Production Of The Recombinant MICA08Protein And The Impact Of Prospective MICA Antibodies On Rat Renal Transplantation

With the introduction of new and potent immunosuppressive agents, aimed at limitingthe effects of T-cell mediated immune responses to the graft, have controlled the acutecellular rejection successfully and decreased early allograft loss. However, recent analyseshave shown that the initial improvement has not been translated into long-termimprovement of graft survival. Initially, studies focused on the effect that HLA antibodieshave on graft survival. Although HLA antibodies are responsible for a large part ofantibody-mediated damage, but rejection can occur in the absence of HLA antibodies,these suggest that except of HLA antibodies they have other antibodies lead to graft losses.MICA (major histocompatibility complex class I–related chain A, MICA) gene islocated within the MHC class I region on chromosome6. The MICA genes are highlypolymorphic and encode MICA antigens, which have been found to be expressed inepithelial cells. Functionally, MICA neither builds β2-microglobulin nor has a role inpeptide binding for antigen presentation. MICA is a ligand that activating the receptor ofNKG2D, a common activating natural killer cell receptor, which is expressed on essentiallyall natural killer (NK) cells, as well as on γδ T and CD8(+) T cells. The engagement ofMICA and NKG2D, strongly activates NK cells and CD8+T cells, enhancing their cytolyticactivity and cytokine production and cause injury of allograft. Meanwhile, the engagementof NKG2D can induce B cells to produce antibodies and involve in antibodies-mediatedimmune responses.Generally, lymphocytes are devoid of surface MICA antigen. Therefore, crossmatching with lymphocytes obtained from the blood does not work for the detection of antibodies against MICA. In recent years, with the progress of the MICA antibodies testingtechnology, MICA antibodies have been found in transplant recipients, before graftrejection, as well as in eluates from rejected grafts in many organ transplantation centers.In basic and clinical research of transplant immunology, many investigators have showngrowing interest in studying the impact that the MICA antibodies have on solid organgrafts. Several studies suggest that antibodies to MICA were associated with graft rejectionand decreased survival, and such negative effects were obvious in patients well matchedfor HLA and in recipients with little or no HLA antibodies. Although some studies suggestthat MICA antibodies can cause allograft vascular endothelial cell injury and result inallograft dysfunction by complement-mediated cytotoxicity. However, the exact immunemechanisms of MICA antigens in acute and chronic rejection and the effective interventionmeasures are still unclear. It is also lack of animal models studies about MICAantibodies-mediated rejection.Therefore, we assume that the prospective MICA antibodies in rat models wereinduced by immunization with recombinant MICA﹡008external domain, which includesthe α1, α2, and α3domains and produces by insect-baculovirus expression system. Theeffects of the prospective MICA antibodies have on graft were observed in rat renaltransplantation models, and explored the value of new immunosuppressive rituximab in thetherapy of MICA antibodies-mediated rejection. The thesis is divided into three parts:PartⅠ: Production of the recombinant MICA08protein using insect-baculovirusexpression systemObjective: To construct recombinant human MICA﹡008external domain (α1, α2, and α3)in Sf-9cells using Bac-to-Bac baculovirus expression system.Methods: The human MICA﹡008external domain gene was cloned into a transfer vectorpFastBac-1to form the recombinant plasmid pFastBac-MICA﹡008, which wastransformed into E.coli.DH10Bac. By transposition, a recombinant baculovirus vectorBacmid-MICA﹡008was obtained. The recombinant Bacmid-MICA﹡008was identified by blue-white selection and confirmed by DNA sequencing. The recombinantBacmid-MICA﹡008was transfected into Sf-9cells by lipidbody, and then therecombinant soluble MICA08protein was expressed in Sf-9cells. Soluble MICArecombinant protein fused with a His-tag were purified by nickel-affinity agarose, andanalyzed by Western blot and Coomassie blue staining.Results: The P3supernatants were harvested by centrifugation at day4after infection, andthe recombinant soluble MICA08protein was purified by nickel affinity agarose with ayield of1.4-1.5mg/L culture medium. The recombinant soluble MICA08protein wasidentified by western-blot and Coomassie blue staining showed a specific band about37kDa, which consistent with the target protein.Conclusions: Recombinant MICA08protein was successfully expressed in Sf-9cells withbaculovirus expression system, and the protein could