| Objective: After a complete spinal cord transection,all axon projection connections to target neurons above and below the lesion site are also interrupted.Spontaneous regrowth of the transversed axon at the lesion site fails,preventing reconnection and reinnervation with the target neuron.Therefore,promoting axon regrowth and neuron reconnection is the key to repairing spinal cord injury.This study aims to investigate the molecular mechanism of axon regeneration promoted by Netrin-1 after complete spinal cord transection,and investigate the effect of simultaneous administration of Netrin-1 and its receptor DCC on axon regrowth,axon reconnection and motor function recovery,as well as its relationship with Ng R1-Rho A-ROCK signaling pathway.Method: Construct expression Netrin-1 Interfering RNA(si RNA)/scramble RNA(NC),Netrin-1 overexpression /scramble RNA(NC)and DCC interfering RNA(si RNA)/scramble RNA(NC),DCC overexpression /scramble RNA(NC)lentiviruses.The model of complete spinal cord T10 transection in rats was established and the rats were divided into 14 groups:Sham group,SCT injury group,SCT+Netrin-1 overexpression group,SCT+Netrin-1 overexpression group,SCT+Netrin-1 underexpression group,SCT+Netrin-1 underexpression group,SCT+Netrin-1 underexpression group,SCT+DCC overexpression group,SCT+DCC underexpression group,SCT+DCC low table Reach group,SCT+DCC low-expression control group,SCT+Netrin-1/DCC co-overexpression group,SCT+Netrin-1/DCC co-overexpression group,SCT+Netrin-1/DCC co-underexpression group,SCT+Netrin-1/DCC co-underexpression group,SCT+Netrin-1/DCC co-underexpression group,The corresponding Netrin-1 was injected with a microinjector at the site of spinal cord injury And DCC lentivirus.After complete spinal cord transection,BBB score was performed and paraffin sections were taken for immunofluorescence staining at 1,7 and 14 days.Axon regeneration was detected by GAP43+(nerve growth related protein),neuroplasticity was evaluated by SYP+(synaptophysin),and neuron apoptosis was detected by Tunel staining.The expressions of Ng R1,Rho A,ROCK1,ROCK2,GAP43 and SYP were detected by q RT-PCR and Western Blot.Results: 1.The model of complete transection of spinal cord injury in rats was successfully constructed.After complete transection at the T10 level,the hind limb of the rat was paralyzed immediately.BBB scores presented at 0,4,8,12,and 24 hours after surgery were.2.Successful construction and verification of Netrin-1 and DCC overexpression and underexpression lentiviruses and their respective controls.The constructed lentivirus-mediated overexpression vector was successfully transfected into 293 T cells,and the stable overexpression cell lines were prepared and verified.The low expression lentivirus produced by screening out effective interfering fragments effectively transfected PC12 cells and stably expressed labeled RNA in vitro.3.Behavioral scores after total spinal cord transection showed that from day 7after SCI,the hind limb motor function of rats in Netrin-1 overexpression group,DCC overexpression group and Netrin-1/DCC co-overexpression group began to improve significantly compared with the control group.Compared with the control group,the recovery of hind limb motor function was significantly slower in the low expression group.4.Netrin-1 increased expression of Netrin-1 and DCC in the acute stage of SCI began to play a role in promoting axon regeneration and reducing the rate of cell apoptosis.5.PCR and Western Blot results showed that both Netrin-1 and DCC were overexpressed/underexpressed and negatively regulated with Ng R1 pathway factors starting from 7 days after total transection.Conclusion: Netrin-1 and DCC overexpression/underexpression lentivirus began to express stably in vivo 3 days after injection and affected axonal regeneration and recovery of motor function after spinal cord injury in rats.Netrin-1 promotes nerve regeneration by binding to its receptor DCC and inhibiting the Ng R1-Rho A-ROCK signaling pathway. |
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