慢病毒介导的稳定表达人GM-CSF、IL-4的EA.hy926细胞株的建立

Construction and Identification of Cell Line to Coexpress GM-CSF and IL-4 Mediated by Lentiviral System

目的:获得能够稳定表达人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)的内皮细胞株。方法:构建重组慢病毒表达载体,在293FT细胞中进行病毒的包装,用获得的高滴度慢病毒感染EA.hy926细胞株,建立能够稳定、高效表达hGM-CSF、hIL-4的EA.hy926细胞株。结果:EA.hy926感染效率达90%以上,长期传代后阳性率仍维持在90%以上;实时定量PCR和ELISA方法证实感染后细胞hGM-CSF、hIL-4在mRNA水平的表达分别是未感染细胞的25倍和9946倍,在蛋白水平的表达分别是未感染细胞的145倍和23倍。结论:构建的细胞系可以稳定表达细胞因子GM-CSF、IL-4。

Objective： To establish the EA.hy926 cell line expressing granulocyte-macrophage colony stimulating factor（GM-CSF） and interleukin 4（IL-4） stably mediated by lentiviral system.Methods： The recombinant lentiviral plasmid carrying human GM-CSF and IL-4 was transfected into 293FT cells with lentiviral packaging plasmids.Then the infectious lentiviral was obtained,which infected the EA.hy926 cells.The expression of GM-CSF and IL-4 was assayed by qPCR and ELISA.Results： The GM-CSF and IL-4 expression at mRNA level of infected EA.hy926 cells were 24 and 9946 fold respectively for the FLSC not infected by the lentivirus,and then at the protein level were 145 and 23 fold.Conclusion： The infected EA.hy926 cells can stably express GM-CSF and IL-4.