Zhao Xin, Xing Hai-Yan, Tian Zheng, Tang Ke-Jing, Wang Min, Rao Qing, Wang Jian-Xiang, Zhang Feng-Kui
State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, Tianjin Province, China.
State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, Tianjin Province, China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2014 Jun;22(3):612-6. doi: 10.7534/j.issn.1009-2137.2014.03.007.
This study was aimed to explore the role and mechanism of IFN-γ in the regulation of hemopoiesis in mice. Murine IFN-γ fragment was amplified from murine splenic cells with RT-PCR and plasmid pCDH1-mIFN-γ-EF1-copGFP (pCDH-mIFN-γ-GFP) was constructed. Plasmids pCDH-mIFN-γ-GFP and pCDH1-EF1-copGFP (pCDH-GFP) together with packaging plasmids pPACK-A, pPACK-B and pPACK-C were respectively transfected into 293T cells by using a method of calcium phosphate precipitation to produce lentivirus. Bone marrow mononuclear cells (BMMNC) from male C57BL/6J mice were transfected with the lentiviral vector pCDH expressing mIFN-γ and green fluorescent protein (GFP). The cells were cultured in M3434 semi-solid medium for colony formation assay and transplanted into lethally-irradiated mice through caudal vein injection, and the peripheral blood cell counts and GFP were monitored regularly after transplantation. The results showed that lentiviral vector pCDH-mIFN-γ-GFP was constructed successfully and 293T cells transfected with mIFN-γ secreted mIFN-γ. Transfection of mIFN-γ into BMMNC decreased colony formation, colony number of the mIFN-γ group was significantly less than that of the control group. The recovering of circulating blood cell parameters in mIFN-γ transplantation group was significantly later than control group. GFP positive cells could be detected in the peripheral blood at 8 weeks after transplantation. It is concluded that mIFN-γ may inhibit the colony-forming capacity of transduced BMMNC and delay the hematopoietic reconstitution.
本研究旨在探讨干扰素-γ（IFN-γ）在小鼠造血调控中的作用及机制。采用逆转录聚合酶链反应（RT-PCR）从小鼠脾细胞中扩增出小鼠IFN-γ片段，并构建质粒pCDH1-mIFN-γ-EF1-copGFP（pCDH-mIFN-γ-GFP）。采用磷酸钙沉淀法将质粒pCDH-mIFN-γ-GFP和pCDH1-EF1-copGFP（pCDH-GFP）分别与包装质粒pPACK-A、pPACK-B和pPACK-C共转染至293T细胞中以产生慢病毒。将表达mIFN-γ和绿色荧光蛋白（GFP）的慢病毒载体pCDH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，并通过尾静脉注射移植到经致死剂量照射的小鼠体内，移植后定期监测外周血细胞计数和GFP。结果显示成功构建了慢病毒载体pCDH-mIFN-γ-GFP，转染mIFN-γ的293T细胞分泌mIFN-γ。将mIFN-γ转染至BMMNC中可降低集落形成，mIFN-γ组的集落数显著低于对照组。mIFN-γ移植组循环血细胞参数的恢复明显晚于对照组。移植后8周在外周血中可检测到GFP阳性细胞。结论是mIFN-γ可能抑制转导的BMMNC的集落形成能力并延迟造血重建。
