应用扩增的Treg预防异基因骨髓移植后移植物抗宿主病的实验研究

目的：探讨扩增后的CD4+CD25+调节T细胞(regulatory T cell ,Treg)输注对异基因骨髓移植(allo-BMT)后小鼠移植物抗宿主病(GVHD)的影响。方法：利用免疫磁珠法分选小鼠Treg细胞； 以抗鼠 CD3ε 单抗、抗鼠CD28 单抗 、鼠重组 IL-2及辐射过的Balb/C小鼠脾细胞为共刺激因子，扩增TREG细胞；建立C57BL/6→BALB/c小鼠allo-BMT模型,接受移植小鼠随机分为3组,移植骨髓后6-8小时后分别予尾静脉输注扩增的Treg细胞、CD4+CD25-T细胞和RPMI 1640培养液(空白对照)。以移植后GVHD表现,肝、脾、小肠组织病理形态、生存期为观察指标并进行组间比较。结果：经免疫磁珠法分选可获得高纯度(91.80±2.15)%及具较强活力(97.58±1.23)%的Treg细胞；移植后(四周左右)的扩增后Treg细胞移植组小鼠肝、脾、小肠GVHD病理损害较同期CD4+CD25-T细胞移植组与空白对照组小鼠有一定程度的减轻。3组小鼠移植后平均存活时间分别为（61. 45±27. 88）天、(18.58±12. 39)天和(26. 37±15. 65)天, 扩增后Treg细胞移植组小鼠平均存活时间较CD4+CD25-T细胞、空白对照组小鼠延长(P<0.05)。结论：allo-BMT后输注增殖的Treg细胞可以减轻移植后GVHD程度,延长小鼠生存时间。

Influence of Expanded Treg Cells on GVHD after Allogeneic Bone Marrow Transplantation in Mice

Objective To explore the influence of expanded regulatory T cells (Treg cells) infused after allogeneicbonemarrow transplantation (allo-BMT) on GVHD.Methods Magnetic activated cell sorting (MACS)method was used to separate CD4+CD25+T and CD4+CD25-T cells from spleens of C57BL/6 mouse, and then the purity and activity of CD4+CD25+T cells were analyzed with flow cytometer. an allo-BMT model of C57BL/6→BALB /c mice was established, After stimulated with mouse anti-CD3ε Ab, mouse anti-CD28 Ab, rm IL-2 and irradiated spleen cells of C57BL/6 mouse, The recipient mice were randomly divided into three groups: expanded CD4+CD25+T cells, CD4+CD25-T cells and RPM I1640 culture medium. These cells and RPM I 1640 were infused into recipient mice by caudalveins at about 6 to 8 hours after allo-BMT respectively. Incidence of GVHD, pathological lesion of liver,spleen, small intestine, survival time in the recipients were observed after allo-BMT. Results (1) After sorting, CD4+CD25+T cells purity and cell vitality rate were (91.80±2.15)% and (97.58±1.23)%, respectively.(2). It was found that GVHD pathologic manifestations of the liver, spleen and small intestine in expanded Treg cells group were slighter in a certain extent than those in other two groups at about four weeks after allo-BMT. The mean survival time in three groups was (61. 45±27. 88)days, (18.58±12. 39)days and (26. 37±15. 65)days after allo-BMT, respectively, which in the expanded Treg cells group was significantly longer than that in other two groups (P<0. 05). Conclusion CD4+CD25+T cells could be expanded by mouse anti-CD3ε Ab, mouse anti-CD28 Ab, rm IL-2 and irradiated allogenic spleen. It is concluded that expanded Treg cells infusion can mitigate the GVHD and prolong survival time after allo-BMT in mice.