Xu Si-Miao, Yang Yang, Zhou Mi, Zhao Xue-Jiao, Qin Yu, Zhang Pei-Ling, Yuan Rui-Feng, Zhou Jian-Feng, Fang Yong
Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Oct;21(5):1126-32. doi: 10.7534/j.issn.1009-2137.2013.05.008.
This study was purposed to establish a retrovirus-mediated murine model with MLL-AF9 leukemia, so as to provide a basis for further investigation of the pathogenesis and therapeutic strategy of MLL associated leukemia. Murine (CD45.2) primary hematopoietic precursor positively selected for expression of the progenitor marker c-Kit by means of MACS were transduced with a retrovirus carrying MLL-AF9 fusion gene. After cultured in vitro, the transduced cells were injected intravenously through the tail vein into the lethally irradiated mice (CD45.1). PCR, flow cytometry and morphological observation were employed to evaluate the murine leukemia model system. The results showed that MLL-AF9 fusion gene was expressed in the infected cells, and the cells had a dramatically enhanced potential to generate myeloid colonies with primitive and immature morphology. Flow cytometric analysis revealed that the immortalized cells highly expressed myeloid lineage surface markers Gr-1 and Mac-1. Moreover, the expression levels of Hoxa9 and Meis1 mRNA were significantly higher in the MLL-AF9 cells than that in control. The mice transplanted with MLL-AF9 cells displayed typical signs of leukemia within 6-12 weeks. Extensive infiltration leukemic cells was observed in the Wright-Giemsa stained peripheral blood smear and bone marrow, and also in the histology of liver and spleen. Flow cytometric analysis of the bone marrow and spleen cells demonstrated that the CD45.2 populations expressed highly myeloid markers Gr-1 and Mac-1. The leukemic mice died within 12 weeks. It is concluded that the retrovirus-mediated murine model with MLL-AF9 leukemia is successfully established, which can be applied in the subsequent researches.
本研究旨在建立一种逆转录病毒介导的MLL-AF9白血病小鼠模型，为进一步研究MLL相关白血病的发病机制和治疗策略提供依据。通过MACS对表达祖细胞标志物c-Kit的小鼠（CD45.2）原代造血前体细胞进行阳性选择，并用携带MLL-AF9融合基因的逆转录病毒进行转导。体外培养后，将转导细胞通过尾静脉注射到经致死剂量照射的小鼠（CD45.1）体内。采用PCR、流式细胞术和形态学观察对小鼠白血病模型系统进行评估。结果显示，MLL-AF9融合基因在感染细胞中表达，且这些细胞产生具有原始和未成熟形态的髓系集落的能力显著增强。流式细胞术分析显示，永生化细胞高表达髓系谱系表面标志物Gr-1和Mac-1。此外，MLL-AF9细胞中Hoxa9和Meis1 mRNA的表达水平明显高于对照组。移植了MLL-AF9细胞的小鼠在6-12周内出现典型的白血病症状。在瑞氏-吉姆萨染色的外周血涂片和骨髓中，以及肝脏和脾脏的组织学检查中均观察到白血病细胞的广泛浸润。对骨髓和脾脏细胞的流式细胞术分析表明，CD45.2群体高表达髓系标志物Gr-1和Mac-1。白血病小鼠在12周内死亡。结论是成功建立了逆转录病毒介导的MLL-AF9白血病小鼠模型，可应用于后续研究。
