【摘要】 为探索与自身免疫病发病密切相关的B细胞活化因子(BAFF)基因高水平转录的启动序列及IFN-γ等炎性细胞因子对其活性的影响,以人BAFF基因转录起始点上游-1 349 bp至-329 bp的片段为靶序列,取长度不等的片段作为启动子与含氯霉素乙酰基转移酶(CAT)报告基因的质粒组成5个重组体,脂质体转染法转染上述重组体至人骨髓白血病细胞株:HL-60,CAT-ELISA测定细胞CAT表达水平以比较各重组体的启动子活性;同时加入细胞因子IL-10、IFN-γ、IL-4、重组人BAFF蛋白(rBAFF),以测定其对BAFF启动序列的影响。结果表明,-1 349~-329 bp、-1 349~-743 bp序列具有强启动调控活性,而-1 349~-1 099 bp片段启动活性最低。细胞因子IFN-γ、IL-10和rBAFF均能在一定程度上调人BAFF基因的启动活性,IL-4则一定程度地抑制BAFF基因的启动活性。研究提示,人BAFF基因-1349~-743 bp片段是进一步研究BAFF基因转录相关DNA结合蛋白的重要调控靶序列。细胞因子IL-10、IFN-γ、IL-4、rBAFF可以一定程度的在转录水平影响BAFF合成和分泌。更多还原

【Abstract】 To analyze sequences that may be important for the regulation of human B cell activating factor(BAFF) gene expression,clarify the fragment of high promoter activity of BAFF gene and explore the potential effects of cytokines on promoter activity,a functional human BAFF promoter with 1 020 base pairs(bp) was cloned and investigated with serial 3’-deletion.Five chimeric genes were constructed in which various lengths of sequences extending from-1 349 bp to-329 bp were fused to chloramphenicol acetyltransferase(CAT) reporter gene.These recombinant plasmids were transiently transfected to human HL-60 cell lines with liposomal transfection method.Cytokines including interferon-γ,interleukin-4,interleukin-10 and recombinant human BAFF(rhBAFF) were added to the culture medium of transiently transfected HL-60 cells.The putative promoters activities were tested and compared by CAT measurement in those transfected cells.The results showed that sequences of-1 349 to-329 bp and-1 349 to-743 bp of human BAFF gene had high activities as promoters.IFN-γ,IL-10,rhBAFF showed increased effects while IL-4 showed inhibitory effect on sequence-1 349 to-743 bp as promoter with activities 3.5-fold,2.3-fold,1.4-fold and 72% of control respectively.The results indicated that the 1 020 bp fragment of the BAFF promoter would serve as a valuable mechanistic tool for better understanding of the regulation of BAFF expression,and the high potential sequence-1 349 to-743 bp is of great significance in the futher study searching for specific cis-elements and consensus DNA-binding proteins in BAFF-producing cells.The enhancement effect of IFN-γ,IL-10,rhBAFF and inhibition effect of IL-4 on BAFF synthesis are associated with their transcriptional regulation.The promoter-reporter construct may also be used as a screening tool to identify inhibitors of BAFF expression.更多还原