Effect Of Lentiviral-mediated Nanog Gene Transfer On The Spinal Cord Synaptic Plasticity In Rat With Peripheral Nerve Injury

Background and purpose: Neuropathic Pain (NPP) is an intractable pain syndromecaused by peripheral or central somatosensory nervous system injury or diseases,which is characteristiced by spontaneous pain, hyperalgesia and allodynia. Thepathogenesis of NPP is still not clarified and the treatment effect is not satisfactory, soit is still the research focus. At present, the domestic and foreign researches generallythink that inhibit nuclear factor-κB (NF-κB) activation can alleviate the mechanicalunusual feeling and heat pain allergy of animal, therefore suppression of NF-κBactivation is a potential NPP treatment strategy. In addition, in the study ofself-renewal and differentiation mechanism of embryonic stem cells, the researchersfound that Nanog binds to NF-κB proteins and inhibits their transcription activity andability of promoting differentiation to maintain pluripotency of embryonic stem cells.The findings provide a new method to inhibit NF-κB activity. Our previousexperiment have found that the expression of NF-κB was significantly weakened inbone marrow mesenchymal stem cells (BMSCs) transduced by Nanog. Lentiviralvector can effectively integrate exogenous gene into the host chromosomes to achievethe persistent express and have good gene therapy effect, so it has wide applicationprospects. To explore the mechanism and effect of Nanog gene transfer on peripheralnerve injury NPP, we construct the lentiviral vector carring Nanog gene, observe invitro the influence of Nanog gene transfer on microglia inflammatory responseinduced by lipopolysaccharide, finally, observe the influence of Nanog gene transfer on the spinal cord synaptic plasticity in rat model with peripheral nerve injuryneuropathic pain. The study consists of four parts. Materials and methods: After double digestion (BamHI/SalI) of cloning plasmidpUC57-Nanog and lentiviral vector plasmid pNL-IRES2-EGFP, the purpose genefragments were collected using gel extraction. Then, the fragments were connected byT4ligase. After DH5α e. coli was transformed, the connection product wasbesmeared on LB solid culture medium plate containing antibiotics for12-16hours at37℃. The bacterial colonies were selected and identified by PCR. The correctbacterial colonies were augmented, and then the small extraction of plasmid wasperformed. Finally, the plasmid was identified by double digestion and sequencing.Results: The recombinational lentiviral vector plasmid was identified right by doubledigestion and sequencing.Conclusion: The recombinational lentiviral vector plasmid was constructedsuccessfully, which laid a foundation for our further study. Materials and methods: Lentivirus was packaged in293T cells cotransfected withlentiviral vector plasmid (pNL-Nanog-IRES2-EGFP), packaging plasmid (pHELPER)and coat plasmid (pVSVG). The virus supernatant was collected, centrifuged andfiltered by0.45um filter, then it was mixed with concentrated reagent containingpolyethylene glycol (PEG). After the mixture was placed at4℃overnight, it wascentrifuged. The precipitation was suspended with PBS, subpackage and preserved at-80℃. The titer of lentivirus was tested using dilution method. Nanog mRNA andprotein expression levels were detected using RT-PCR and Western Blot.Results: Lentivirus was successfully produced in293T cells cotransfected withpNL-Nanog-IRES2-EGFP, pHELPER and pVSVG. After concentration, the titer oflentivirus was above1×10~7TU/mL. The expressions of Nanog mRNA and protein inlentiviral product were confirmed with RT-PCR and Western Blot test. Conclusion: High titer lentiviral vector carrying Nanog gene was produced, whichlaid a foundation for next in vitro and in vivo study. SD rat pups under aseptic conditions. After the meningeal and blood vessels wereremoved, the cortex tissue was chopped, then digested with pancreatic enzyme andfiltered with200mesh mesh filter. Cell suspension was cultured in incubator. Oneighth day, the microglia was separated and purified using hydrochloric acid lidocaine.The3-8generation cells