骨髓间充质干细胞治疗再生障碍性贫血模型鼠的机制

Mechanism of bone marrow mesenchymal stem cells for treating model mice with aplastic anemia

背景:骨髓间充质干细胞在体外抑制再生障碍性贫血T淋巴细胞的活化与增殖,并有支持骨髓造血作用。目的:观察骨髓间充质干细胞对再生障碍性贫血模型鼠存活率的影响,探讨其治疗再生障碍性贫血的机制。设计、时间及地点:随机对照动物实验,于2009-01/04在安徽医科大学第一附属医院中心实验室完成。材料:清洁级雌性BALB/C小鼠40只,8～10周龄,体质量16～18g,作为受体。清洁级DBA/2小鼠5只,6～10周龄,体质量15～19g为淋巴细胞供体。方法:取BALB/c小鼠骨髓进行间充质干细胞体外增殖培养,传至3～5代的细胞用于实验。建立免疫介导再生障碍性贫血小鼠模型。40只BALB/C小鼠随机分为3组,治疗组(n=15):造模后将1×106个骨髓间充质干细胞输注到治疗组小鼠体内进行治疗;模型组(n=15):仅造模不进行任何治疗;对照组(n=10):不进行任何干预。主要观察指标:分别于第1,2,3,4周观察各组小鼠的存活率;流式细胞仪及酶联免疫吸附法测定骨髓间充质干细胞治疗后第28天各组小鼠T淋巴细胞亚群及血浆肿瘤坏死因子α的表达。结果:①模型组小鼠3只于第2周内死亡,7只于第3周内死亡,2只于第4周内死亡,28天存活率20%;治疗组小鼠4只于第3周内死亡,28天存活率73%。②与模型组相比,治疗组和对照组CD4的表达增高[(27.12±4.23)%,(32.18±6.73)%,(38.44±7.88)%,P<0.01];CD8和肿瘤坏死因子α表达均降低[CD8:(34.42±5.13)%,(27.33±5.09)%,(23.31±5.24)%,P<0.01;肿瘤坏死因子α:(47.27±10.11),(12.34±5.87),(9.31±3.77)ng/L,P<0.01]。结论:骨髓间充质干细胞可以提高再生障碍性贫血小鼠存活率,其治疗机制可能与调节T淋巴细胞亚群及抑制T淋巴细胞分泌的肿瘤坏死因子α有关。

BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） inhibit activation and proliferation of T cells in vitro and support hematopoiesis of bone marrow. OBJECTIVE： To observe the effect of BMSCs on survival rate of mice with aplastic anemia and to explore the mechanism for treating aplastic anemia. DESIGN, TIME AND SETTING： A randomized controlled animal experiment was performed at the Central Laboratory of the First Affiliated Hospital of Anhui Medical University from January to April 2009. MATERIALS： A total of 40 female BALB/c mice, clean grade, 8 10 weeks old, weighing 16 18 g, were used as recipients; another 5 DBA/2 mice, clean grade, 6-10 weeks old, weighing 15 19 g, were used as donors. METHODS： BMSCs were extracted from BALB/c mice. The third-passage cells were used to establish aplastic anemia injury. A total of 40 BALB/c mice were randomly divided into three groups： treatment （1×10^6 BMSCs, n=15）, model （modeling alone, n=15）, and control （without any intervention, n=10） groups. MAIN OUTCOME MEASURES： Survival rate was observed at 1, 2, 3, and 4 weeks after operation; T lymphocyte subgroup and tumor necrosis factor e （TNF-o） expression were measured at day 28 postoperatively using flow cytometry and enzyme linked immunosorbent assay. RESULT： （1） In the model group, three mice died at 2 weeks, seven at 3 weeks, and two at 4 weeks, and the survival rate was 20% at day 28 postoperatively. In the treatment group, four mice died at 3 weeks, and the survival rate was 73% at day 28 postoperatively. （2） As compared with the model group, CD4 expression increased in both treatment and control groups [（27.12±4.23）%, （32.18±6.73）%, （38.44±7.88）%, P 〈 0.01]; CD8 and TNF-α expression decreased [CDS： （34.42±5.13）%, （27.33±5.09）%, （23.31±5.24）%, P 〈 0.01; TNF-e： （47.27±10.11 ）, （12.34±5.87）, （9.31±3.77） ng/L, P 〈 0.01]. CONCLUSION： BMSCs enhance survival rate of mice with aplastic anemia, and the mechanism may be related to regulating T lymphocyte subgroup and inhibiting secretion of TNF-α.