The Effects Of IDO Gene Modified BMSCs In Rat Composite Tissue Allograft Rejection

BackgroundSevere burn and trauma are often caused by various industrial accidents and frequentnatural disasters. Due to the limited amount of autologous tissues for reconstruction, thetreatment of such patients was extremely difficulties in the clinical work. The compositetissue allotransplantation has been widely accepted because it can provide fine functionand appearance of for reconstruction. However, as known to all, after composite tissueallotransplantation long-term useage of immunosuppressive drugs may cause various sideeffects, including opportunistic infections and even malignant tumor. Besides,the inevitable chronic rejection post transplantation has greatly hindered the clinicalapplication of allo-transplantation. Therefore, exploring novel method that suppress theimmune rejection to reduce or avoid the use of immunosuppressive drugs, is one of themajor clinical problems which need to be solved.Although BMSCs have low immunogenicity and can negatively regulated theimmunity, they could not inhibit the allograft rejection completely. IDO has the effects of promoting necrosis and inhibiting proliferation on T cells. Meanwhile, it could induce theregulatory T cells. However, under normal circumstances, there is no or low expressionlevel of IDO in BMSCs, which makes the immunoregulatory capacity of BMSCssuppressed. Accordingly, this study is designed to construct the overexpression vector ofIDO and investigate its effects on the proliferation of allogeneic T lymphocyte in vitro.Then, the IDO-BMSCs were transfused into the recipient mice and the composite tissueallograft transplantation was performed simultaneously. Evaluation the influence ofIDO-BMSCs on graft rejection/tolerance formation. This study is aim not only to find anew approach for the immune rejection after composite tissue allotransplantation, but alsoto provide the basis for the study of applied research in transplantation rejection usingBMSCs as cell carrier.Materials and Methods1. BMSCs were isolated from rat bone marrow of juvenile BN rats by whole bonemarrow culture method. The stable amplification system was established by theobservation of cell morphology and biological characteristics, purification, andamplification reaction. The surface antigens of BMSCs and differentiation capacitywas further identificated.2. Construc IDO (GFP) lentivirus vector, transfect rat BMSCs with different titer, andthen select the optimal infection index MOI value. Determination of the transfectionefficiency and IDO activity in culture system. Detect the change in the expressionlevels of mRNA and protein in IDO-BMSCs simultaneouly. Validate the effects ofIDO-BMSCs on the proliferation of T cells by establishment of the mixture culturesystem of IDO-BMSCs and allogeneic T lymphocytes.3. Establish the model of allograft composite tissue transplantation and observed thechange of limb survival time under de-escalation treatment of cyclosporinA(CsA) intervention. Observe effects of IDO-BMSCs transfusion on thelimb survival time by frozen section after the input of CM-Dil labeled BMSCsthrough rat tail vein. Result1. Primary BMSCs adhered after36hours. They showed colony growth after3-5daysculture and grow up to80%confluency after7days. The cells growed faster afterpassage and reach to monolayer after3days. The cultured BMSCs have the consistentphenotypic identification with stem cells as well as the multilineagedifferentiation ability induced by specific culture medium.2. IDO lentiviral vector was constructed successfully and could reach the optimalinfection efficiency of80.7%in BMSCs, at MOI=10. The mRNA and proteinlevels of IDO was over-expressed stably, with the obviously higher biological activityin supernatant compared with control group （IDO-BMSCs,61.3±5.4uM vsGFP-BMSCs,5.0±0.9uM&BMSCs,5.3±0.6uM, p＜0.01）. The IDO-BMSCscould inhibited the proliferation of allogeneic T lymphocyte in the mixedlymphocyte culture system, with the statistically significant difference compared withthe control group（p＜0.05）3. A rat allogeneic limb transplantation model was established successfully. Besides, weconfirmed that de-escalation cyclosporin A(CsA) treatment could result in a longersurvival time of allogeneic limbs after transplantation. In addition, the IDO-BMSCscould significantly prolong the survival time of graft limbs and reduce rejectioncompared with the control group（IDO-BMSCs,14.5±0.8days vs BMSCs,11.5±0.6days&saline,9±0.3days，n=6， p<0.05）.ConclusionIsolation and culture of BMSCs with whole bone marrow culture method could obtainthe BMSCs with stable phenotype and good ability of differentiation. CM-Dil labelprovided a clear tracing of BMSCs in vivo. BMSCs tranfected with IDO-Lentivirusshowed a stable expression of IDO and the corresponding biological activity.IDO-BMSCs could suppress proliferation of allogeneic T cell in vitro. Meanwhile, theycan significantly prolong graft survival time and reduce rejection reaction in vivo.