CD4^+CD25^+Treg在儿童急性免疫性血小板减少性紫癜发病机制中的作用

Role of CD4^+CD25^+ regulatory T cells in children with acute immune thrombocytopenic purpura

目的研究CD4+CD25+调节性T细胞(CD4+CD25+Treg)在儿童急性免疫性血小板减少性紫癜(AITP)发病机制中的作用。方法以25例AITP患儿的外周血为标本,30例健康儿童为正常对照。采用三色单克隆抗体直接标记法检测细胞膜表面抗原;检测胞浆/核抗原时先标记膜表面抗原,固定破膜后再标记胞浆/核抗原;应用多参数流式细胞仪进行Treg细胞的数值检测和结果分析;酶联免疫吸附法检测患儿和正常儿童血清中TGF-β和IL-10水平。结果约60%的CD4+CD25+细胞为FoxP3阳性,而90%以上的CD4+CD25hi表达FoxP3。AITP患儿与健康儿童CD4+CD25hi、CD4+CD25+FoxP3+细胞数和CD4+CD25+FoxP3+与CD4+CD25+FoxP3-的比值分别为(0.95±0.27)%对(1.08±0.37)%、(4.54±1.31)%对(5.28±1.52)%和(0.61±0.26)%对(0.73±0.29)%,两组间差异均无统计学意义(P均>0.05);AITP患儿和正常儿童血清的TGF-β水平分别为(9.44±2.78)ng/ml对(25.23±3.42)ng/ml,两者差异有统计学意义(P<0.05);两组间IL-10水平差异无统计学意义。AITP患儿的成熟B淋巴细胞显著高于正常儿童[(19.90±7.42)%对(12.02±3.82)%],P<0.05;NK细胞、CD3+T细胞和CD3+CD4+/CD3+CD8+数值在两组间差异无统计学意义(P均>0.05)。结论数量正常但TGF-β分泌不足的Treg细胞,可能在儿童AITP发病中发挥一定的作用。

Objective To explore the role of CD4＋CD25＋ regulatory T cells （Treg） in children with acute immune thrombocytopenic purpura （AITP）. Methods 25 patients with AITP were recruited in this study and 30 healthy children without immunologic diseases or infectious disorders were studied as control. Peripheral blood mononuelear cells （PBMC） were isolated by Ficoll density gradient centrifugation and were stained with anti-CD4-PE-Cy5, anti-CD25-FITC, anti- CD62L-PE or anti-FoxP3-PE or anti-CD152-PE monoclonal antibodies. For cell surface staining, all Abs were added and cells were stained for 20 minutes. When performing intraeellular staining for FOXP3 or CD152, cell-surface staining was first completed, and then the cells were fixed and permeabilized for intracellular staining. Stained cells were examined by three-color flow cytometric analysis using a FACS Altra cell sorter. The serum levels of transforming growth factor-β（TGF- β） and interleukin-10 （IL-10） were assayed by enzyme-linked immunosorbent assay kits. Results Only about 60% CD4＋ CD25＋ T cells were positive for FoxP3＋, while an overwhelming majority of CD4＋CD25＋ T cells （over 90% ） expressed FoxP3. There were no significant differences in CD4＋CD25＋, CD4＋ CD25＋FoxP3＋ , and the ratio of CD4＋CD25＋FoxP3＋ T ceils to activated effective CD4＋CD25＋FoxP3- T cells between AITP group and the control group [（0.95± 0.27）% vs （ 1.08 ± 0.37）%, （4.54±1.31）% vs （5.28 ± 1.52）%, and （0.61 ± 0.26） vs （0.73 ± 0.29）, respectively, P〉 0.05]. The level of TGF-β in AiTP group decreased significantly compared with that in normal children （（9.44± 2.78） ng/ml vs （25.23±3.42） ng/ml, P 〈 0.05）. In contrast, the level of serum IL-10, only measured in five patients, was not different from normal reference value. The percentage of B lymphocyte was significantly higher in AITP group-than that in the control group （（19.90±7.42）% vs （12.02 ± 3.82）%, respectively, P 〈 0.05）, with no significant differences in proportions of NK cells, CD3＋ T cells, and the ratio of CD3＋CD4＋ to CD3＋CD8＋ T cells between the two groups （ （ 15.06 ± 8.25）% vs （18.43 ± 7.71）%, （52.64± 8.45）% vs （55.53 ±11.90）%, （0.97± 0.37） vs （1.12 ± 0.32）, respectively, P〉 0.05）. Conclusions The results suggested that insufficient secretion of TGF-β might be one of the mechanisms that caused immune regulation dysfunction in children with AITP despite normal percentage of Treg cell.