次黄嘌呤单核苷酸脱氢酶抑制剂对人外周髓样树突状细胞功能影响的研究

The impact of inosine monophosphate dehydrogenase inhibitor on human peripheral myeloid dendritic cell

目的 探讨次黄嘌呤单核苷酸脱氢酶抑制剂（inosine monophosphate dehydrogenase inhibitor,IMPDHI）对人外周髓样树突状细胞（myeloid dendritic cells,MDC）功能的影响.方法 新鲜外周血单个核细胞（PBMC）来源于健康志愿者（n=15）,实验组加入IMPDHI,流式细胞仪分析MDC表面共刺激因子、黏附分子、趋化因子受体等的表达水平.Transwell小室实验中,加入不同的趋化因子,经Lin-1/CD11c/HLA-DR染色后,流式细胞仪计数,以迁移细胞的百分比表示其迁移能力.分离血树突状细胞抗原-1＋（blood dendritic cell antigen-1＋,BDCA-1＋）细胞,流式细胞仪测定BDCA-1＋细胞中FTTC标记的右旋糖酐的荧光值.混合淋巴细胞培养后,流式细胞仪测定同种异体CD4＋T淋巴细胞在G0期的比例.结果 细胞表面标志：与对照组相比,实验组MDC表面的CD40、CD62L、HLADR、CD54、CD80、CD83和CD86的表达水平明显下降（P＜0.05） 趋化因子受体的表达水平：与对照组相比,实验组MDC表面的CCR1表达水平明显升高（17.02±3.23 vs 30.63±9.13,P＜0.05）,CCR3的表达水平（10.26±2.25 vs 5.81±0.97,P＜0.05）和CCR7的表达水平（9.56±1.84 vs5.18±0.60,P＜0.05）明显下降 迁移功能：实验组MDC对趋化因子CCL2、CCL3、CCL4、CCL7、CXCL12的趋化能力明显增强（P＜0.05） 吞噬能力：实验组MDC的吞噬能力明显强于对照组（P＜0.05） 刺激同种异体CD4＋T淋巴细胞增殖的能力：实验组中MDC诱导同种异体CD4＋T细胞分裂、增殖的能力几乎完全受到抑制.结论 IMPDHI抑制外周MDC的成熟,增强其吞噬能力和炎性趋化的能力,抑制其刺激同种异体CD4＋T淋巴细胞增殖、应答的能力.

Objective To study the effect of inosine monophosphate dehydrogenase inhibitor （IMPDHI） on maturation, migration, endocytosis and allostimulatory properties of human peripheral myeloid dendritic cell （MDC）. Methods PBMC from healthy donors were isolated. MDC were cocultured with PBMC and exposed to mycophenolic acid （MPA） for 48 h. The expression of co-stimulatory and adhesion molecules as well as chemokine receptors on MDC was analyzed by flow cytometry. In separate experiments,MDC were cultured with or without MPA, and their endocytosis function was estimated by means of FITC dextran uptake. MDC migration experiments were performed in Transwell chambers. Inflammatory chemo kines were added to the lower chambers and MDC numbers were analyzed by flow cytometry. MPA treated （48 h） BDCA-1 ＋ DC served as stimulator cells in MLR. Allogenic healthy CD4 T responder cells were labeled with fluorescent dye CFSE and measured by flow cytometry. Results Maturation： compared to the control group, the expression of CD40, CD62L, HLA-DR, CD54, CD80, CD83 and CD86 on MDC in study group were significantly down-regulated （ P 〈 0.05 ）. Chemokine receptor and migration： compared to control group, the expression of CCR1 on MDC in study group was up-regulated significantly （17.02 ±3.23 vs 30.63 ± 9.13, P 〈 0.05 ）, the expression of CCR3 （ 10.26 ± 2.25 vs 5.81 ± 0.97, P 〈 0.05 ） and CCR7（9.56 ± 1.84 vs 5.18 ±0.60, P 〈0. 05） on MDC were down-regulated significantly in the study group.MDC in study group showed enchanced migratory response to inflammatory chemokine CCL2, CCL3, CCL4,CCL7, CXCL12 （P〈0.05）. Endocytotic capacity： the capacity of endocytosis in study group was signifi cantly higher than that in control group（ P 〈 0.05 ）. Llostimulatory capacity： MPA-treated MDC exhibited a markedly reduced ability to stimulate allogenic CD4＋ T cell proliferation. Conclusion Treatment of MDC with MPA exhibited an immature phenotype, a propensity to migrate in response to inflammatory chemokines, increased endocytotic capacity and impaired allogenic ability of MDC.