基因工程化Treg细胞对小鼠异基因骨髓移植后急性移植物抗宿主病的作用

Influence of mouse genetic engineering regulatory T cells infusion on post-allogeneic bone marrow transplantation acute graft-versus-host disease in mice

目的探讨输注慢病毒载体介导的鼠基因工程调节性T淋巴细胞（Treg细胞）对小鼠异基因骨髓移植后移植物抗宿主病（GVHD）的影响。方法利用慢病毒载体介导，将鼠Foxp3基因转导人BALB／c小鼠的CD4^=CD25-T淋巴细胞，即为基因工程Treg细胞。建立小鼠异基因骨髓移植模型，在移植时联合输注基因工程Treg，通过受鼠移植后生存期、GVHD临床评分、组织病理学改变、供者T淋巴细胞激活、炎性细胞因子浓度等指标评价其防治GVHD的作用。结果单纯照射组、移植对照组、工程Treg组和空载体对照组小鼠存活时间分别为（8．8±0．6）、（36．7±2．5）、（51．6±4．0）和（34．1±2．3）d，工程Treg组小鼠存活时间明显长于其他各组（P〈0．05）。移植对照组及空载体对照组小鼠肝脏、皮肤和小肠病理切片均存在GVHD病理改变，工程Treg组长期存活小鼠的肝脏、皮肤和小肠常规病理切片结构基本正常，未见GVHD病理表现，该组GVHD评分明显低于移植对照组及空载体对照组。移植后3d（＋3d）、＋4d工程Treg组受鼠脾脏中供者T淋巴细胞数明显低于移植对照组和空载体对照组（P〈0．05），且＋4d工程Treg组受鼠脾脏供者T淋巴细胞中IFN-γ^＋细胞比例较后两组明显降低（P〈0．05）。在＋21～＋28d移植对照组、工程Treg组、空载体对照组受鼠血清IFN-γ、IL-2和TNF-α浓度达高峰，工程Treg组在＋21d时IFN-γ、IL-2和TNF-α浓度高峰较其他两组为低（P〈0．05）。结论小鼠异基因骨髓移植时联合输注基因工程Treg细胞可通过减少受鼠移植后供者T淋巴细胞的早期扩增、激活以及炎性细胞因子的分泌有效减少GVHD的发生，减轻其严重程度。

Objective To explore the influence of the lentiviral vector mediated mouse genetic engineering regulatory T cells （Treg） infusion on post-allogeneic bone marrow transplantation （allo-BMT） acute graft-versus-host disease （GVHD） in mice. Methods Lentivirus-mediated Fm＇khead box 1〉3 （Foxp3） gene was transducted into BALB/c mice CD4^＋ CD25 - T cells （Treg） to construct engineered Tregs in vitro. An al- lo-BMT model of BALB/c to C57BL/6 mice was established. After irradiation, the recipients were injected with donor cells plus the genetic engineering Tregs. Survival time, clinical GVHD score, histopathologieal findings, activation of donor T cells or serum levels of inflammatory cytokines were observed after allo-BMT. Results The mean survival times for radiation alone group（ Gp Ⅰ ）, transplantation control group（ Gp Ⅱ ）, engineering Treg infusion group（ Gp Ⅲ） and empty vector control group （ Gp Ⅳ ） were （ 8.8 ± 0.6 ） d, （ 36.7 ± 2.5 ） d, （51.6 ± 4.0） d and （ 34.1 ± 2.3 ） d, respectively. The survival time was significantly longer in Gp Ⅲ than in other groups （ P 〈 0.05 ）. Histopathological finding in several target organs （ skin, liver and small intestine） confirmed the presence of severe GVHD in Gp Ⅱ and Gp Ⅳ, while no histological signs of GVHD were observed in long survival recipients in Gp Ⅲ, and clinical GVHD scores in Gp Ⅲ were significantly lower than that in Gps Ⅱ and Ⅳ. The numbers of donor T ceils and the percentage of IFN-producing donor T cells in the spleen of recipients in Gp UI were significant lower than those in Gps Ⅱ and IV at days 3 and 4, and at day 3 after transplantation, respectively （P 〈 0.05）. The serum levels of IFN-γ, IL-2 and TNF-α were increased at day 21 to 28 after transplantation in all groups. The peak concentrations of IFN-γ, IL-2 and TNF-α in Gp Ⅲ were significantly lower lhan those in Gps Ⅱ and IV control groups at day 21 （ P 〈 0.05 ）. Conclusion Co-injectlon of genetic engineering Treg can efficiently prevent recipients from lethal GVHD after allo-BMT in mice by inhibiting the early activation and expansion of donor T cells and reducing the serum levels of inflammatory cytokines.