Effect Of Human Umbilical Cord Mesenchymal Stem Cell Exosomes And Their Mediated LncRNAs On Degenerated Nucleus Pulposus Cells

Objective: To explore the effects of human umbilical cord mesenchymal stem cells(h UC-MSCs)exosomes and LncRNA on degenerated Nucleus pulposus cells(NPCs).Through high-throughput gene sequencing,we screened out LncRNA MEG3,a gene associated with NPCs degeneration,and overexpressed LncRNA MEG3 to further explore the mechanism of the target gene in the process of NPCs degeneration.Methods: NPCs were extracted from degenerated nucleus pulposus by enzyme digestion.The cells were identified by toluidine blue staining and type II collagen immunofluorescence staining.PKH26 staining experiment proved the transmembrane ability of exosomes.CCK-8,q RT-PCR and flow cytometry were used to verify the effects of h UC-MSCs exosomes on cell proliferation,inflammatory reaction,synthesis and degradation of extracellular matrix of NPCs.After that,the cells in exosome intervention group and exosome negative group were sequenced by high throughput gene,and LncRNA and m RNA were screened according to Fold Change ≥ 2 as the differential screening standard.Through Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG),the biological functions of differentially expressed genes were enriched and analyzed,and the target gene LncRNA MEG3 was further screened out to verify its mechanism of action.LncRNA MEG3 was overexpressed in NPCs by transfecting plasmid vectors,and cell apoptosis,inflammatory response and ECM synthesis and degradation were further evaluated by q RT-PCR and flow cytometry.Results: The NPCs extracted in this study were irregular in shape,spindle-shaped or polygonal,and were positive for toluidine blue staining and type II collagen staining,indicating that cells could secrete proteoglycans and type II collagen.The results of PKH26 exosome staining and CCK-8 experiment indicate that h UC-MSCs exosomes can be ingested by NPCs and can promote their proliferation.The results of q RT-PCR show that the expressions of IL-1β,MMP-13,caspase-3 and other genes related to inflammatory reaction,ECM degradation and apoptosis in NPCs are significantly decreased compared with the exosomes negative group.Through chip analysis,we detected 9877 differentially expressed LNC RNAs and 1224 differentially expressed m RNA,and screened them according to Fold Change ≥ 2 standard.Compared with the control group,there were 826 significantly up-regulated and 832 significantly down-regulated LNC RNAs,84 significantly up-regulated and 87 significantly down-regulated m RNA in exosomes intervention group,respectively.GO analysis was conducted by using up-regulated differential genes in the chip.In CC and MF items,Extracellular matrix,Caspase binding and other items were significantly enriched,and GO analysis was carried out by down-regulated differential genes in the chip.In BP items,regulation of infection response and acute infection response were significantly enriched,indicating that the inflammatory reaction,apoptosis and ECM-related genes of nucleus pulposus cells treated with exosomes were greatly affected.After screening out the desired target gene LncRNA MEG3,it was overexpressed in NPCs by plasmid transfection,and further verified by q RT-PCR and flow cytometry.The results showed that after overexpression of target gene,apoptosis level,inflammatory response and degradation of ECM were significantly reduced.Conclusion:1.In IDD,h UC-MSCs exosomes can be absorbed by NPCs,and can promote the proliferation of NPCs,inhibit the apoptosis of NPCs,reduce the inflammatory reaction of cells,and inhibit the degradation of ECM.2.The results of gene chip show that LncRNA mediated by h UC-MSCs exosomes plays an important role in the influence of HUC-MSCs exosomes on degenerated NPCs.3.LncRNA MEG3 overexpression experiments have proved that the effect of h UC-MSCs exosome-mediated LncRNA MEG3 on degenerative NPCs may be due to its effects on inflammation,apoptosis,ECM degradation,etc.