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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。
Objective: To investigate the effect of Diammonium Glycyrrihizinate (DG) on the expression of CD25-T and CD25 + T cells in B cell non-Hodgkin’s lymphoma (B-NHL) Immunomodulatory effect. Methods: Flow cytometry was used to detect the proportion of HCV-associated CD4 + CD25 + T cells in CD4 + T cells and the proportion of CD25 + cells in total PBMC. The results were compared with those of HCV infected and healthy subjects. CD25- and CD25 + cells were sorted by immunomagnetic bead sorting (MACS) and incubated with non-sorted peripheral blood mononuclearcells (PBMC) for 72 hours with CFSE, The cell analyzer set APC CD3 positive cells as a gate to detect proliferation of CD25-T cells, unsorted T cells, CD25 + T cells, and proliferation of CD25-T cells and CD25 + T cells after intervention with glycyrrhizin Happening. Results: Flow cytometric analysis showed that the proportion of CD4 + CD25 + CD4 + cells and CD25 + cells in healthy B-NHL patients and HCV-infected patients showed a gradually increasing relationship (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). The percentages of cleaved phase (M 1 + M 2) were highest at 72 h after incubation with CFSE (74.4% ± 1.2%, 63.1% ± 5.4, respectively) for unclassified T cells followed by CD25 + T cells %, 47.2% ± 5.9%, P <0.05). The percentages of M1 division of CD25-T cells in CD25-T cells were significantly increased after diammonium glycyrrhizinate intervention (57.7% ± 4.2%, 46.5% ± 5.6%, P <0.05, respectively). Glycyrrhizic acid diammonium intervention CD25 + T cells compared with the non-intervention of the percentage of mitotic M1 division of CD25 + T cells decreased (14.7% ± 1.3%, 22.1% ± 4.1%, P <0.05). CONCLUSION: The percentage of CD4 + CD25 + and CD25 + cells in HCV-associated B-NHL group is significantly higher than that in HCV-infected group, indicating that T-cell immunosuppression exists and the degree of inhibition is more serious than that of HCV group. The proliferation of CD25-T cells after CD25 + cells were removed was more active than that of T cells without CD25 + removal, indicating that CD25 + cells in HCV-associated B-NHL cells can inhibit CD25-T cell proliferation. However, the proliferation of mitosis of HCV-associated B-NHL CD25 + T cells was decreased by DG, while the cleavage phase of CD25-T cells was increased, indicating that DG could inhibit the proliferation of CD25 + T cells and inhibit the proliferation of CD25 + T- -T cell proliferation in a positive immunomodulatory role.