| Objictive : Spinal Cord Injuries are a common clinical problem, which caused complete and incomplete paraplegia below injured level. OECs has bigeminal character of Schwann and Astrocytes showen by that it can cross the transition zone between PNS and CNS and enter into CNS from PNS, so it can compensate the defect of Schwann cells and fibroblasts because they are non-CNS origin and are limited in PNS. On one hand, OECs can pretect the regeneration of axon from the influence of inhibitors by the production of Chondroitinase and remeylinization for axons; on the other hand , OECs can product NTs such as NGF, BDNF, NT-3, NT-4 et al. So OECs may be better candidates for biobogical implantation, but the number of regenerating axoms distal from the injury site was still limited partly because of the limited neurotrophic factor expression and differences in the capacity of various axonal populations to regenerate through OECs implants have been reported. Neurotrophin-3 is one of the important factors in the regenerative milieu and plays variety of physiological effects on the development of nervous system. Many studies are focused on the usage of NT-3 to stimulate nerve regeneration, but the safe and efficient delivery of it to the site of injury is still problematic because of the short half-life and barrier between brain and vessel. Gene transfer technique provides a new strategy for the applicationof NT-3 and allows the insertion of NT-3 gene into OECs, which would then produce NT-3 continuously and exert the physiological action. The objective of this study is to constructe rat PcDNA3.1(+)-NT3-6his recombinant eukaryotic expression plasmid and investigated its transfection and expression in OECs.Methods: Coding sequence of NT3 was amplified by PCR from gene got from rat liver cells and cloned into PcDNA3.1(+) eukaryotic expression vector. Analysis by restricting enzyme digestion and DNA sequencing were carried out to demonstrate the sequence of plasmid. The recombinant PcDNA3.1(+)-NT3-6his plasmid was then transfected in vitro to OECs mediated by using Iiperfectmine2000. The expression of NT3 protein was determined by RT-PCR and Western Blot in PcDNA3.1(+)-NT3-6his plasmid transfected OECs. At last, supernatant of OECs transfected by PcDNA3.1(+)-NT3-6his were co-culture with human neuroblastoma cell line SY-N-SH to observe the biological activity ofNT-3.Results: Analysis by restricting enzyme digestion of recombinant PcDNA3.1(+>NT3-6his showed two fragments: 780bp and 5.4Kb. DNA sequencing revealed that NT3 cloning was successful. The recombinant plasmid can express NT3 mRNA and protein in eukaryotic OECs. And the secreted NT-3 in OECs supernant can prompt the survival and the longer processes of human neuroblastoma cell line SY-N-SH.Conclusion: OECs may be better candidates for neurotrophic factor delivery since they can reduce the problem of regenerating axons to re-enter the distal part of the spinal cord and have advantages over the use of Schwann cells and fibroblasts. Previous studies have demonstrated that OECs can be transferred by BDNF gene. In current study, We successfully transfected PcDNA3.1(+)-NT3-6his into OECs, Then it canenlarge the production of NT-3 in spite of the low efficiency of transfection. And the secreted NT-3 has biological activity to prompt the survival of human neuroblastoma cell lineSY-N-SH and the longer processes were observed. The experiment made a basis for gene therapy of spinal cord injury by the use of gene modified OECs.
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