选择性去除同种异体反应T细胞异基因小鼠骨髓移植的实验研究

发布时间：2018-11-01 20:26

【摘要】：目的:探讨选择性去除供鼠(C57BL/6~(H-2b))细胞即移植物中表达CD25+、CD69+活化抗原的同种异体反应T 细胞减轻异基因小鼠骨髓移植急性移植物抗宿主病(GVHD)的可能性。方法:以C57BL/6~(H-2b)小鼠为供鼠, BALB/c~(H-2b)小鼠为受鼠,建立MHC 完全不相合的急性致死性GVHD 模型。通过流式细胞仪检测初次单向混合淋巴细胞反应(受鼠BALB/c 脾单个核细胞为刺激细胞,供鼠C57BL/6 脾单个核细胞为反应细胞,)细胞CD25+、CD69+表达情况,应用磁性细胞分离系统去除分选法在表达高峰去除CD25+、CD69+同种异体反应T 细胞,获得阴性细胞。通过MTT 法在不同时间点(第1、2、3、4、5 天)检测再次混合淋巴细胞反应的细胞增殖情况(新鲜相同刺激原、无关刺激原、肿瘤刺激原加入阴性细胞进行再次单向混合淋巴细胞反应)。移植处理组为阴性细胞(2.0*106)与C57BL/6 骨髓细胞(5.0*106)共输注给致死剂量照射的BALB/c小鼠;移植对照组为相同条件下初次单向混合淋巴细胞反应的细胞(2.0*106)与C57BL/6 骨髓细胞(5.0*106)共输注,比较急性GVHD 的发生率、发生程度、造血重建及存活时间情况。结果:将供鼠C57BL/6~(H-2b)脾单个核细胞(2.0*10~6)混合其骨髓细胞(5.0*10~6)通过尾静脉输注给致死剂量照射的受鼠BALB/cH-2d小鼠可以成功地构建急性GVHD 模型,100%发生GVHD,病理组织表现重度急性GVHD 病理改变。初次混合淋巴细胞反应提示CD69+、CD25+的表达率高峰分别为第48 小时、72 小时(峰值分别为21.5±1.1%,56.4±3.4%),结合其余时间点实验数据提示初次混合淋巴细胞反应的第48 小时为选择性去除CD25+、CD69+的最好时机。再次单向混合淋巴细胞反应显示明显降低了对相同刺激原的反应性(29.02%～64.17%, 48.82%±13.71%),保留大部分对无关刺激原的反应性(49.61%～75.69%,65.47%±9.84%)和肿瘤刺激原的反应性(61.14～68.62%,64.83%±3.65%)。在构建的急性GVHD 模型(C57BL/6 →BALB/c)中,处理组急性GVHD 发生率下降(10 只中3 只死于急性GVHD),病理形态学分析提示未发生或发生轻度急性GVHD;而对照组均在24 天内死于急性GVHD,病理形态学分析提示为中重度急性GVHD。
[Abstract]:Aim: to investigate the possibility of selective removal of allogeneic T cells expressing CD25 and CD69 activated antigen in donor rat (C57BL / 6H-2b) cells to alleviate acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation. Methods: C57BL / 6H-2b mice were used as donor mice and BALB/c~ (H-2b) mice as recipient mice to establish acute lethal GVHD model which was completely incompatible with MHC. Flow cytometry was used to detect the expression of CD25 and CD69 in primary unidirectional mixed lymphocyte reaction (splenic mononuclear cells of recipient rat BALB/c and donor spleen mononuclear cells of C57BL/6). The CD25 and CD69 allogeneic T cells were removed at the peak of expression by magnetic cell separation system and negative cells were obtained. The cell proliferation of remixed lymphocyte reaction was detected by MTT method at different time points (1 ~ 2 ~ 3 ~ 3 ~ 4 ~ 4 ~ 5 days) (fresh same stimulator, no stimulant). Tumor stimulator was added to negative cells for reunidirectional mixed lymphocyte reaction. In the transplantation treatment group, negative cells (2.0 ~ 106) and C57BL/6 bone marrow cells (5.0 ~ 106) were injected into BALB/c mice irradiated with lethal dose. In the control group, the primary unidirectional mixed lymphocyte reaction cells (2.0 ~ 106) and C57BL/6 bone marrow cells (5.0 ~ 106) were co-infused under the same conditions. The incidence and degree of acute GVHD were compared. Hematopoietic reconstitution and survival time. Results: the donor C57BL / 6 ~ (H-2b) splenic mononuclear cells (2.0 ~ 10 ~ (6) mixed with bone marrow cells (5.0 ~ 10 ~ (6) were successfully injected by tail vein to irradiated BALB/cH-2d mice with lethal dose. Build an acute GVHD model, The pathological changes of severe acute GVHD occurred in 100% of patients with GVHD,. The primary mixed lymphocyte reaction showed that the peak expression rates of CD69 and CD25 were at 48h and 72h (21.5 卤1.1 and 56.4 卤3.4%, respectively). Combined with the experimental data of other time points, 48 hours of primary mixed lymphocyte reaction was the best time to selectively remove CD25 and CD69. The reunidirectional mixed lymphocyte reaction showed that the reactivity to the same stimulant was significantly decreased (29.02 卤64.17, 48.82% 卤13.71%), and most of the reactivity to the unrelated stimulator was retained (49.61%, 75.69%). 65.47% 卤9.84% and 61.14% 卤9.84%, 64.83% 卤3.65%, respectively. In the acute GVHD model (C57BL/6 BALB/c), the incidence of acute GVHD in the treatment group decreased (3 out of 10 died of acute GVHD), pathomorphology analysis showed no or mild acute GVHD;). In the control group, all died within 24 days of acute GVHD, pathomorphology analysis showed moderate and severe acute GVHD..
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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