调节性T细胞在小鼠抗肾小球基底膜型肾小球肾炎模型中的免疫调节机制研究

Immunoregulation mechanism of Treg cells in anti-GBM glomerulonephritis model of mice

目的探讨在小鼠抗肾小球基底膜型肾小球肾炎(抗GBM肾炎)模型的肾炎进展中调节性T细胞(regulatory T cell,Treg)免疫调节机制,为早期干预免疫反应,减轻病理损害提供理论依据。方法将C57BL/6小鼠随机分为2组即正常对照组和抗GBM肾炎模型组。在建模不同时间点即建模的第7、14、21、28天将小鼠处死后收集并检测血清中的肌酐(serum creatinine,SCr)、尿素氮(blood urea nitrogen,BUN)及尿蛋白含量,随机尿中尿蛋白/肌酐比值(albumin-to-creatinine ratio,ACR)变化;留取肾脏标本检测病理改变。流式细胞术(fluorescence-activated cell sorting,FACS)检测脾脏中Treg细胞表达情况;免疫印迹法(Western blot)检测肾脏组织中DNA结合抑制因子3(Id3)和Foxp3等蛋白表达情况。结果与正常对照组小鼠相比,抗GBM肾炎模型组小鼠肾脏病理示GBM不规则弥漫增厚甚至部分断裂、系膜细胞和基质增生、新月体形成;免疫荧光示鼠IgG和兔IgG在基底膜呈线性沉积;SCr、BUN、尿蛋白和ACR增加明显(P<0.05);抗GBM肾炎模型组中细胞和蛋白水平检测到的相关因子均呈规律性表达:与正常对照组相比,随着病情进展抗GBM肾炎模型组中Th17细胞的表达比例呈现先上升后下降的趋势,而与此同时Treg细胞则从第7天开始即出现升高的趋势,差异具有统计学意义(P<0.05或P<0.01);抗GBM肾炎模型组小鼠肾脏组织中Foxp3及Id3的蛋白表达均较正常对照组明显增加,差异均具有统计学意义(P<0.05或P<0.01);Id3的蛋白表达与Treg细胞特异性转录因子水平相关。结论在抗GBM肾炎的免疫进程中,Treg发挥重要作用,这种作用可能与转录调节因子Id3有关。

Objective To explore the immunoregulation mechanism of Treg cells in anti-GBM glomerulonephritis model of mice, and provide theoretical basis for early intervention of immune response and alleviating pathologic damage. Methods C57BL/6 mice were randomly divided into nephritic group and normal control group. The anti-GBM glomerulonephritis model was established in the mice of the nephritic group. At different time points of 7th, 14th, 21st and 28th day, mice were sacrificed. The serum was collected, and creatinine （SCr）, blood urea nitrogen （BUN）, urine albumin and albumin-to-creatinine ratio （ACR） were tested. Renal pathological changes were observed. Treg cells in the spleen were examined by fluorescence-activated cell sorting （FACS）. Western blotting was used to detect the expression of Id3 and Foxp3 protein in the renal tissues. Results The irregular thickening and breakage of GBM, glomerular mesangial cells, matrix proliferation, and crescent formation were observed in nephritic group. Immunofluorescence showed rabbit IgG and mouse IgG line- arly deposited along the GBM in nephritic group. SCr, BUN, urine albumin and ACR increased significantly in nephritic group as compared with normal control group （P〈0. 05）. The anti-GBM glomerulonephritis model group of cells, and protein expression levels of relevant indicators show the law： FACS showed Th17 cells declined after increased, while Tregs increased significantly from 7th day （P 〈0. 05 or P〈0. 01） ; The protein expression levels of Id3 and the transcription factors Foxp3 in anti- GBM glomerulonephritis group were increased significantly（P〈0. 05 or P〈0. 01 ）. The expression levels of Id3 protein were positive correlated with the Tregs＇ transcription factors. Conclusions In the immune process of anti-GBM nephritis, Treg plays an important role, which may be related to the transcriptional regulator Id3.