脐带间充质干细胞对再生障碍性贫血患者Treg细胞的调节影响

Modulation of umbilical cord blood mesenchymal stem cells on Treg cells in the patients with aplastic anemia

目的 探讨脐带间充质干细胞（UC-MSCs）对再生障碍性贫血（AA）患者调节性T细胞（Treg细胞）数目、功能及其上淋巴细胞功能相关分子-1（LFA-1）表达的影响.方法 分离2015年5月至2016年7月济南军区总医院血液科收治的20例初治的非重型再生障碍性贫血（NSAA）患者及同期门诊体检的10名健康对照者外周血单个核细胞（PBMCs）,流式细胞术分别检测PBMCs单独培养、PBMCs＋UC-MSCs共培养条件下Treg细胞比例及其细胞上LFA-1的表达变化;磁珠分选AA患者Treg细胞及CD4＋ CD25-T细胞,CFSE标记CD4＋ CD25-T细胞,分别检测Treg细胞、Treg细胞与UC-MSCs共培养后对CD4＋ CD25-T细胞分裂能力的影响.结果 AA组淋巴细胞上LFA-1平均免疫荧光强度明显高于健康对照组[（71.4±10.1）比（52.5±8.7）,P=0.002],但Treg细胞上的LFA-1表达与健康对照组差异无统计学意义（P=0.199）.与AA患者PBMCs单独培养相比,UC-MSCs可明显降低AA患者Treg细胞上LFA-1的比例[（20.96±1.76）％比（44.26±1.19）％,P=0.012],升高AA患者Treg细胞的比例[（5.33±1.14）％比（1.94±0.65）％,P=0.003].AA患者Treg细胞、Treg细胞与UC-MSCs共培养后对CD4＋ CD25-T细胞分裂能力的影响,差异无统计学意义（P =0.290）.结论 AA患者淋巴细胞上LFA-1平均免疫荧光强度明显增高,可能是参与AA发生的一个重要机制.UC-MSCs可明显降低AA患者Treg细胞上LFA-1的表达比例,提高Treg细胞比例,但对单个Treg细胞的功能可能无明显改善.

Objective To research the modulation of Umbilical cord blood mesenchymal stem cells on the number and function of Treg cells in the patients with aplastic anemia,as well as the expression of LFA-1 on Treg cells.Methods A total of 20 newly diagnosed NSAA patients were collected from May 2015 to Jun 2016 in Department of Hematopathy,General Hospital of Jinan Military,and 10 healthy volunteerswere recruited as controls.Separation of the patients and controls with peripheral blood mononuclear cells were divided into two groups,including PBMCs culture alone,PBMCs co-culture with UC-MSCs,application of flow cytometry detect respectively the proportion of the Treg cells and the expression of LFA-1 on Treg cells under different culture conditions.The Treg cells and CD4 ＋ CD25-T lymphocyte were separated by magnetic ccll sorting （MACS） system,CFSE label CD4 ＋ CD25-T lymphocyte,comparing the inhibitive function of Treg cells on CD4 ＋ CD25 T lymphocyte with or without co-culture with UC-MSCs.Results The intensity of fluorescence expression of LFA-1 on T lymphocyte in aplastic anemia increased obviously（（71.4 ± 10.1）vs（52.5 ± 8.7）,P =0.002）,but the LFA-1 expressed on Treg cells had no significant difference （P =0.199）.After co-cultured with UC-MSCs,the proportion of LFA-1 on Treg cells in aplastic anemia reduced greatly （（20.96 ± 1.76） % vs （44.26 ± 1.19） %,P =0.012）,at the same time,UC-MSCs increased the proportion of Treg cells obviously （（5.33 ± 1.14）%vs（1.94 ±0.65）%,P =0.003）,but the effect of Treg cells ou the mean frquency of dividing CD4 ＋ CD25-T lymphocyte had no significant difference with or without co-culture with UC-MSCs （P =0.290）.Conclusions The intensity of fluorescence expression of LFA-1 on lymphocyte in aplastic anemia increases obviously,indicating the possible pathogenesis of AA.UC-MSCs inhibit the expression of LFA-1 on Treg cells and enhance the proportion of Treg cells,but UC-MSCs doesn＇t directly improve the immunosuppression of single Treg cells.