The Study Of CTLA4-Ig Recombinant Lentiviral Vector Directed Expression To Draining Lymph Nodes

Ethnology, the rapid development of transplant surgery, plastic surgeonsalso stepped up exploration in the field of allogeneic composite tissuetransplantation. However, the CTA transplantation has lagged far behind thevisceral organ transplantation; the reasons why it is backward are its complexcomposition and antigenic heterogeneity. Since1998, Dubernard first successfulimplementation of the hand transplant, until2007the world has completed20cases of hand transplantation, eight cases of face transplant. According to theliterature review, the first case CTA patient in France experienced2acuterejections in the postoperative. In our institute the second patient of the worldexperienced3acute rejections. Increasing the dosage of immunosuppressiveagents, rejection could be controlled, but the price has been a variety of difficultcomplications, acute renal failure, hypertension, hyperglycemia, leukopenia, andso on. Therefore, how to minimize the risk of immunosuppressive agents hasbecome a top priority in the field of CTA transplantation. Allogeneic composite tissue transplantation rejection episodes follow thegeneral rules of the immune response. Namely, first, the foreign alloantigenimmune recognition, and then entering the activation phase, the resultingimmune rejection of the graft, therefore, selective blocking any one link, arelikely to induce transplantation tolerance.CTLA4-Ig could block CD28-B7pathway to induce immune tolerance.Dendritic cells are the main antigen-presenting cells. it has high level of surfaceexpression of MHC-Class II molecules and costimulatory molecules of CD80,CD86and stimulate the way through the second signal to activate naive T cells.Mature dendritic cells have the characteristics of the lymphatic homing;therefore, the mature dendritic cell is a cell reinfusion treatment program idealalternative cell.Objectives:To research the expression efficiency of CTLA4-Ig directly injected intolymph node, the related gene expression which was infected CTLA4-Igrecombinant Lentiviral Vector in mature dendritic cells, and local injection oftransfection of mature dendritic cells and observing the expression of CTLA4-Igin the draining lymph nodes. Proposing the new concept of local cell therapy toinduce immune tolerance or lymph nodes directly gene therapy, reducing therisk of systemic immunosuppressive agents for allogenic composite tissuetransplantation.Method:1. Injecting CTLA4-Ig recombinant Lentiviral Vector to Rat lymph node toobserve the expression of CTLA4-Ig in the lymph nodes.2. Adherent cultured inducing mature dendritic cells in vitro, taking the morphological identification, the activity and phenotype identification, andfinally identifying immunological function in vitro.3. Study CTLA4-Ig recombinant Lentiviral Vector transecting rat maturedendritic cells in vitro, and measuring the expression time, peak expressionof CTLA4-Ig.4. Study the Mature dendritic cells’ directed expression of CTLA4-Igrecombinant Lentiviral Vector transfection in lymph node drainage in ratsin vivo.Result:1. Real time fluorescence quantitative PCR and the frozen section showed thatthere was no expression of CTLA4-Ig in the lymph nodes in the blankcontrol and negative control group. CTLA4-Ig recombinant LentiviralVector injection within the lymph node group has significant CTLA4-Igexpression.2. Cultured mature dendritic cell colony dispersion, suspension, anddistribution more uniform could be seen by Optical microscopy. A largenumber of cell surface fold with thickness and process ranging fromthickness could be seen by Scanning electron microscope. Flow cytometryshowed that DC function-associated antigen MHC II, CD80, and CD86werehighly expressed in the surface of mature dendritic cells. Function tests todetect the secretion of IL-12capacity showed that the mature DCs arestronger than immature ones, mixed lymphocyte reaction showed its strongability to stimulate T cell proliferation. Living cells (80.34±1.25)%3. After76h of Lentiviral transfection of mature dendritic cells, it showed thatthere were observed fluorescence expressions under an inverted fluorescence microscope. the fluorescence expression increased with time, cellmorphology turned to be senescent. The cells turned to be senescentobviously after7days of transfection. Q-PCR showed that there wereCTLA4-Ig expression at the mRNA level after3days of the CTLA4-Igrecombinant Lentiviral Vector mature dendritic cells transfection, Westernblot detection showed the same results as Q-PCR4. Injecting slowly of CTLA4-Ig recombinant Lentiviral Vector harvested after3days of transfection of DCs away from the rat axillary the inherent lymphnodes at2cm subcutaneous then took a tissue after three days, the Q-PCRshowed that it could be detected the CTLA4-Ig expression after injection ofCTLA4-Ig recombinant Lentiviral Vector transfection DCs in lymph nodes.Conclusion:1. CTLA4-Ig recombinant Lentiviral Vector could be injected into lymph nodedirectly, but the range is limited and the damage is more.2. Cell therapy is feasible preliminary, but it is necessary to research for furtherexperiental studies in the allogeneic composite tissue transplantation model.