骨髓源性间充质干细胞联合来氟米特对小鼠T淋巴细胞的免疫调节作用  

The immunoregulatory effects of bone marrow-derived mesenchymal stem cells combined with lefiunomide on mice T-lymphocytes

作　　者：裘影影[1] 李晶[1] 殷玉俊[1] 汤郁[1] 尤海燕[1] 朱伟[2] 焦志军[1] 

机构地区：[1]江苏大学附属医院风湿科,镇江212001 [2]江苏大学医学技术学院血液学实验室

出　　处：《中华风湿病学杂志》2008年第7期469-472,共4页Chinese Journal of Rheumatology

基　　金：江苏省科技厅社会发展项目（BS2005043）

摘　　要：目的探讨骨髓源性间充质干细胞（BMSCs）在体外联合来氟米特（LEF）对小鼠T淋巴细胞的免疫调节作用。方法直接贴壁筛选法分离培养小鼠BMSCs，流式细胞分析（FCM）鉴定BMSCs的纯度。用EZ—SepTMMouse1x分离异体BALB／c小鼠的脾淋巴细胞，在刀豆球蛋白A（ConA）诱导的同时，先经LEF处理，洗去LEF后，脾淋巴细胞再和BMSCs共培养。分组：A组：脾淋巴细胞；B组：脾淋巴细胞＋BMSCs；C组：LEF处理的脾淋巴细胞；D组：LEF处理的脾淋巴细胞＋BMSCs。用四甲基噻唑蓝（MTT）比色法检测各组T淋巴细胞的增殖情况，流式细胞术分析各组T淋巴细胞的活化及凋亡情况，定量反转录-聚合酶链反应（RT—PCR）检测各组T淋巴细胞的白细胞介素（IL）-2、IL—10基因水平。结果在体外，BM—SCs处理组（B组）T淋巴细胞的A570nm值为0．578±0．042，LEF处理组（C组）A57nm值为0．502±0．040，两组A570nm值均显著低于对照组（A组A570nm值为0．778±0．035（P〈0.01）。BMSCs联合LEF组（D组A570nm值为0．218±0．033，显著低于BMSCs处理组及LEF处理组（P〈0．01）。BMSCs联合LEF对CD3＋CD69＋、CD3＋CD28＋表达、IL-2基因表达均无明显影响。BMSCs单独或联合LEF组T淋巴细胞凋亡率分别为（2．29±0．32）％、（4．22±0．98）％，较A组T淋巴细胞凋亡率（8．08±1．20）％均明显下降。BMSCs处理组和LEF处理组T淋巴细胞IL—10基因相对表达分别为：0．098±0.039、0．054＋0．022明显低于A组（IL—10基因相对表达为1．000）（P〈0．01），MSCs联合LEF组IL—10基因相对表达为：0．023＋0．015，显著低于BMSCs处理组及LEF处理组（P〈0．01）。结论BMSCs联合LEF对小鼠T淋巴细胞有一定程度的协同免疫调节作用。Objective To investigate the immunoregulatory effects of bone marrow-derived mesenchyreal stein cells （BMSCs） combined with leflunomide （LEF） on mice T-lymphocytes in vitro. Methods BMSCs from BALB/c mice were isolated and expanded. The purity of BMSCs was identified by flow cytometry （FCM）. The BALB/c mice＇s spleen lymphocytes were isolated by using EZ-SepTM Mouse 1X. Under ConA stimulation, spleen lymphocytes were pretreated with LEF, then washed and co-cultured with BMSCs. We set up four groups to investigate in this study： group A, spleen lymphocytes alone; group B, spleen lymphocytes with BMSCs; group C, LEF-pretreated spleen lymphocytes alon6 and the group D, LEF-pretreated spleen lymphocytes with BMSCs. T-lymphocytes proliferation was assessed by MTI＇. FCM was used to analysis T-lymphocytes apoptosis and surface markers of CD69 and CD28. The mRNA expression of interleukin （IL）-2, IL-10 were detected by real-time RT-PCR. Results In vitro, the T-lymphocytes＇values of A570nm were significantly lower in group B and group C, compared with group A （group B vs group C vs group A, 0.578±0.042 vs 0.502± 0.040 vs 0.778±0.035, P〈0.01）, while the value of A570nm in group D was 0.218±0.033, which was also obviously lower than that in group B and group C （P〈0.01）. There were no suppressing effects on T-lympho- cytes＇activation and expression of IL-2 had been observed. The proportion of apoptotic T-lymphocytes in group B and group D were （2.29±0.32）% and （4.22±0.98）%, which was significantly lower than that in group A （8.08±1.20） （P〈0.01）. The expression of IL-10 in group B and C were also lower than that in group A （group B vs group C vs group A, 0.098±0.039 vs 0.054±0.022 vs 1.000, P〈0.01 ）. Either, the expression of IL-10 in group D was 0.023±0.015, whieh was obviously lower than that in group B and group C （P〈0.01）. Conclusion BMSCs combined with LEF show synergistic immunoregulatary effects on miee＇s T-lymphoeytes.

关 键 词：间质干细胞 T淋巴细胞 来氟米特 免疫调节 

分 类 号：R96[医药卫生—药理学]