MDS铁过载小鼠模型建立及鉴定

Establishment and Identification of MDS Mouse Model with Irom Overload

目的:建立一种MDS铁过载小鼠模型,研究铁过载对MDS的影响。方法:通过逆转录病毒,将外源突变基因RUNX1-S291fs插入小鼠骨髓单个核细胞基因组,然后移植给经60Coγ射线照射的C57BL/6小鼠。8周后,腹腔注射铁剂,建立MDS铁过载小鼠模型。移植24周后,取小鼠外周血、骨髓、股骨、肝脏和脾脏进行鉴定:对外周血和骨髓细胞行瑞氏染色观察形态特征;对股骨、肝脏和脾脏组织行HE染色检测病理变化;对肝脏、脾脏和骨髓行普鲁士蓝染色观察铁沉积情况;应用流式细胞术检测小鼠骨髓细胞抗原表达情况;对骨髓单个核细胞和脾组织蛋白行免疫印迹实验,确认RUNX1-S291fs基因转入并表达蛋白。移植后对小鼠定期监测血常规指标和移植细胞嵌合率。结果:相比空白质粒对照小鼠,RUNX1-S291fs突变小鼠外周血白细胞计数、血红蛋白水平和血小板计数均降低;外周血和骨髓细胞显示病态造血;肝脏和脾脏明显增大;在股骨、肝脏和脾脏可见组织结构异常;骨髓细胞表面抗原表达异常;骨髓细胞和脾组织有RUNX1-S291fs蛋白表达。相比生理盐水对照小鼠,铁剂注射小鼠普鲁士蓝染色可见骨髓、肝脏和脾脏组织有明显铁沉积。结论:注射铁剂的RUNX1-S291fs突变组小鼠合并有MDS和铁过载的病理特征,成功构建了MDS铁过载小鼠模型,为研究铁过载对MDS的影响奠定了基础。

Objective： To establish a MDS mouse model with iron overload and to study the effect of iron overload on MDS. Methods： The exogenous mutant gene RUNX1-S291 fs was inserted into the mice bone marrow mononuclear cell＇s genome in mice by retrovirus and transplanted into C57 BL/6 mice irradiated by 60 Co γ-ray. After 8 weeks,intraperitoneal injection of iron was performed to establish an MDS mouse model with iron overload. After 24 weeks of transplantation,the peripheral blood,bone marrow,femur,liver and spleen of mice were taken,then the morphological characteristics of peripheral blood and bone marrow cells were observed by Wright＇s staining; the liver,spleen and bone marrow were stained with Prussian blue to observe the iron deposition. The surface antigens of bone marrow cells were detected by flow cytometry. Bone marrow mononuclear cells and spleen tissue proteins were detected by Western blot to confirm the transfection of RUNX1-S291 fs gene and expression of protein. The blood routine and transplanted cell chimeric rate of mice were monitored periodically. Results： Compared with the empty plasmid control mice,levels of leukocyte and hemoglobin as well as platelet were decreased in RUNX1-S291 fs mutant mice; the peripheral blood cells and bone marrow cells showed pathological hematopoiesis; the liver and spleen enlarged significantly; the tissue structure of femur,liver and spleen was abnormal; the expression of bone marrow cell surface antigens was abnormal. Bone marrow cells and spleen tissue expressed the RUNX1-S291 fs protein. Compared with the controlled mice injected with normal saline,iron deposition occurred in the bone marrow,liver and spleen stained with Prussian blue in the mice injected with iron agent. Conclusion： Mice engineered to carry exogenous mutant gene RUNX1-S291 fs and injected with iron showed pathologic features of MDS and iron overload,resulting in establishing MDS iron overloaded mouse model successfully,which lays a foundation for studying the effect of iron overload on MDS.