泛T淋巴细胞结合照射诱导的再生障碍性贫血小鼠模型的优化

目的:建立适用于研究T淋巴细胞功能的获得性再生障碍性贫血(acquired aplastic anemia,AA)动物模型以及应用于再生障碍性贫血发病机制及治疗研究的动物模型。方法:对X射线联合淋巴细胞注射诱导的获得性再生障碍性贫血小鼠模型进行优化。AA组:富集C57BL/6J小鼠的脾脏泛T细胞,按细胞数5×10⁶/只经尾静脉注射至分别经3、4和5 Gy X射线照射的CB6F1小鼠体内;单纯照射组(total body irradiation,TBI):经尾静脉注射与AA组相同体积的PBS至分别经3、4和5 Gy X射线照射的CB6F1小鼠体内;对照组:经尾静脉注射与AA组相同体积的PBS至CB6F1小鼠体内;检测外周血血常规,计数骨髓有核细胞数,骨髓病理切片检测骨髓造血,流式细胞术检测骨髓T细胞分布比例、骨髓细胞凋亡和脾脏T细胞的分化程度。结果:相较于4、5 Gy照射的AA组,3 Gy照射的AA组生存期明显延长;经3、4、5 Gy X射线照射联合泛T淋巴细胞注射均可成功诱导受体鼠外周血红细胞、白细胞、血小板数严重减少,骨髓有核细胞严重减少,骨髓造血衰竭,骨髓中T淋巴细胞占比明显增加,CD4⁺T和CD8⁺T细胞均增加但以CD8⁺T细胞为主,并促进T细胞从初始T细胞向效应记忆T细胞分化。结论:3、4和5 Gy X射线的照射联合5×10⁶泛T细胞注射均可通过引起T淋巴细胞功能亢进,成功构建AA动物模型;较4、5 Gy照射剂量,3 Gy照射组生存期明显延长,外周血血象与AA临床患者表现更接近。

Optimization of Mouse Model of Aplastic Anemia Induced by Pan T Lymphocytes Combined with Irradiation

Objective:To establish an acquired aplastic anemia animal model for investigating the function of T lymphocyte and the pathogenesis and treatment of aplastic anemia(AA).Methods!To establish the acquired aplastic anemia mouse model through the X-ray irradiation in combination with lymphocytes injection.AA Group:the purified Pan T lymphocytes from the spleen of C57 BL/6 J mice were enriched and injected to the mice through tail vein(5×10⁶),the CB6 F1 mice were exposed to 3,4 and 5 Gy X-ray irradiation;TBI Group:the CB6 F1 mice were exposed to3,4 and 5 Gy X-ray irradiation,and were injected with the same volume of PBS buffer;Control group:the CB6 F1 mice were only injected with the same volume of PBS buffer.The peripheral blood routine was examined and the number of nucleated cells in bone marrow were calculated;the hematopoiesis changes in bone marrow was examined;flow cytometry was used to examine the distribution of T lymphocytes in bone marrow,and it also used to examine the apoptosis of bone marrow cells and the differentiation of spleen T lymphocytes.Results:Compared with 4,5 Gy irradiated mice in AA groups,the survival time of 3 Gy irradiated A A groups was significantly prolonged.3,4 and 5 Gy X-ray irradiation combined with Pan T lymphocyte injection could successfully induced severe reduction of red blood cells,blood neutrophils,and platelets,severe reduction of bone marrow nucleated cells,severe bone marrow hematopoietic failure,and the significant expansion of T lymphocytes ratio in the bone marrow.CD4⁺and CD8⁺T cells were both increased,but mainly on CD8⁺T cells,and could promote the differentiation of T cells from naive T cells to effector memory T cells.Conclusion:3,4 and 5 Gy X-ray irradiation combined with 5×10⁶pan-T cell injection could successfully induce acquired aplastic anemia through T lymphocyte hyperfunction.Compared with 4,5 Gy irradiated AA group,the 3 Gy irradiated AA group shows significantly longer survival time,and the peripheral blood routine profile closely resembles the clinical manifestations of AA patients.