致敏小鼠调节性T细胞对其致敏水平及免疫功能的影响

Influence of Recipient Treg Cells on the Level of Donor Reactive Antibody and Immune Function of Sensitized Mice

【目的】探讨致敏小鼠调节性T细胞对其致敏水平及免疫功能的影响。【方法】免疫磁珠法从致敏小鼠脾细胞中分选出CD4+CD25+Treg细胞,流式细胞术检测分选后细胞纯度、主要表面标记及Foxp3基因的表达。实验分组:正常对照组、致敏对照组和输注组,输注组以不同方式向致敏小鼠体内输注CD4+CD25+Treg(5×105/只·次),流式检测在处理后的第21天检测各组血清中与供者反应性抗体结合的供者脾脏细胞百分比、CD4+CD25+Treg、记忆性T细胞和效应性T细胞的比例。ELISA法检测各组小鼠血清中Th1类细胞因子(IFN-r、IL-2)和Th2类(IL-4、IL-10)细胞因子水平。【结果】分选出CD4+CD25+Treg细胞纯度平均达到87%,细胞活性大于97%,高表达Foxp3基因。致敏对照组的CD4+CD25+Treg细胞、记忆性T细胞、效应性T细胞和血清IFN-r水平较正常对照组升高,致敏对照组的血清IL-4、IL-10水平较正常对照组降低,差异有统计学意义(P<0.05);输注组CD4+CD25+Treg、记忆性T细胞比例、血清IL-4、IL-10水平较致敏对照组高;输注组效应性T细胞比例、血清IFN-r水平较致敏对照组低,且差异有统计学意义(P<0.05)。输注组小鼠血清中与供者反应性抗体结合的供者脾脏细胞百分比与致敏对照组的比较,差异无统计学意义(P>0.05)【结论】成功分选出致敏后的调节性T细胞,致敏促使免疫细胞免疫记忆,Th1细胞活化增强,Th2细胞活化受到抑制。向致敏动物体内输注CD4+CD25+Treg可降低细胞免疫,诱导Th1/Th2细胞因子向Th2偏移,但供者反应性抗体水平未降低。

[Objective] The study was aimed to investigated the effect of recipient Treg cells on the level of donor reactive antibodies and immune function of sensitized mice.[Methods] Sensitized mice CD4^＋CD25^＋ regulatory T cells were isolated from mouse splenocytes by MACS.Phenotype of the T cell lines and expression of the FOXP3 was determined by FACS.The BALB/c mice were divided into 4 groups：①Mice were sensitized and applied 5 × 10^5 Treg on 14 days as infusion A group.② Mice were sensitized and applied 5 × 10^5 Treg on 8 days and 14 days as infusion B group.③ Mice were sensitized and applied equal volume of PBS as sensitized control group.④Normal mice blank control group.On 21 days,serum was separated from the sensitized mice,the level of reactive antibodies were measured by flow cytometry,and the level of Th1 （IFN-r,IL-2） and Th2 （IL-4,IL-10） type cytokines were tested by ELISA.Spleen cells were obtained from recipients and were used for the tests of memory T cells,effect T cells and regulatory T cells by flow cytometry.[Results] The average purity of CD4^＋CD25^＋Treg cell was 87%,the activity was more than 97%,and the expression of FOXP3 was high.Compared with normal control group,the ratio of CD4^＋CD25^＋Treg cells,memory T cells,effect T cells and the level of IFN-r were higher in sensitized control group,the level of IL-4,IL-10 were lower in sensitized control group,the difference was statistically significant （P 〈 0.05）.Compared with sensitized control group,the ratio of CD4^＋CD25^＋Treg cells,memory T cells and the level of IL-4 and IL-10 were higher in infusion group,the ratio of effect T cells and the level of IFN-r were lower in infusion group,the difference was statistically significant （P 〈 0.05）.The percentage of combined cells occurring antigen-antibody reaction was no significant difference between sensitized control group and infusion group （P 〉 0.05）.[Conclusion] CD4^＋CD25^＋Treg with high purity were isolated by immune magnetic sands.Sensitization promoted recipients to produce immune memory,and increased activation of Th 1 cells while inhibited proliferation of Th2 cells.After infusing CD4^＋CD25^＋Treg,the cellular immunity was decreased and the level of donor reactive antibodies were not reduced in sensitized recipients.