Transfection Of Antisense-B7-1 Into Liver Prolongs Survival Of Rat Liver Graft

Background:End stage liver disease is highly prevalent in China.Liver transplantation is the only available curative medical procedure for patients.Liver transplantation has been rapidly growing and evolving with improvement in surgical technique and admistration of immunosuppressive drug.Immunal rejection remains the major problem in liver transplantation.Despite the impressive results of preventing acute rejection,current anti-rejection drugs reduce systemic immunity nonselectively and increase the side effect of toxicity and risk of cancer on the long term.The high expenditure also limited its application,With the development of molecular biology,gene therapy is the therapy of choice for allograft transplantation in the past 10 years,Blockade of costimulatory molecules to induce immunotolerance is an altemative strategy in liver transplantation.The success of gene therapy in the transplant medicine is strongly dependent on the choice of the target gene which plays a key role during liver transplantation. Important advances have emerged in our understanding of the molecular events of rejection underlying liver transplantion.Costimulatory pathways have been shown to play a crucial role in immune responses of liver transplantation.Two distinct signals are required for full T-cell activation.The first signal is evoked by the T-cell receptor after engagement with the major histocompatibililty complex-peptide complex expressed on the surface of antigen-presenting cells(APCs).The second costimulatory signal is provided by cell surface receptors after binding to their ligands, B7-1(CD80)and/or B7-2(CD86)on the surface of APCs which stimulates T-cell proliferation,cytokine secretion and maintains the T-cell response.Then evoked T cells express CTLA4 which is the homologous receptor of CD28.Binding of B7 and CTLA4 inhibits T-cell activation,T-cell receptor alone without costimulatory signal leads to a state of specific T-cell anergy.The two major approaches of gene transfer for organ transplantation can be defined as:â‘ Increasing expression of the associated gene of inhibiting immune response.Secreted proteins produced by the graft after gene transfer could produce local immunosuppressive effects,thus avoiding generalized immunosuppression.The success of these strategies obtains promising results which acquires graft tolerance or at least a reduction in the toxicity of immunosuppressors.Gene transfer has only been described for adenovirus-mediated CTLA4Ig in transplantation of liver,pancreas and kidney.Local production of CTLA4Ig can inhibit B7/CD28 interactions and reduce their systemic effects,â‘¡Antisense technology has been recognized as a reliable tool to inhibit anti-graft immune factors.Antisense oligonucleotides and antisense gene are novel therapeutic agents that reduce the number of specific mRNAs available for translation of the encoded protein.Poston proved that antisense ICAM-1 gene therapy prevented chronic graft rejection in cardiac allografts.Antisense genes are capable of making specific contacts to other RNA/DNA molecules by Watson-Crick base pairing for degradation of the target gene.Among the costimulatory pathways,B7-CD28 is a major receptor-ligand pair.Blockade of the B7-1-CD28 signaling pathway induces a state of immunotolerance in allografts transplantation in the absence of immunosuppressive drugs or at least a reduction in the required doses of immunosuppressors.The eukaryotic expression vector of antisense B7-1 was constructed which should facilitate the study of antisense B7-1 in inhibition of rejection in liver transplantation.Objectives:To construct an antisense rat B7-1 cloning vector which can be used to investigate the role of antisense B7-1 in inhibition of rejection in liver transplantation.Methods:1.Designing a pair of primers which contain two restriction sites,Xho I and BamH I,the 426 bp region of rat B7-1 cDNA was amplified by PCR from pcDNA3-B7-1.The purified PCR products and PMD18-T cloning vector were ligated,transformed and indentified,and an intermediary plasmid was acquired.2.Both PMD18-T-B7-1 and pcDNA3B(-)expression vector were excised using Xho I and BamH I,then B7-1 gene was ligated reversely into the multiclone site of pcDNA3B(-)which clone site of BamH I and Xhol I was in an antisense orientation compared to B7-1 sequence.3.The constructed vectors were further identified by PCR,restriction endonuclease analysis and DNA sequencing.Results:1.The intended gene fragment was about 420 base pairs as expected shown by electrophoresis of enzyme digested recombinant vector and PCR products with recombinant vector as template.2.The sequencing result showed a 420 bp fragment in pcDNA3B which was in in accordance with the known B7-1 gene.The insert direction was identified by DNA sequencing.Conclusions:The recombinant plasmid pcDNA3B-B7-1 was constructed successfully.The availability of the pcDNA3B-B7-1 should facilitate anti-rejection gene therapy in allograft transplantation.