| Natural CD4~+CD25~+ regulatory T cell(Treg)have been attracting wide attention in the field of organ transplantation,for their response suppression in immune,and are considered as one of effective cell in inducing immune tolerance after organ transplantation,so far there isn't any report about their clinical application,the most important reason may be it's quantity limitation. Although some efforts to expanding the Treg have been done in vitro mimicing the condition in vivo have been successful partly,these don't take any advantage to clinical research,for it need a long time culture,the high cost,and the strict experimental condition.Foxp3 is a transcription factor,belonging to the forkhead family,which is crucially important for the development and function of Treg.Recently someone has shown that ectopic Foxp3 expression can pheno- typically and functionally convert effector T cells to regulatory T cells.The transduction domain(PTD),part of transactivator(TAT)protein encoded by the human immunodeficiency virus type 1,is capable of mediating heterologous protein transduce into the cellular plasma almost all eukaryotic cells freely.Our group have successfully constructed the prokaryotic expression vector of pET28a-PTD-mFoxp3,the fusion protein was expressed and purified efficiently.The results of flow cytometry indicated that His-PTD-mFoxp3 fusion protein could transduce into EL-4 efficiently.MLR assay indicated that the fusion protein could inhibit the proliferation of T cells.Objective:To confirm the ability of anti-rejection of the PTD-mFoxp3 fusion protein on the the survival of heterotopic heart and lung transplantation in rats.Methods:1.The PTD-mFoxp3 fusion protein was purified and renatured by the kit.2.We copied the model of heterotopic abdominal heart and lung transplantation in rat via single blood vessel established by Lee, transplanting the donor heart form BN rats to the receptor Lewis rats.3.We injured the PTD-mFoxp3 fusion protein into the recipient rats via vena caudalis respectively the day before the transplantation,and continuous the recipient rats the same way for 7 days.We observed the influence of the fusion protein on the survival periods time of the heterotopic heart and lung transplantation in rats and by the pathological appearance of the allografts.Results:1.The fusion protein was purified and renatureed efficiently.2.We transplanted donor heart and lung of BN rats to Lewis,and succeed to establish the heterotopic abdominal heart and lung transplantation in rat throngh elevateing the technique of microsurgery and improving the protectant method of the heart and lung.The achievement ratio of operation is more than 85 percent.3.The general state of the recipient 3 days after transplatation:the general state of ciclosporin and PTD-mFoxp3 fusion protein groups were better than the blank and no mFoxp3 groups.The Wilcoxon rank sum test was used to compare the band,was considered significant deviation. (p＜0.01).4.The pathological examination of the graft 5 days later the operation indicated that there were a great quantity lymphocytes infiltration and necrosis in the graft of blank and no relation protein group,The Inflammatory band is 4 according the stanford stand.The ciclosporin and PTD-mFoxp3 fusion protein group had been found that the cardiac muscle are milder hydroncus,or focal damaged.The Inflammatory band is 1_A or betwin 2_B.The Wilcoxon rank sum test was used to compare the band,was considered significant deviation(p＜0.01).5.The allograft survival time of ciclosporin group was 20±2.34 days, the PTD-mFoxp3 fusion protein group was 19.4±2.70 days,the blank group was 8.2±1.92 days,the mFoxp3 group was 7.4±1.14 days.The analysis of variance show that there are a significant deviation between them(p＜0.001).Conclusion:1.For studying the influence of the PTD-mFoxp3 fusion protein on organ transplantation in vivo,we established heterotopic abdominal heart and lung transplantation in rats successfully,and confirmed that it was a stable and reliable heart and lung transplantation model in rat.2.We found that inject the PTD-mFoxp3 fusion protein into the recipient rats via vena caudalis could prolong the survival time of heterotopic abdominal heart and lung transplantation in rats,and abated the Inflammatory reaction in the transplantation.These results suggest that PTD-mFoxp3 fusion protein has the clinical application perspective in inducing immune tolerance after organ transplantation. |
PDF Full Text Request