Silencing Effect Of PSUPER Recombinant Vectors Targeting CⅡTA Gene Of Rat Schwann Cells

Peripheral nerves long defects is a difficult clinical problem to reconstructive surgery.To date,the standard for bridging peripheral nerve defects involves the use of autografts.Because of the the limited availability of autografts,and the associated morbidity at the donor site,its clinical practice is limited.Tissue engineering of artificial nerve involves the isolation of seed cells and the choice of a biocompatible,degradable matrix,or scaffold,and the cultivation of the viable cells seeded into the scaffold.Schwann cells play an important role in peripheral nerve regeneration,is the common seed cell of artificial nerve.However,host rejection often impede the survival of schwann cells,thus affect the nerve regeneration.Mosahebi's research demonstrated that the increase in the expression of MHCⅡat 3 weeks in the conduits containing allogeneic SC corresponded to an increase in T-lymphocyte as well as macrophage infiltration.Furthermore,there was a corresponding reductionin X-gal staining at 3 weeks pointing to a rejection process of allogeneic SC.Therefore,MHC classⅡgene is a important factor affecting schwann cell's survival.Immune suppressing agents have been used to prolong the survival of schwann cells,but some investigators have described extensive loss of regeneratedaxons in the allograft when immunosuppression was withdrawn.RNA interference is initiated by the Dicer enzyme,which processes double-stranded RNA into 21～23-nucleotide small interfering RNAs.The siRNAs are incorporated into a multicomponent nuclease,RISC.Then the antisense strand of the siRNA is used by an RNA interference silencing complex to guide mRNA cleavage,so promoting mRNA degradation.Therefore,RNA interference can inhibit the expression of specific gene which may cause host rejection in the allotransplantation,diminish the immunogenicity of allograft cells.CⅡTA(ClassⅡTransactivator)is referred to as the master regulator of MHC-Ⅱtranscription.It is important for both constitutive expression of MHC-Ⅱin professional APCs as well as cytokine-induced expression of MHC-Ⅱin a variety of other cell types.Allograft survival of CⅡTA hearts dramatically prolonged as compared with wild-type grafts.So CⅡTA is an ideal target gene to inhibit the expression of MHC classⅡgenes.The construction of siRNA expression vector is the only way of long-term research.After the vectors are transfected into mammalian cells,depend on one of the polymerase-Ⅲgene promoter,the resulting transcript of the recombinant vector is predicted to fold back on itself to form a 19-base pair stem-loop structure,then the stem-loop precursor transcript is quickly cleaved in the cell to produce a functional siRNA.The study construct pSUPER recombinant vectors targeting CⅡTA gene, then transfect the vectors into rat schwann cells.The vectors can specifically suppress the gene expression of CⅡTA gene,significantly decrease the immunogenicity of rat schwann cells.The study provide a new method to create a seed cell bank of artificial nerves.