Abstract: Objective    To study the effect of stem cells from apical papilla（SCAP）on methylation status of Foxp3 locus in CD4+ T cells，clarifying the role of SCAP in promoting the conversion of CD4+ T cells to regulatory T cells（Tregs）. Thus this study can provide theoretical foundation and experimental basis for further exploring the mechanism of immunoregulatory role of the dental-derived stem cells. Methods    Human SCAP and mouse spleen CD4+ T cells were isolated and identified respectively. SCAP and CD4+ T cells were co-cultured at a ratio of 1∶5 for 72 h via transwell in vitro（co-culture group），and CD4+ T cells cultured alone were used as the control group. Flow cytometry was used to detect the ratio of Tregs. Hydroxymethylated DNA immunoprecipitation-polymerase chain reaction（hMeDIP-PCR）was used to evaluate the expression level of 5-hydroxymethylcytosine（5hmC）at Foxp3 locus in CD4+ T cells. Enzyme-linked immunosorbent assay（ELISA）was used to measure the expression level of the Tregs′ effector interleukin-10（IL-10）in the co-culture supernatant. Results    Primary cultured SCAP expressed mesenchymal stem cell surface markers （CD105，CD90，CD44，CD29）， and were negative for hematopoietic stem cell markers （CD45，CD34）. Compared with the control group，the ratio of Tregs and the expression level of 5hmC at Foxp3 locus in CD4+ T cells was elevated by SCAP in the co-culture group. The level of anti-inflammatory cytokine interleukin-10（IL-10）in the co-culture supernatant was increased by SCAP as well. The differences were statistically significant（P < 0.001）. Conclusion　SCAP can up-regulate the expression level of Foxp3，and induce DNA demethylation of Foxp3 locus in CD4+ T cells by paracrine factors，which promotes CD4+ T cells to convert to Tregs and then plays a role in immunoregulation.

Key words: stem cells from apical papilla, SCAP；CD4+ T cells；regulatory T cells, Tregs；Foxp3；DNA demethylation

摘要： 目的    研究根尖牙乳头干细胞（stem cells from apical papilla，SCAP）对CD4+ T细胞内Foxp3位点甲基化状态的影响；明确SCAP对CD4+ T细胞向调节性T细胞（regulatory T cells，Tregs）转化的作用，为深入探究牙源性干细胞发挥免疫调节的作用机制提供理论依据和实验基础。方法    分别提取人SCAP、小鼠脾脏CD4+ T细胞并鉴定，应用transwell将SCAP与CD4+ T细胞以1∶5的比例在体外共培养72 h（共培养组），以单独培养的CD4+ T细胞为对照组，应用流式细胞术检测Tregs比例；DNA羟甲基化免疫沉淀-聚合酶链反应技术检测CD4+ T细胞内Foxp3位点5-羟甲基胞嘧啶（5-hydroxymethylcytosine，5hmC）表达水平；酶联免疫吸附测定法检测共培养后上清液中Tregs效应因子白细胞介素10（interleukin-10，IL-10）的表达水平。结果    原代培养的SCAP表达间充质干细胞表面标记物CD105、CD90、CD44、CD29，不表达造血干细胞表面标记物CD45、CD34。与对照组相比，SCAP上调共培养组中Tregs比例及CD4+ T细胞内Foxp3位点5hmC表达水平，同时上清液中抗炎因子IL-10水平升高，差异均有统计学意义（P < 0.001）。结论    SCAP通过旁分泌作用上调CD4+ T细胞内Foxp3表达水平，诱导Foxp3位点发生DNA去甲基化，促使CD4+ T细胞向Tregs转化，进而发挥免疫调节作用。

关键词: 根尖牙乳头干细胞, CD4+ T细胞, 调节性T细胞, Foxp3, DNA去甲基化