论文部分内容阅读
目的:探讨IFN-γ和NF-κB抑制剂对人BAFF-R基因启动子活性的影响。方法:以人全血基因组DNA为模板,PCR方法扩增得到BAFF-R基因转录起始点上游5’端-1562~+261 bp的片段,与荧光素酶报告基因载体pGL3-Basic连接构建8个序列缺失质粒。将重组质粒瞬时转染人多发性骨髓瘤细胞株KM3,通过检测荧光素酶的相对活性,确定启动子活性最强的区域;分别用NF-κB抑制剂(BAY11-7082)和IFN-γ对活性最强的启动子进行干预,测定并比较荧光素酶的相对活性;同时RT-PCR检测干预前后BAFF-R基因mRNA的表达水平。结果:IFN-γ对BAFF-R基因启动子活性有促进作用并且可以上调BAFF-R mRNA的表达,NF-κB抑制剂(BAY11-7082)对BAFF-R基因启动子活性有抑制作用并且可以下调BAFF-R mRNA的表达。结论:IFN-γ和NF-κB信号通路参与了BAFF-R基因的转录调控,为进一步研究BAFF-R的调节机制提供了基本的依据。
Objective: To investigate the effects of IFN-γ and NF-κB inhibitors on the promoter activity of human BAFF-R gene. Methods: Human whole blood genomic DNA was used as a template to amplify the 5’-end to -1562 to +261 bp upstream of the BAFF-R gene transcription initiation site by PCR and ligated with luciferase reporter gene vector pGL3-Basic to construct 8 Sequence deletion plasmid. The recombinant plasmids were transiently transfected into human multiple myeloma cell line KM3, and the most active region of the promoter was determined by detecting the relative luciferase activity. NF-κB inhibitor (BAY11-7082) and IFN-γ The most active promoter was intervened to determine and compare the relative luciferase activity. At the same time, the mRNA expression of BAFF-R before and after intervention was detected by RT-PCR. Results: IFN-γ promoted the promoter activity of BAFF-R gene and up-regulated the expression of BAFF-R mRNA. The inhibitor of NF-κB (BAY11-7082) inhibited the promoter activity of BAFF-R gene and down-regulated BAFF-R mRNA expression. CONCLUSION: The IFN-γ and NF-κB signaling pathways are involved in the transcriptional regulation of BAFF-R gene, providing a basic basis for further study on the regulatory mechanism of BAFF-R.