Ex Vivo Transfection Of Adctla4ig To Regulate Rejection Response After Cta And Its Mechanism Research

Allograft transplantation of facial tissue is emerging as a potential treatment for complex facial tissue defects caused by trauma, tumor resection, or congenital abnormalities. However, large doses and long-term administration of immunosupressantes may lead to complications such as opportunistic infections, organ impairments, and malignancies. Thus, an effective method to minimize the need for immunosuppressantes, especially during the early stages after transplantation, is urgently needed. The immunosuppressive effects of cytotoxic T-lymphocyte-associated antigen-4 immunoglobulin (CTLA4Ig) have been confirmed in numerous animal models. In this paper, we investigated whether ex vivo perfusion of an adenoviral vector carrying the CTLA4Ig gene (AdCTLA4Ig), can induce the long-term survival of microvascular free flaps allografts. What's more, the mechanisms behind the inhibition effects of the local expressed CTLA4Ig were studied too. Purpose: Confirm the efficacy of ex vivo transfection method. Test the impact of ex vivo transfection method on immune rejection response. Choose the most proper perfusion time. Test the efficacy of local expression of CTLA4Ig gene on the survival of rat free flap allografts and investigate the mechanisms behind the inhibition effects of the local expressed CTLA4Ig.Methods: 1. Vascularized groin ?aps were transplanted from BN to Lewis rats after 0, 1, 2, or 3 h of storage and the allografts in each group were evaluated daily. Biopsy samples taken from the each group on postoperative d 2–8 were graded for signs of acute rejection. Biopsy samples taken from each group on postoperative d 6 were stained for chemokine receptor CXCR3.2. Brown Norway rat groin flaps were transplanted to Lewis rat recipients. The donor flaps were perfused ex vivo with AdCTLA4Ig via afferent artery before transplantation. The distribution and duration of CTLA4Ig transgene expression in the flaps were assessed by immunohistochemical staining and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) after transplantation. ELISA was employed to detect the doses of CTLA4Ig protein in the serum. Real-time PCR was employed to detect the expression of IL-4 and IFN-γmRNA in the perfused allografts. Mixed lymphocyte reaction was performed to test the antigen specific immune response.3. Flow cytometry was used to detect the variations of CD4~+25~+Foxp3~+ T cells in recipients second lymph organs. Real-time PCR was employed to detect the mRNA expression of IDO in the AdCTLA4Ig-perfused free flap allografts and AdEGFP-perfused free flap allografts.Results: 1. When the ischemia time was greater than 4 hours, the survival time of the grafts was significantly shorter (7.0±0.8 days) than that of the grafts that did not undergo cold ischemia (9.0±1.2 days). Histological evaluation showed acceleration of activated lymphocyte infiltration in the Isc-4h group compared with the Isc-1h group. Furthermore, the proportion of CXCR3-positive cells in the Isc-4h group was significantly higher than that in the allo-0h group on days 6 after transplantation.2. Immunohistochemical staining and RT-PCR demonstrated expression of CTLA4Ig transgene in the AdCTLA4Ig-perfused free flap allografts. The doses of CTLA4Ig protein in recipients serum was below 25ng/ml. AdCTLA4Ig-perfused free flap allografts survived significantly longer compared with survival of AdEGFP-perfused free flap allografts. When combined with a short course of rapamycin, the survival time of AdCTLA4Ig-perfused free flap allografts was remarkably prolonged. The histological finding of AdCTLA4Ig-perfused free flap allografts was normal while the histological finding of AdEGFP-perfused free flap allografts showed signs of epidermolysis. The IL-4 and IFN-γmRNA expression was significantly lower in the AdCTLA4Ig-perfused free flap allografts than the AdEGFP-perfused free flap allografts. The MLR results indicate that ex vivo transfer of AdCTLA4Ig induces antigen specific unresponsiveness.3. AdCTLA4Ig perfusion didn't change the proportion of CD4~+25~+Foxp3~+ T cells in second lymph organs. The IDO mRNA expression was significantly increased in the AdCTLA4Ig-perfused free flap allografts than that in the AdEGFP-perfused free flap allografts.Conclusions: When ischemic time was over 3 hours, the immune response is accelerated. Therefore, the best perfusion protocol is one hour's perfusion plus one hour's incubation. By using this method, we demonstrated that a singular ex vivo perfusion of AdCTLA4Ig gene induced efficient transduction of the flaps and promoted the remarkably longer survival of flap allografts. When combined with a short course of rapamycin, the survival of flap allografts were enormously extended. The up-regulation of IDO other than expansion of CD4~+25~+Foxp3~+ T cells may be responsible for the immune inhibition effect of local expressed CTLA4Ig.