Lentivirus-Mediated Overexpression Of MiR-29a Promotes Axonal Regeneration And Functional Recovery After Spinal Cord Injury In Rats Via PTEN/AKT/mTOR Pathway

Part ?:Improved Allen 's method to establish contusion model of spinal cord injury model in rats.Objective:To establish contusion model of spinal cord injury model in rats by improved Allen's method,and to observe the BBB score and histological differences after spinal cord injury in the sham operation group and spinal cord injury group.Methods:A rat model of thoracic 10 contusion spinal cord injury was established.Changes in motor function of the hind limbs after spinal cord injury were evaluated by BBB scoring method,and histological changes of each group after spinal cord injury were observed by HE staining.Results:After spinal cord contusion in thoracic 10 rats,massive hemorrhage,necrosis and obvious swelling cell occurred in the spinal cord injury group.In the sham operation group,the nerve cells were evenly distributed,normal in shape,the intercellular matrix was evenly distributed,and the nerve fibers were orderly arranged.The difference of BBB score between the spinal cord injury group and the sham operation group was statistically significant(p<0.05).Conclusion:1.The contusion model of spinal cord injury in rats was successfully established;2.BBB score of the sham operation group was significantly higher than that of the spinal cord injury group.Part ? Expression of PTEN/Akt/mTOR signaling pathway related proteins after spinal cord injury in ratsObjective:To elucidate the expression,time distribution and role of PTEN/AKT/mTOR signaling pathway related proteins PTEN,p-AKT and p-S6 in spinal cord injury of rats.Methods:72 adult rats with SPF level and body weight of 200±20 g were used to establish a spinal cord injury model of contusion type in the injury group using modified Allen's method,and only laminectomy was performed in the sham operation group.After modeling,samples were collected at 2W,4W,6W and 8W in each group,and the expression of PTEN/AKT/mTOR signaling pathway related proteins in spinal cord tissues were detected by Western blot.Results:Compared with the sham operation group,the expression level of PTEN in the spinal cord injury group decreased significantly at 2W,4W,6W and 8W after spinal cord injury.The expression levels of p-AKT and p-S6 increased significantly at the corresponding time points.Conclusion:PTEN,an extremely important regulatory site in the PTEN/AKT/mTOR signaling pathway,was significantly reduced after spinal cord injury in rats,and the downstream molecules p-AKT and p-S6 were significantly increased.This suggests that this signaling pathway may play an important role in the development of spinal cord injury in rats.Using PTEN as a target,miRNA inhibition of PTEN may have a protective effect on spinal cord injury.Part ? Lentivirus-mediated overexpression of miR-29a promotes axonal regeneration and functional recovery after spinal cord injury in rats via PTEN/Akt/mTOR pathwayObjective:To establish a rat model of spinal cord injury with contusion using modified Allen's method,and to investigate the role and potential mechanism of the recombinant lentivirus vector lv-miR-29a in the repair of spinal cord injury.Methods:Normal adult SD rats were selected to establish a model of thoracic T10 spinal cord injury.Intraoperative injection of recombinant lentivirus vector lv-miR-29a,empty lentivirus vector lv-eGFP and normal saline into the head and caudal side of spinal cord injury were performed.BBB score was performed at 2W,4W,6W and 8W after surgery.PTEN,NF-200 were detected by qRT-PCR,and the expressions of related proteins PTEN,AKT,p-AKT,S6,p-S6 and NF-200 in the PTEN/AKT/mTOR signaling pathway were detected by Western blot.Meanwhile,immunofluorescence staining was performed to observe the regeneration of nerve fibers and synaptic regeneration.Results:Compared with lv-eGFP group and normal saline group,the expression of PTEN mRNA in lv-miR-29a group decreased by 79±4.0%,85±3.0%,75±3,0%and 65±2.0%respectively at 2,4,6 and 8 weeks after surgery(P<0.001).Western blot showed that the expression of PTEN was down-regulated by 77±3.0%,67±5.5%,67±2.0%and 60±2.5%(P<0.001).The levels of p-AKT and p-S6 were higher than those of lv-eGFP group and normal saline group(p<0.001).The mRNA and protein levels of NF-200 were significantly increased(P<0.001).However,there was no significant difference between lv-eGFP group and normal saline group(P>0.05).The results of immunofluorescence showed that the NF-200 and synapse elements in the 8W lv-miR-29a group were 4.0±0.2 times and 2.9±0.1 times of those in the other two groups,respectively.At 2,4,6 and 8 weeks after surgery,the BBB score of lv-miR-29a was significantly increased compared with the other two groups(P<0.001)Conclusion:Overexpression of miR-29a significantly inhibits the expression of PTEN,enhances the PTEN/AKT/mTOR signaling pathway,promotes axon regeneration and synapse formation,and promotes functional recovery after spinal cord injury...