细胞因子IFN-γ、IL-10通过转录调控促进人骨髓白血病细胞HL-60对B细胞活化因子的表达

Effect of HL-60 cells stimulated by IFN-γ and IL-10 to express B cell activating factor via transcriptional pathway

目的探讨常见炎性细胞因子IFN-γ、IL-10和IL-4对人骨髓白血病细胞HL-60中B细胞活化因子（BAFF）表达的影响及其可能的分子调控机制，为研究BAFF异常表达调控在骨髓白血病发病过程中的作用提供实验依据。方法以细胞因子IFN-γ、IL-10和IL-4刺激体外培养的HL-60细胞1～3d后，流式细胞术、ELISA和荧光定量RT-PCR方法检测BAFF的核酸和蛋白表达含量的变化。并对人BAFF基因上游的启动子序列（-1349～-329bp）进行5’系列基因缺失研究，组成5个不同长度的含报告基因的重组体，瞬时转染至HL-60细胞，经上述细胞因子干预后，报告基因测活技术观察各重组体启动转录活性的变化。结果细胞因子IFN-γ、IL-10干预后，HL-60细胞中BAFF的核酸和蛋白表达含量均明显升高（P〈0．05）；IFN-γ、IL-10均能明显上调人BAFF基因启动子的转录活性（P〈0．05），且其在转录水平的有效调控区域为序列-929～-719bp。结论细胞因子IFN-γ、IL-10可能通过上调BAFF基因转录来促进HL-60细胞对BAFF核酸和蛋白的表达。

Objective To explore the effect of IFN-γ,IL-10 and IL-4 on B cell activating factor （BAFF） expression in human HL-60 cells, a kind of myeloid tumor cell lines, and its possible regulation mechanism. Methods Cultured human HL-60 cells were treated with IFN-γ, IL-10 and IL-4 for 1-3 days. The expression of membrane-bound BAFF on HL-60 cells was examined by flow cytometry, the amount of soluble BAFF was detected by ELISA assay, and the level of BAFF mRNA was tested by real-time PCR method. A functional 1021 bp fragment of the 5＇-flanking region of the human BAFF gene （-1349 to -329 bp） was cloned and investigated with serial 5＇-deletion. The 5＇-deleted promoters were recombinated with chloramphenicol acetyhransferase （CAT） as reporter gene. These five recombinant plasmids were transiently transfected to HL-60 cells with liposomal transfection method. Promoters activities were determined by CAT reporter gene assay（CAT-ELISA） in those transfected cells treated with different cytokines. Results The results showed that the expression of membrane-bound BAFF, soluble BAFF and BAFF mRNA in human HL-60 cells were significantly elevated （P〈0.05） after incubated with IFN-γ and IL-10. In addition, IFN-γ and IL-10 showed significantly （ P 〈 0.05 ） increased effects on promoter activity in human BAFF gene. And the cytokines-responsive sequences were located between -929 and -719 bp of the BAFF promoter region. Conclusion The enhancement of IFN-γ and IL-10 on BAFF expression and synthesis were regulated by promoter activation. Our in vitro studies also raise the possibility to investigate the mechanisms regulating BAFF expression in other tumor cells of myeloid origin under pathological circumstances.