Intrathecal RMSCs Modified Human Interleukin-10 Gene Therapy For Vincristine Induced Neuropathic Pain In Rats

Part One Construction of hIL10 Gene Recombinant Adenovirs Vector and Its Expression on Mesenchymal Stem CellsObjectives To obtain intact and errorless human interleukin-10(hIL10) gene. And construct adenovir(Ad) vector carrying the hIL10 gene, and then infect the rat mesenchymal stem cells (rMSCs) to get hIL10 gene-modified rMSCs.Methods The hIL-10 gene was constructed by PCR with pSNAV2.0-hIL10 recombinant plasmid as template, enzyme digestion and ligation, and inserted the obtained hIL10cDNA fragment into shuttle vector pDC316-IRES-EGFP-lacZalpha linearization with PmeI and supercoil adenovirus skeleton plasmid pAdMax for homologous recombination.293 cells was transfected with the obtained recombinant adenovirus vector pAd/ hIL10 in mediation of liposme. The replication defective recombinant adenovirus were propagated by repeat infection of 293 cells and purified by ion exchange method, then the number of viral particles was counted and the purity and titer were determined. After the the rat mesenchymal stem cells(rMSCs) were infected by the acquired Ad, the expression of hIL10 was observed with fluorine microscopy, flow cytometry and Western-blotting method.Results Both recombinant shuttle plasmid pDC316-hIL10-IRES-EGFPand recombinant adenovirus vectorAd-hIL10-IRES-EGFP were correctly constructed, and proved by PCR ,restriction analysis and sequencing. After propagation and purification ,the virus particle count , OD260/OD280 and titer of recombinant adenovirus were3.2×1011VP/mL,2.0 and1.1×1010CCID50/mL respectively. The light green fluorescence in infected rMSCs was observed under fluorine microscopy, and the expression of EGFP and hIL10 protein were in a multiplicity of infection(MOI)and time-dependent manner.Conclusions Ad vector carrying the hIL10 and EGFP gene were constructed successfully.The hIL10 gene-modified rMSCs could express hIL10 to a higher degree,which laid a foundation for further study on adenovirus vector mediated immunosuppressive therapy withhIL10 gene. Part Two Roles of spinal glia and proinflammatory cytokines in rats with vincristine induced neuropathic painObjective To investigate the roles and mechanism of spinal glia and proinflammatory cytokinesIL-1β,IL-6 and TNFαin the rat model of neuropathic pain induced by vincristine.Methods SD rats were randomly divided into two groups(model group and control group).The rat neuropathic pain model was established by repeated peritoneal injection of vincristine. The day of first injection of vincristine was defined as the first day. The rats of model group were peritoneally injected vincristine in the dose of 0.1mg·(kg·d)-1 on the days of the first day to the fifth day and the eighthday to the tweleveth day. The rats of control group were peritoneally injected with normal saline(NS) in the same volume . The thermal hyperalgesia (PWL) and mechanical hyperalgesia (PWT) of the rats were measured, and the expression levels of IL-1β, IL-6 and TNFαin the spinal cord of neuropathic pain models and controls were measured with histochemical staining and reverse transcription polymerase chain reaction (RT-PCR).Results The peritoneal injection of vincristine led to progressive thermal and mechanical hyperalgesia along time course. Histochemical staining showed that the GFAP and OX-42 optical density ratios of the spinal dorsal horn were increased with time course after vincristine injection.The levels of IL-1β,IL-6 and TNFαwere up-regulated in the spinal cord with time course.Conclusion These results demonstrated that the rat neuropathic pain model could be successfully established by repeated peritoneal injection of vincristine. In this model, vincristine induced spinal microglia and astrocyte activations, which promoted the release of IL-1β,IL-6 and TNFα, thus contributing to the maintenance of neuropathic pain. Part Three Intrathecal MSCs modified human interleukin-10 gene therapy for vincristine induced neuropathic pain in ratsObjectives To transplant hIL-10 gene-modified MSCs to the rats with vincristine induced neuropathic pain by intrathecal injection, then observe the viability of MSCs in the subarachnoid space and the analgesic effect, and explore the mechanism of vincristine induced neuropathic pain in rats.Methods Sixty model rats were randomly divided into three groups(n=20, each): hIL10-MSCs group, no-load-MSCs group and control group, and received intrathecal (IT) injection of 30uL hIL10-MSCs suspension(1×106cells/mL), 30uL no- load-MSCs suspension(1×106cells/mL)and 30uL normal saline(NS) respectively in corresponding groups. Paw withdrawl threshold(PWT)and paw withdrawl latency(PWL) were measured on the 3, 7 and 14 days after IT injection.The viability of hIL10-MSCs and no-load-MSCs , the activation of glial cell, and the expression of hIL10, TNFa ,IL-1βand IL-6 in the lumbar segment of spinal cord were identified with immunohistochemical staining , fluorescence excitation , ELISA and Western- blotting respectively.Results PWT and PWL were significantly increasd in rats treated with hIL10-MSCs compared with that of control group and no-load-MSCs group (p<0.05), whereas there was not difference found between no-load-MSCs group and control group(p>0.05). The green fluorescence was seen in the hIL10-MSCs group and the no-load-MSCs group on the 7 and 14 days after IT injection, but no green fluorescence was found in the control group. The expression of hIL10 was found in the hIL10-MSCs group, but not in the no-load-MSCs and control group. Compared with control group and no-load-MSCs group, the activity of gliaI cells and the expression of TNFa,IL-1βand IL-6 were significantly decreased in hIL10-MSCs group(p<0.05).There was not difference found between no-load-MSCs group and control group(p>0.05).Conclusion Intrathecal MSCs modified human interleukin-10 gene therapy can ease pain induced by vincristine, this effect may depend on the inhibition of the activity of spinal gliaI cells and reduction of the release of TNFa ,IL-1βand IL-6 in the lumbar segment of spinal cord by expressing hIL-10 protein of MSCs modified human interleukin-10 gene living in the subarachnoid space for long time.