The Research Of The Effect And Mechanism Of HucMSCs-EVs Promoting The Repair Of Spinal Cord Injury Through MiR-29b-3p/PTEN Signaling Pathway

Objective: To explore the mechanism of HucMSCs-EVs promoting the repair of spinal cord injury through miR-29b-3p/PTEN signaling pathway.Methods:1.HucMSCs were cultured.Flow cytometry was used to detect the marker on the surface of HucMSCs.After induction differentiation,the adipogenic,osteogenic and chondrogenic differentiation abilities of HucMSCs were identified.EVs secreted by HucMSCs were isolated by ultracentrifugation,and EVs were detected by electron microscope,NTA and Western blot(WB).2.The spinal cord injury model of SD rats was established by "Forceps operation".The expression of miRNA-29b-3p was detected by RT-qPCR.The targeting relationship between miRNA-29b-3p and PTEN was verified by dual-luciferase reporter.HucMSCs were transfected with miRNA-29b-3p inhibitor and NC respectively.Animal experiments were divided into 8 groups:sham group,SCI group,EVs group,GW group,EVs NC group,EVs inhibitor group,EVs+ad NC group and EVs+ad PTEN group.EVs were labeled by PKH26 to observe the distribution in the spinal cord.The motion ability of rats were evaluated by BBB score and Rivlin plate test.HE staining and Nissl staining sections were made to observe the injury of spinal cord tissue.NeuN staining,NF200 staining and GFAP staining were used to detect the injury of spinal cord.The apoptosis of spinal cord cells was detected by TUNEL method.The expression of miRNA-29b-3p in each group was detected by RT-qPCR.The expressions of PTEN,p-AKT and p-m TOR were detected by Western blot.3.The morphology of rat spinal cord neurons was observed by optical microscope.Neurons were identified by NSE staining and DAPI nuclear staining.EVs were labeled with PHK26 to observe the uptake of EVs by neurons.The neuron apoptosis model was established by LPS.Cell experiments was divided into 10 groups:LPS group,Blank group,LPS+GW group,LPS+EVs group,LPS+EVs-miR-NC group,LPS+EVs-miR-inhi group,LPS+EVs-miR-inhi+Si-NC group,LPS+EVs-miR-inhi+SiPTEN group,LPS+EVs-miR-inhi+Si-PTEN group,LPS+EVs-miR-inhi+Si-PTEN+DMSO group and LPS+EVs-miR-inhi+Si-PTEN+LY group.The cell viability of each group was detected by MTT assay.Apoptosis was detected by flow cytometry.The expressions of caspase3,cleaved caspase-3,caspase9,Bcl2,PTEN,PI3 K,AKT and p-AKT were detected by Western blot.The activities of caspase3 and caspase9 were detected by caspase activity kit.The expression of IL-6 and IL-1β were detected by enzyme-linked immunosorbent assay.The expression of miRNA-29b-3p and PTEN in each group was detected by RT-qPCR.Result:1.HucMSCs used in this study met the standards,and HucMSCs EVs were successfully prepared.2.SCI rat model can be successfully established by "Forceps operation".HucMSCs-EVs promoted the repair of SCI in rats.HucMSCs-EVs alleviated spinal cord neuronal injury in rats.HucMSCs-EVs promoted the repair of SCI by carrying miR-29b-3p.miR-29b-3p targeted PTEN in spinal cord tissues of rats.Overexpressing PTEN reversed the repair effect of HucMSCs EVs on SCI rats.HucMSCs-EVs activated the AKT/m TOR pathway during SCI repair in rats via the miR-29b-3p/PTEN axis.3.HucMSCs-EVs were successfully isolated and identified.HucMSCs-EVs reduced LPS-induced neuronal apoptosis.EVs carried miR-29b-3p into cells.Knockdown of miR-29b-3p partially reversed the inhibitory effects of EVs on LPS-induced neuronal apoptosis.miR-29b-3p targeted PTEN.EVs carried miR-29b-3p reduced LPSinduced neuronal apoptosis by silencing PTEN.EVs reduced neuronal apoptosis by activating the PI3K/AKT pathway via the miR-29b-3p/PTEN axisConclusion:HucMSCs-EVs can reduce spinal cord injury and promote the repair of nerve function through miR-29b-3p/PTEN axis.