INVESTIGATION ON THE REDUCTION OF IMMUNOSUPRESSANTS IN LONG TERM SURVIVED POST RENAL TRANSPLANT PATIENTS.

辅助性T细胞产生的白细胞介素2（IL-2）在移植后的免疫调节中起重要作用，IL-2基因调控元件具有AP-1和NF-AT两个结合位点。最近有报道称，与正常T细胞相比，无能T细胞的转录因子AP-1和NF-AT的结合活性降低和下调。本研究采用电泳迁移率变动分析法检测了4例正常志愿者外周血淋巴细胞AP-1和NF-AT的DNA结合活性，4例肾移植受者存活超过5年，4例存活少于5年，通过Ficoll梯度离心并培养，用改良的Dignam等的方法从人胚肾细胞中提取细胞核， ...更多信息 将IL-2基因启动子区的AP-1和NF-AT结合共有序列的末端标记的DNA片段与核提取物反应。采用电泳迁移率改变法和BAS-2000生物图像分析仪检测AP-1和NF-AT的DNA结合复合物。4例正常人显示AP-1和NF-AT的DNA结合活性处于中等水平，而4例长期存活的肾移植受者显示AP-1和NF-AT的DNA结合活性明显降低。此外，在这些淋巴细胞中检测PMA和PHA的刺激作用;而4例长期存活的受者AP-1和NF-AT的结合活性均未增强，4例正常志愿者中有2例增强。研究AP-1和NF-AT的结合活性，可为免疫抑制效果的评价提供依据，从而建立更有效的免疫抑制方案。少

Interleukin-2 (IL-2) produced by helper T cells has been showm to play an important role in the immunomodulation after transplantation and regulatory elements of IL-2 gene have two AP-1 and NF-AT binding sites. Recently it has been reported that binding activities of transcription factor AP-1 and NF-AT and NF-AT of anergic T cell are decreased and down-regulated as compared with normal T cells. In this study, we have assessed DNA-binding activities of AP-1 and NF-AT by electrophoretic mobility shift assay in posttransplant recipients in order to evaluate the immunomodulatory effect of the immunosuppressants.Lymphocytes were prepared from perlpheral blood of four normal volunteers, four renal transplant recipients surviving for more than five years and four that surviving less than 5 years by gradient centrifugation over Ficoll and cultured for four hours at 37ﾟC with or without stimulation of PMA and PHA.Nuclear extracts were prepared from lympocytes by the modified method of Dignam et … More al. Endlabeled DNA fragments of AP-1 and NF-AT binding consensus sequences of IL-2 gene promoter regions were reacted with nuclear extracts. Electrophoretic mobility shift assays were performed and BAS-2000 Bioimage analyzer was used for the detection of DNA binding complex of AP-1 and NF-AT.Four normal volunteers showed DNA-binding activities of AP-1 and NF-AT in moderate level, but four long term surviving renal transplant recipients showed much lower level of the activities. Furthermore, a stimulatory effect of PMA and PHA was examined in these lymphocytes ; whereas all of four long term surviving recipients showed no enhancement of binding activities of AP-1 and NF-AT,two of four normal volunteers showed enhancements.In this paper, we have shown that the DNA-binding activities of AP-1 and NF-AT in immumosuppressive state, are decreased and not enhanced even after the stimulation of PMA and PHA.Investigations of the binding activivies of AP-1 and NF-AT will provide us the estimation of immunosuppressive effect, leading to establishment of more usuful immunosuppressive regimen. Less