats,randomly divided into 5 experimental groups:The sham operation group,control group,PBS control group,miR control group,miR-1 33b intervention group,each experimental group has 6 rats.The aneurysm clipping T10 in rats spinal cord for10s,in order to establish the experimental animal model of spinal cord injury.24 hours after the model was established by tail vein injection respectively using PBS,mir-control,miR-1 33b interference,four days after SCI,the rats were sacrificed respectively.Remove the T1 0 rat spinal cord tissue for specimens,using real-time PCR method to detect the expression of miR-1 33b in spinal cord.Usingwestern blot method to detect the expression of target gene RhoA.Determine the expression of p-erk1/2 p-STATB,STAT3,erkl/2,p-CREB and CREB by Western blot,to find the effection of miR-1 33b in axon regeneration pathway.Western blot and immunofluorescence method to determine the expression of NF、GAP43、MBPand GFAP,understand the effection of miR-13 3b on the recovery of nerve function.Results:PCR results showed that:The expression of miR-133b in control group compared with sham group,the expression decreased significantly(p<0.001);compared with PBS group and control group,the expression of miR-133b has no significant difference(P>0.05);compared with miR-con group and PBS group the expression of miR-133b increased,but the results were not statistically significant(p>0.05);compared with miR-con group and miR-133b group expression of miR-133b were significantly increased(P<0.001).Western blot results showed that:The expression of RhoA:in miR-con group decreased than in control group,but has no significant difference(p>0.05);miR-133b group compared with mir-con group decreased significantly(p<0.05).miR-con group of p-erkl/2/erk1/2 levels than those of group control has no statistical difference(p>0.05);miR-1 33b group has significantly different than in miR-con group(p<0.05).the p-CREB/CREB levels in miR-con group has no statistical difference than those in group control(p>0.05);compared with miR-133b group and miR-con group they were significantly different(p<0.01).the p-STAT3/STAT3 levels in miR-con group has no statistical difference than those in group control(p>0.05);miR-133b group has significantly different than in miR-con group(p<0.05).The expression of NF in miR-con group has no significant difference compared with control group(p>0.05),miR-133b group has significantly different than in miR-con group(p<0.05).The expression of GAP-43 in miR-con group significantly higher than in control group(p<0.05),miR-133b group compared with mir-con group,there were obvious difference(p<0.05).The expression of GFAP in miR-con group significantly higher than in control group(p<0.05),miR-1 33b group compared with mir-con group,there were obvious difference(p<0.01).The expression of MBP in miR-con group significantly higher than in control group(p<0.05),miR-133b group compared with mir-con group,there were obvious difference(p<0.01).Conclusion:1.After injection of exosomes derived from miR-133b modified MSCs,the expression of miR-1 33b was significantly in spinal cord tissue of SCI rats;2.miR-133b influence axon regeneration pathway,increased the expression of NF,GAP-43,GFAP and MBP;3.exosomes derived from miR-133b modified MSCs,promote the recovery of neural function in SCI rats.