CD4+CD25+调节性T细胞治疗反复性流产的动物实验研究

目的:通过动物实验,探讨CD4~+CD25~+调节性T细胞用于反复性流产免疫治疗的效果。方法:密度梯度离心法从正常昆明白新生鼠的血液和能正常生育的BALB/c雄鼠血液中分别分离单核淋巴细胞,再用免疫磁珠法筛选出CD4~+CD25~+调节性T细胞;以CBA/J雌鼠×DBA/2J雄鼠交配组合作为反复自然流产的动物模型,于孕期给予鼠尾静脉注射不同种类的淋巴细胞;采用独立样本t检验对各组得到的数据进行分析和处理。结果:注射CD4~+CD25~+T细胞组小鼠CD4~+CD25~+T细胞在CD4~+T细胞中的占比为(17.49±0.60)%,高于注射单核淋巴细胞组的(14.68±0.83)%、无菌PBS组的(9.54±0.85)%和不注射组的(9.28±0.68)%(t=4.754,P0.05;t=13.242,P0.05;t=15.621,P0.05)。CD4~+CD25~+调节T细胞中的Foxp3蛋白相对表达量(5.85±0.45)高于注射单核淋巴细胞组(2.86±0.54)、无菌PBS组(1.08±0.16)和不注射组(1.00±0.00)(t=7.276,P0.05;t=17.227,P0.05;t=18.635,P0.05)。在第4天和第8天分别注射调节T细胞得到了较好的效果,胚胎吸收率为(4.92±0.08)%,低于注射单核淋巴细胞组的(13.07±0.06)%、注射无菌PBS组的(23.11±0.12)%和不注射组的(25.47±0.11)%(t=-2.603,P0.05;t=-4.012,P0.05;t=-4.700,P0.05)。结论:给反复性流产小鼠于孕期进行多次CD4+CD25+T细胞注射,免疫治疗效果好于传统的注射单核淋巴细胞。

Animal study on CD4+ CD25+ regulatory T cells for treating female mouse with recurrent spontaneous abortion

Objective:To explore immunotherapy effectiveness of the CD4+ CD25+ regulatory T cells for treating fe male mouse with recurrent spontaneous abortion (RSA) by animal experiments.Methods:Mononuclear lymphocytes were isolated from the blood (instead of cord blood) of new-born baby of KunMing Bai mouse or BALB / c male mouse with normal birth ability (as unrelated third party blood source) by density gradient centrifugation method.The CD4+ CD25+ regulatory T cells were selected by magnetic-activated cell sorting from mononuclear cells of cord blood cells.CBA/J female mouse copulated with DBA/2J male mouse was utilized as RSA animal model.Pregnant RSA mice were injected different types of lymphocytes through tail vein.Independent sample t-test was used to analyze the data from each group.Results:The proportion of CD4+ CD25+ T cells in CD4+ T cells of injected CD4+ CD25+ regulatory T cells group was (17.49 ± 0.60) ％,which was statistical significant higher than that of mononuclear lymphocyte injection group (14.68±0.83) ％,sterile PBS group (9.54±0.85) ％ or no injection group (9.28±0.68) ％,(P＜ 0.05,t values was 4.754,13.24 and 15.62,respectively).The Foxp3 relative protein expression level of CD4+ CD25+ regulatory T cells injected group was 5.85±0.45,which was also significant higher than that of mononuclear lymphocyte injection (2.86±0.54),sterile PBS group (1.08± 0.16) or no injection group (1.00± 0.00) (P＜ 0.05,t values were 7.27、17.23 and 18.64,respectively).Finally,two times of CD4+CD25+ T cell injected group at the 4th and 8th day had well effect for RSA mouse,and embryo sorption rate was 4.92±0.08％,which significant lower than that of two times of mononuclear lymphocyte injected group (13.07 ± 0.06) ％,sterile PBS group (23.11 ± 0.12) ％,or no injection group (25.47 4 0.11) ％,(P ＜ 0.05,t values was-2.60,-4.01 and-4.01,respectively,).Conclusion:Pregnant mouse with RSA injected CD4+ CD25+ T cells several times for immunotherapy can get better effectiveness than that of pregnant mouse injected traditional mononuclear cells.