多种细胞因子对转化生长因子β_1转录的调控作用

Regulatory effect of cytokines on promoter activity of human transforming growth factor-β_1 gene

目的:探讨细胞因子TNF-α、IFN-γ、IFN-α、PDGF-BB、bFGF对TGF-β1启动活性的影响。方法:以人基因组DNA为模板,PCR扩增获得含有人TGF-β1基因启动子序列的-1328～+812bp片段,与氯霉素乙酰基转移酶(CAT)报告基因构建重组体phTGF2.14,将其瞬时转染大鼠nFSC细胞,加入细胞因子进行干预,测定CAT活性。结果:10ng/mlTNF-α可以使重组体的CAT活性升高3.24倍;浓度为1000U/ml的IFN-γ、IFN-α使phTGF2.14的CAT活性分别降低至对照的(42±12)%、(58±6)%;浓度为10ng/ml的PDGF-BB、bFGF对TGF-β1基因启动子的活性没有影响;IFN-γ、IFN-α和TNF-α联合应用TGF-β1基因启动子的活性分别为对照的1.32和1.46倍。结论:TNF-α可促进TGF-β1基因启动子的活性;IFN-γ、IFN-α则对TGF-β1基因启动子的启动活性有抑制作用;IFN-γ、IFN-α可以阻断TNF-α对TGF-β1基因启动子的促进作用;PDGF-BB、bFGF对TGF-β1基因启动子的启动活性无影响,此研究为进一步研究细胞因子在纤维化疾病的相互作用机制提供了依据。

Objective：To study the effect of the cytokines, tumor necrosis factor-α（TNF-α）, interferon-γ（IFN-γ）, interferon-α （IFN-α）, platelet-derived growth factor BB（PDGF-BB）, basic fibroblast growth factor（ bFGF）, on promoter activity of human transfor- ming growth factor-[31 （TGF-[31） gene. Methods：A construct of phTGF2.14 containing sequence from - 1 328 bp to ＋812 bp of human TGF--β1; gene,linking with chloramphenicol acetyltransferase（CAT） as reporter gene, was transiently transfected into rat hepatic stellate cell line nFSC. The cells were subsequently treated with TNF-α, IFN-γ, IFN-α, PDGF-BB, bFGF, and the CAT activity was assessed 48 hours after stimulation with each cytokines. Results：TNF-α of 10 ng/ml can increased the CAT activity of phTGF2.14 to 3.24 fold compared to control. IFN-β1 and IFN-α at 1 000 U/ml decreased CAT activity to （42 ±12）% and （58 ：t：6）% of control respectively. PDGF- BB,bFGF of 10 ng/ml had no effect on promoter activity of human TGF-β1 gene;Combined application of IFN-γ,IFN-α and TNF-α,the promoter TGF-β1 gene were 1.32 and 1.46 fold compared with control,respectively. Conclusion：These data indicate that TNF-α can increase the promoter activity of human TGF-β1 gene, but IFN-γ, IFN-α can downregulate the CAT activites of phTGF2.14, and IFN-γ, IFN-α can interdict the upregulate effect of TNF-α on phTGF2.14. We did not find PDGF-BB,bFGF have any effect on TGF-β1 promoter. These provided an essential evidence for study the interaction mechanism of cytokines in fibrogenisis diseases.