were used for the stimulation and intervention study in vitro.The groups were divided as normal control group, LPS stimulation group and threetransfection groups in different pNL-Nanog-IRES2-EGFP concentrations (1.0,2.0,and3.0) after LPS stimulation. At indicative time, the cell viability was measuredusing MTT. The mRNA expression levels of β-actin, Nanog, IL-1β, TNF-α and IL-6were detected using RT-PCR. Protein expression levels of IL-1β, TNF-α and IL-6were measured using ELISA.Results: Nanog and LPS had no significant effect on the viability of microglial cellsin cultrue. Nanog can reduce LPS-induced expression levels of IL-1β, TNF-α andIL-6mRNA and protein in rat primary microglial cells in vitro.Conclusion: The in vitro observation confirms that Nanog can inhibit the release ofpro-inflammatory cytokines induced by LPS stimulation, weaken the cell inflamma-tion reaction and give play to anti-inflammatory effect. Materials and methods: Male SD rats, four to five months old, weight180-200gwere randomly divided into5groups (n=8in each group): the sham group, the bCCIgroup, the bCCI+saline group, the bCCI+LV-EGFP group and the bCCI+LV-Nanog-EGFP group. On first day after bCCI,2μL saline, or LV-EGFP, or LV-Nanog-EGFPwas injected into the bilateral L4-5spinal cord segment of the bCCI+saline group, thebCCI+LV-EGFP group and the bCCI+LV-Nanog-EGFP group respectively. Mecha- nical, thermal and cold pain thresholds were determined on pre-operation day andpost-operation every3days. The rats were euthanized on postoperative90th day. Thespinal cord was removed and the tissue slice was made. The expression of greenfluorescent protein in spinal cord dorsal horn was observed under fluorescencemicroscope. The number of neurons was counted after toluidine blue staining. Theexpression levels of synaptophysin (Syn), P65, mu opioid receptor (MOR) andcholecystokinin-8(CCK-8) were observed after immunohistochemistry. The changesof synapse number and its proportion to neurons were analysed using stereologyimaging system.Results: Compared with sham group, after operation, the mechanical, thermal andcold pain thresholds of bCCI group decreased on4th day, reached to the lowest on13th,16th and21th day (P<0.05) respectively. Mechanical pain threshold and thermalpain threshold returned to baseline in45th and33th day respectively. However, coldpain threshold was still far from the baseline on postoperative90th day. There was nosignificant improvement (P>0.05) on pain threshold beteween bCCI+saline group,bCCI+LV-EGFP group and bCCI group. The bCCI+LV-Nanog-EGFP group hadhigher pain threshold when compared with bCCI group (P<0.05).The result oftoluidine blue staining demonstrated that there was no significant difference amonggroups on the number of neurons in spinal cord dorsal horn. The result ofimmunohistochemical staining revealed that the bCCI group, bCCI+saline group andbCCI+LV-EGFP group had significantly increased expression of Syn and P65andsignificantly decreased expression of MOR and CCK-8when compared with those ofsham group (P<0.05). There was no significant difference (P>0.05) found on aboveindexes with immunohistochemical staining among bCCI+saline group, bCCI+LV-EGFP group and bCCI group, while there was significant difference (P<0.05)found beteween the bCCI+LV-Nanog-EGFP group and bCCI group. The result ofstereology image analysis system analysis displayed that bCCI group, bCCI+salinegroup and bCCI+LV-EGFP group had higher number of synapse and its proportion toneurons when compared with those of sham group (P<0.01). There was no significantdifference (P>0.05) among bCCI+saline group, bCCI+LV-EGFP group and bCCI group on the number of synapse and its proportion to neurons, while bCCI+LV-Nanog-EGFP group was significant lower than bCCI group (P<0.05).Conclusion: Lentiviral-mediated Nanog gene directly injected into the spinal corddorsal horn reduce the expression of Syn and P65, increase the expression of MORand CCK-8, reduce the number of neurons synapses and its proportion with neurons,improve pain threshold level and has the function for NPP treatment. It may be aneffective gene treatment strategy.