Li Zheng, Hu Shao-Yan, Cen Jian-Nong, Chen Zi-Xing
Department of Hematology, The Affiliated Children Hospital of Soochow University, Jiangsu Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Jun;21(3):576-80. doi: 10.7534/j.issn.1009-2137.2013.03.008.
In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.
为研究慢病毒载体应用于急性髓系白血病的潜力，采用磷酸钙共沉淀法将重组载体Venus-C3aR转染至293T包装细胞。收集所有病毒原液并转染至HL-60细胞，通过流式细胞术检测HL-60细胞中的绿色荧光蛋白（GFP）表达。采用逆转录-聚合酶链反应（RT-PCR）和流式细胞术鉴定转染后HL-60细胞中C3aR1的表达水平。使用细胞计数试剂盒-8（CCK-8）法检测慢病毒对HL-60的毒性，并观察细胞分化能力。结果表明，慢病毒载体对HL-60细胞的转染效率超过95%，满足进一步研究的需求。用重组慢病毒载体转染后，HL-60细胞上C3aR1表达增加。转染前后，细胞的增殖和分化变化不大。结论是，慢病毒载体对急性髓系白血病（AML）细胞具有高效转染能力，可整合到HL-60细胞基因组中实现目的基因的稳定表达。同时，慢病毒载体不影响HL-60细胞的增殖和分化能力。
