甘草酸二铵对于HCV相关性B-NHLCD25^-和CD25^+T细胞增殖影响研究

Differential proliferation of CD25^- and CD25^+T cells in HCV-associated B-NHL intervened by Diammonium Glycyrrihizinate

目的:本研究初步探究甘草酸二铵(Diammonium Glycyrrihizinate,DG)对HCV相关性B细胞非霍奇金淋巴瘤(B-cellnon-Hodgkin's lymphoma,B-NHL)CD25-T细胞、CD25+T细胞的免疫调控作用。方法:应用流式细胞分析仪检测HCV相关性B-NHL CD4+CD25+T细胞占CD4+T细胞的比例、CD25+细胞占总PBMC比例,并与单纯HCV感染患者、健康人检测结果相对照。应用免疫磁珠分离法(MACS)分选获得CD25-和CD25+细胞,将两者和未分选的外周血单个核细胞(peripheral blood mononuclearcell,PBMC)以CFSE标染孵育72小时后,应用流式细胞分析仪将APC CD3阳性细胞设定为门检测CD25-T细胞、未分选T细胞、CD25+T细胞的增殖情况,及甘草酸二铵干预后的CD25-T细胞、CD25+T细胞增殖情况。结果:应用流式细胞分析仪检测健康人、单纯HCV感染者和HCV相关性B-NHL患者在CD4+CD25+占总CD4+细胞比例和CD25+占总细胞比例上均呈现逐步递增的关系(分别为33.94%±2.18%,57.95%±1.77%,70.24%±12.75%,P<0.05;18.16%±2.23%,33.45%±1.32%,54.69%±8.66%,P<0.05)。CFSE标染后孵育72小时检测(M1+M2)分裂相百分比呈现CD25-T细胞最高、未分选T细胞其次、CD25+T细胞最低的表现(分别为74.4%±1.2%,63.1%±5.4%,47.2%±5.9%,P<0.05)。甘草酸二铵干预后CD25-T细胞较未干预CD25-T细胞M1分裂相百分比增加(分别为57.7%±4.2%,46.5%±5.6%,P<0.05)。甘草酸二铵干预后CD25+T细胞较未干预CD25+T细胞M1分裂相百分比减少(分别为14.7%±1.3%,22.1%±4.1%,P<0.05)。结论:HCV相关性B-NHL CD4+CD25+、CD25+细胞百分比较单纯HCV感染者、健康人明显增加,提示存在T细胞免疫抑制,且抑制程度重于单纯HCV感染。去除CD25+细胞后CD25-T细胞增殖较未去除CD25+的T细胞增殖活跃,说明HCV相关性B-NHL患者的CD25+细胞能够抑制CD25-T细胞增殖。而DG干预后HCV相关性B-NHL CD25+T细胞增殖M1分裂相减少,而CD25-T细胞增殖M1分裂相增加,表明DG对HCV相关性B-NHL具有抑制CD25+T细胞增殖而促进CD25-T细胞增殖的积极免疫调节作用。

Objectives： The study primarily explored the immune regulatory effect of Diammonium Glycyrrihizinate （DG） on HCV-associated B-cell non-Hodgkin＇s lymphoma （B-NHL）. Methods： The percentages of CD4＋CD25＋ T cells in CD4＋ T cells and CD25＋ cells in all peripheral blood mononuclear cells （PBMCs） in patients with HCV-associated B-NHL were detected by flow cytometry in comparison with only HCV-infected patients and health donors. CD25 and CD25＋cells were isolated by magnetic cell sorting （MACS）. Then CD25- and CD25＋ cells intervened by DG or not, and whole PBMCs were stained by carboxyfluorescein diacetate succinimidyl ester （CFSE）, incubated for 72 hours, and then detected by flow cytometry. T cell proliferation was measured at the gate setting ofAPC CD3 positive cells. Results： The percentages of CD4＋CD25＋ T cells in CD4＋ T cells and CD25＋ cells in all PBMCs detected by flow cytometry increased in the sequence of health donor, only HCV-infected patients and HCV-associated B-NHL（33.94%±2.18% vs. 57.95%±1.77% vs. 70.24%±12.75%, respectively, P〈0.05;18.16%±2.23% vs. 33.45%±1.32% vs. 54.69%±8.66%, respectively, P〈0.05）. The MI percentage ofCD25- T cells in HCV-associated B-NHL intervened by DG was higher than of CD25- T cells undone （57.7%± 4.2% vs. 46.5%±5.6%, respectively, P〈0.05）. The M1 percentage ofCD25＋ T cells in HCV-associated B-NH,L intervened by DG was lower than of CD25＋ T cell undone （14.7%±1.3% vs. 22.1%±4.1%, respectively, P〈0.05）. Conclusions： The number of CD25＋ cells including CD4＋CD25＋ cells in the patients with HCV-associated B-NHL were obviously high compared with only HCV-infected patients and health donors. This verifies that T cells in HCV-associated B-NHL are in suppressed condition, which is more serious than in only HCV-infected patients. The cell proliferation of CD25- T cells without CD25＋ cells was more active than the one of all T cells including CD25＋. This demonstrates that CD25＋ ceils can inhibit CD25- T cell proliferation in HCV-associated B-HNL. Interestingly, the M1 percentage of CD25＋ T cell proliferation intervened by DG decreased; the one of CD25 T cell proliferation intervened inversely increased. This shows that DG has the immune regulatory effect of suppressing CD25＋ T cell and promoting CD25 T cell on HCV-associated NHL.