PTD-mFoxp3 Inhibits Skin Allograft Rejection In Mice

Organ transplantation is a definitive therapy for end stage of organ failure, but allograft rejection remains the major obstacle to long-term allograft survival.It has become one of important methods for the transplantation to induce the specific immune tolerance to the donors,graft in recipients.Natural CD4~+CD25~+ regulatory T cells(Treg) have attracted wide attention of the immunologists and surgeons for their ability to induce immune tolerance and maintain self immunological homeostasis,but it is too difficult to obtain enough numbers of Treg for clinic therapy.Foxp3 is a transcription factor,which has been touted as a lineage-specific marker of Tregs.It belongs to the forkhead-winged-helix family. For the more,it is crucially important for the development and function of Treg. Recently,it had been demonstrated that ectopic expression of mFoxp3 or hFOXP3 in mouse na(?)ve CD4~+CD25~-T cells or human T-lymphoma Jurkat cells is sufficient to convert them into phenotypic ally and functionally natural Treg-like cells.The protein transduction domain(PTD),a short basic peptide fragment of transactivator(TAT),has been developed to allow the delivery of exogenous proteins into almost all living eukaryotic cells rapidly and efficiently.PTD doesn’t affect the bioactivities of fusion proteins in the cells. Objective:To investigate the potential inhibit ability to activities lymphocytes proliferation and skin allograft rejection in mice of fusion protein of mouse Foxp3 combining with transduction domain(PTD-mFoxp3).Methods:1.The Expression of the fusion protein of PTD-mFoxp3:The fusion protein of PTD-mFoxp3 was expressed by E.coli Rosetta(DE3) and purified via Bio-Rad Profinity IMAC Ni-Charged Resin.2.The translocation of the PTD-mFoxp3:The PTD-mFoxp3 was identified by Western-blot after transmenbrance.(co-operation with Baoju Ji)3.The cell proliferation assay:The single-cell suspension of lymphocytes was prepared from mouse spleen.Polyclonal activator Con A was added to activate the lymphocytes.Then the cell suspension was incubated with different concentrations of PTD-mFoxp3,PTD-GFP,cyclosporine(CsA) and normal saline(NS) for 48 h,respectively.The bioactivity of inhibiting lymphocytes proliferation of each group was detected by incorporating of CCK-8 in vitro.4.Mouse allo-skin transplantation experiment.Forty C57BL/6 mice were randomly distributed into four groups,with each group having 10 mice and receiving a different treatment including PTD-mFoxp3,CsA,NS and PTD-GFP,respectively.At the same day,the treating regents were injected into C57BL/6 mice abdomen,and continuous injected for another 7 days.One day after the treatment beginning,they were transplanted with Balb/c mice skin.Two mice of each group were selected out randomly for histological observation till 9th day after skin transplantation.The pathological examination of the skin graft was observed.Other mice’s survival time of skin allograft were observed.Results:1.The fusion protein was expressed and purified efficiently.2.The results of western blotting indicated that PTD-mFoxp3 could transduce into EL-4 and located in the nucleus.3.In the lymphocytes proliferation assay,the results disclosed that PTD-mFoxp3 and CsA can inhibite the splenocytes proliferation significantly(P＜0.05) after stimulating by ConA,but there were no significantly difference between CsA and PTD-mFoxp3 in 320 nM,640 nM and 1280 nM(P＞0.05).4.The pathological examination of the graft at 9th day after the operation exporsed that there was a great deal of inflammatory cells infiltrated and some necrosis in the graft of tissue NS and PTD-GFP treated groups,but the inflammatory were milder in PTD-mFoxp3 and CsA groups.5.The allograft average survival time of PTD-mFoxp3 group is 13.6±1.50d, CsA group is 14.2±1.28d,NS group is 10.0±1.07d and PTD-GFP group is 10.5±1.31 days.These results revealed that the bioactivity of PTD-mFoxp3 was similar to CsA,they could prolonged the mean survival time(MST) of skin allograft significantly(P＜0.05,comparing with NS and PTD-GFP groups).Conclusion:PTD-mFoxp3 inhibits the splenocytes proliferation,and prolongs the survival time of allo-skin transplantion grafts in mice.