be used to induce MICA antibodies innext step study.PartⅡ: Microvascular anastomosis training and establish the model of ratrenal orthotopic transplantationObjective: To explore the microsurgery training methods for rapid establish a stable andreliable model of rat renal orthotopic transplantation, which providing basis for the studyexperiments of organ transplant.Methods: For rapid breakthrough the bottleneck of microsurgery technique in rat renalorthotopic transplantation, a staged microvascular anastomosis training program wasdesigned and implemented. The staged microvascular training program consisted of ganzesuture, rat carotid artery anastomosis, rat femoral vessels anastomosis and rat tail arteryanastomosis. To overcome the rat renal transplantation learning curve, harvest andtransplant both kidneys from one donor SD rat isotransplantation (30cases) was performed.During the operation, the renal vessels were reconstructed using end-to-end anastomosis,and the urinary was reconstructed by a bladder patch technique or ureter end-to-end anastomosis. Then SD→Wistar allotransplantation (20cases) was performed to establishan acute rejection model, and the recipients were randomly divided into two groups,rejection group and MMF group (MMF20mg/kg/d gavage,×10d). The blood specimenswere harvested at3,7,14and21days postoperatively, and the blood creatinine wasexamined in serum.Results: After two months intensive staged training, we have completed more than200microvascular anastomosis training, and established a stable and reproducible rat renaltransplantation model, which the survive rate reach up to90%at postoperative7days. Themean operative time of the20cases allotransplantation was72.9±4.18min, and the meanhot ischemia time was25.4±1.63min. The rejection group blood creatinine was increasedat day3after operation, and the MMF group creatinine was increased at day14. Twogroups creatinine reached its peak at day7and day21, respectively, there have significantdifferences when compared to syngraft group (P <0.05).Conclusion: The microsurgical anastomosis skills can be acquired though the stagedmicrosurgical training programs in shout term. And the staged microsurgical anastomosistraining programs can shorten the learning curve in rat renal transplantation. TheSD-Wistar rat kidney transplantation can establish a stable and reproducible acute rejectionmodel, which can be applied in the experiment of renal transplantation research.PartⅢ: Establish the model of prospective specific MICA antibodies in ratand explore its impact have on graftObjective: Establish the model of prospective specific MICA antibodies in rat, and exploreits impact and intervention measures of prospective specific MICA antibodies have ongraft.Methods: The prospective specific MICA antibodies in rat were induced by recombinantMICA08protein and P3Sf-9cells. The effects of the prospective specific MICAantibodies have on graft were observed in rat renal transplantation models. The recipients were treated with MMF and rituximab. Form the perspective of serum antibodies,creatinine and histopathology, explored the effects of the prospective specific MICAantibodies have on graft and the value of new immunosuppressive rituximab in the therapyof MICA antibodies-mediated rejection.Results: During the sensitization rats, which were sensitized by recombinant MICA08protein,the score of prospective specific MICA antibodies was increased from2to6after renaltransplantation(P＜0.05). However, the rats were sensitized by the P3Sf-9cells, the scoreof prospective specific MICA antibodies was increased from6to8after transplantation(P＜0.05). In rituximab intervention group, the CD20lymphocytes were reducedsignificantly when compare to pre-operation(P＜0.05). This indicated that rituximab havesignificant effect in depleting B lymphocytes cells. However, rituximab have no significanteffect in suppress the MICA antibodies development after operation. At day14post-operation, the rat blood creatinine in prospective specific MICA antibodies group washigher than control group (P＜0.05), these suggested that prospective MICA antibodiesmay cause damage to rat grafts. In prospective specific MICA antibodies group,the ratstreated with rutiximab and MMF have low level creatinine at day14, when compare totreated with only MMF. These indicats the new immunosuppressive rituximab may havepotential therapeutic value in the therapy of humoral rejection.Conclusion: The model of prospective donor specific MICA antibodies can be establishedby recombinant MICA08protein and P3Sf-9cells. Renal transplantation in prospectivespecific MICA antibodies rats can induces MICA antibodies development. The newimmunosuppressive rituximab may have potential therapeutic value in therapy of MICAantibodies-mediated rejection.