Xu Fa-Mei, Xing Hai-Yan, Tian Zheng, Tang Ke-Jing, Rao Qing, Wang Min, Wang Jian-Xiang
Chinese Academy of Medical Sciences, Tianjin, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2011 Dec;19(6):1477-81.
This study was aimed to explore the role and mechanism of AML1a in abnormal hematopoiesis in mice. Plasmids pMSCV-FLAG-AML1a-IRES-YFP and pMSCV-IRES-YFP together with envelope-encoding plasmid pECO and packaging plasmid pGP were respectively transfected into 293T cells by using a method of calcium phosphate precipitation to produce retrovirus. Bone marrow mononuclear cells (BMMNC) from male C57BL/6J mice were transfected with the retroviral vector MSCV expressing FLAG-AML1a fusion protein and yellow fluorescent protein (YFP). The cells were cultured in M3434 semi-solid medium for colony formation assay and in M5300 fluid medium containing murine IL-3 (mIL-3), IL-6 (mIL-6) and SCF (mSCF) for long-term culture. The results showed that transfection of AML1a into BMMNC enhanced colony formation, colony size of the AML1a group was significantly larger than that of the control group, and the colonies were mainly composed of CFU-E and CFU-GEMM. In the long-term culture, AML1a-transfected BMMNC showed differentiation block, while the control cells were in a more mature stage. It is concluded that AML1a may block the normal hematopoiesis at the stage of primitive progenitors. At the same time, AML1a also enhances the proliferation activity of primitive progenitor cells.
本研究旨在探讨AML1a在小鼠异常造血中的作用及机制。采用磷酸钙沉淀法将质粒pMSCV-FLAG-AML1a-IRES-YFP和pMSCV-IRES-YFP分别与包膜编码质粒pECO和包装质粒pGP共转染至293T细胞中以产生逆转录病毒。用表达FLAG-AML1a融合蛋白和黄色荧光蛋白（YFP）的逆转录病毒载体MSCV转染雄性C57BL/6J小鼠的骨髓单个核细胞（BMMNC）。将细胞接种于M3434半固体培养基中进行集落形成试验，并接种于含小鼠白细胞介素-3（mIL-3）、白细胞介素-6（mIL-6）和干细胞因子（mSCF）的M5300液体培养基中进行长期培养。结果显示，将AML1a转染至BMMNC中可增强集落形成，AML1a组的集落大小显著大于对照组，且集落主要由红系集落形成单位（CFU-E）和粒-红-巨噬-巨核系集落形成单位（CFU-GEMM）组成。在长期培养中，转染AML1a的BMMNC表现出分化阻滞，而对照细胞处于更成熟阶段。结论为，AML1a可能在原始祖细胞阶段阻断正常造血。同时，AML1a还增强了原始祖细胞的增殖活性。
