TLR5激动剂鞭毛蛋白预防小鼠异基因造血干细胞移植后急性移植物抗宿主病的初步研究

Prophylactic Effect of TLR5 Agonist Flagellin on Acute Graft versus Host Disease after Allogeneic Hematopietic Stem Cell Transplantation and Its Mechanism

本研究旨在探讨Toll样受体(TLR5)激动剂鞭毛蛋白(flagellin)对小鼠异基因造血干细胞移植(allo-HSCT)后急性移植物抗宿主病(aGVHD)的预防作用和可能作用机制。用主要组织相容性抗原完全不合的纯种近交系小鼠〔供体鼠:雄性C57BL∕6鼠;受体鼠:雌性BALB∕c鼠〕建立allo-HSCT的aGVHD动物模型。受鼠随机分为3组:鞭毛蛋白组,于移植前后2次尾静脉注射高纯度(纯度≥95%)的鞭毛蛋白50μl〔5μg∕(只.次)〕预防aGVHD;单纯移植组,移植后仅给予等容量PBS;单纯照射组,仅照射不移植,亦给予等容量PBS。观察比较移植后aGVHD的表现。结果表明,移植小鼠均出现典型的aGVHD症状,单纯移植组小鼠死亡高峰在移植后第4-5天。用鞭毛蛋白作为aGVHD预防方案小鼠的aGVHD症状明显减轻,平均生存时间较单纯移植组显著延长(P<0.05)。三组小鼠移植前外周血白细胞数比较均无显著性差异,但在照射后第14、21天,鞭毛蛋白组较单纯移植组外周血白细胞数显著性升高(P<0.05)。鞭毛蛋白预防组移植小鼠aGVHD病理表现较单纯移植组明显减轻。流式细胞仪检测鞭毛蛋白组与单纯移植组移植前后不同时间点Treg细胞/CD4+T细胞含量结果也表明,移植后2-4周鞭毛蛋白组小鼠的Treg细胞数较单纯移植组明显增加(P<0.05)。结论:鞭毛蛋白对小鼠allo-HSCT后发生aGVHD有预防作用,能减轻其症状和病理损害程度,显著延长其平均生存时间,其机制有可能通过增加移植后小鼠的Treg细胞含量,从而有效改善并减轻移植后小鼠aGVHD。

This study was aimed to investigate the prophylactic effect of Toll like receptor （TLR）5 agonist flagellin on acute graft versus host disease （aGVHD） after allogeneic hematopietic stem cell transplantation （allo-HSCT） and its possible mechemism. The animal model with allo-HSCT aGVHD was established by using purebreed mice （male mouse C57BL/6 as donor, femal mouse BALB/c as recipient） with complete-unidentical major histocompatibitily antigen. The recipient mice were randomly divided into 3 groups： group 1 in which mice were injected with high purity （95%） flagellin before and after allo-HSCT respectively, group 2 in which mice received allo-HSCT without injection of flagellin, group 3 in which mice were radiated alone. The aGVHD features of mice in group 1 and 2 were observed and compared. The results showed that the typical symptoms of aGVHD appeared in tranplanted mice. The death peak of mice in group 2 appeared at day 4 - 5 after transplantation. The aGVHD aymptoms were obviously alleviated and the mean survival time was prolonged significantly in mice group 1 as compared with mice in group 2 （ P 〈 0.05 ）. The comparison of WBC count in peripheral blood of mice in 3 groups before transplantation showed no significant difference （ P 〉 0.05 ）, while WBC count of mice in group 1 and 2 showed the significant difference at days 14 and 21 after transplantion （ P 〈 0.05 ）. The pathological appearences of aGVHD in mice of group 1 were obviously reduced as compared with mice in group 2. The flow cytometric detection of Treg cell/CD4^＋ T cell levels at different time before and after transplantion demonstrated that the Treg cell level in mice of group 1 at weeks 2 - 4 after transplantion significantly increased as compared with mice in group 2 （ P 〈 0.05 ）. It is concluded that flagellin can effectively prevent the aGVHD occurence after allo-HSCT, reduce the symptoms and pathological changes of aGVHD, obviously prolong mean survival time of mice in group 1. The mechanism of flagellin effect may be associated to increase of Treg cell level in mice after allo-HSCT.