骨髓间充质干细胞对小鼠急性移植物抗宿主病的预防及治疗作用

目的 观测小鼠骨髓间充质干细胞(MSC)对急性移植物抗宿主病(aGVHD)的预防及治疗作用,并探讨其干预机制.方法 建立以C57BL/6小鼠为供体、致死剂量照射的BALB/c小鼠为受体的aGVHD动物模型.将小鼠分成6组:PBS组(单纯放射组)、BM组(单纯输注骨髓)、GVHD组(输注骨髓+脾细胞)、MSC-A组(输注骨髓+脾细胞,并于移植当天输注1×10~5 MSC)、MSC-B组(输注骨髓+脾细胞,并于移植当天输注5×10~5 MSC)、MSC-C组(输注骨髓+脾细胞,并于+7 d输注1×10~5 MSC),观测各组的生存状态;绿色荧光蛋白基因(Ad-EGFP)转染MSC,输入aGVHD模型小鼠,观察MSC在靶器官组织中的分布.结果①供体MSC可显著控制小鼠的GVHD症状,延长aGVHD小鼠的平均生存时间:PBS组为(13.5±2.6)d、GVHD组为(11.1±4.0)d,MSC-A组为(26.4±7.7)d、MSC-B组为(22.7±9.2)d,MSC-C组为(22.9±8.2)d,MSC各组与GVHD组比较差异均有统计学意义(P＜0.01),但MSC各组之间无明显差异(P=0.28).病理评估显示MSC可明显减少小肠及脾脏组织中淋巴细胞的浸润,减轻损害.②MSC可明显减少aGVHD环境中Th1类炎性因子的分泌:GVHD、MSC.A、MSC-B、MSC-C各组+14 d外周血上清中IFN-γ的水平分别为(607.9±157.1)、(143.6±37.5)、(117.0±77.8)、(131.4±63.4)ng/L,各组TNF-α的水平分别为(52.31±17.95)、(6.02±3.99)、(5.21±0.28)、(22.39±18.21)ng/L;MSC不影响aGVHD环境中异基因T淋巴细胞的增殖,但促进其凋亡:GVHD、MSC-A、MSC-B、MSC-C各组的CD3+ Annexin V+ PI-细胞率分别为(10.3±6.6)%、(13.5±13.8)%、(19.7±6.0)%、(16.6±7.3)%,其中MSC-B组与GVHD组比较差异具有统计学意义(P＜0.05).③Ad-EGFP成功高效转染MSC,输注入aGVHD模型鼠6 d后,共聚焦显微镜下发现MSC可大量分布至肺及小肠,而肝、脾及肾脏有少量分布.结论 MSC可促进异基因T淋巴细胞凋亡、抑制其二次活化功能、减少Th1类炎症因子的分泌表达、参与修复aGVHD靶器官组织,可有效防治aGVHD.

The role of marrow derived mesenchymal stem cells in the prevention and treatment of acute graftversus-host in mice

Objective To observe the prevention and treatment of acute graft-versus-host disease (aGVHD) by murine marrow mesenchymal stem cells (MSCs) in vivo.Methods Allogeneic aGVHD model was established with lethally irradiated BALB/c recipients receiving allogeneic BM (BMC) and spleen cells (SP) from C57BL/6 with or without mMSCs at different dose and different time posttransplantation.Six groups were set up,group 1 (irradiation control group);group 2 (i.v.BMC only);group 3 (i.v.SP +BMC);group4 (i.v.SP + BMC + 5×10~6/kg mMSC at day 0);group5 (i.v.SP + BMC +2.5×10~7/kg mMSC at day O);group 6 (i.v.SP + BMC +5×10~6/kg mMSC at day 7).The survival was monitored daily,mMSCs infected with adenoviral vector (Ad-GFP) were injected into aGVHD model to observe the distribution of MSCs in vivo.Results ①Addition of donor mMSCs significantly controlled the lethal GVHD.The survival time (day) in group 1 was 13.5±2.6,group 3 11.1±4.0,group 4 26.4±7.7,group 5 22.7±9.2,group 6 22.9±8.2,respectively.The difference between groups 4-6 and group 3 was statistically significant (P＜0.01),but there was no difference among groups 4-6 (P = 0.28);There was less lymphocyte infiltration and architectural disruption in the intestine and spleen of groups 4-6 than that of group 3;②mMSCs significantly reduced IFN-γ and TNF-α in the serum of recipient mouse;the levels of IFN-γ in groups 3,4,5,6 were (607.9±157.1),(143.6±37.5),(117.0±77.8),(131.4±63.4) ng/L,respectively.And of TNF-α were (52.31±17.95),(6.02±3.99),(5.21±0.28),(22.39±18.21) ng/L,respectively,mMSCs had no effect on allogeneic T cell proliferation in GVHD model but increased apoptosis of allogeneic T cells.The percentage of CD3 + Annexin V+ PI-in each group were (10.3±6.6)%,(13.5±13.8) %,(19.7±6.0) %,(16.6±7.3) %,respectively.③After intravenous infusion,large numbers of GFP-MSCs lodged in lungs and intestines while small numbers in the liver,spleen and kidney.Conclusions MSCs has no effect on proliferation but induce apoptosis of allo-reactive T cells;MSCs can inhibit the second activation of allogeneic T cells,significantly reduce the secretion of IFN-γ and TNF-α;MSCs might be able to repair GVHD target tissues by extensive distribution to lungs,intestines,and liver of the animals.