热休克因子1对白细胞介素-10的转录调控作用及其机制

The transcription regulation mechanism of heat shock transcription factor 1 on IL-10

目的探讨热休克因子1（HSF1）对白细胞介素（IL）-10的转录调控作用及其机制。方法合成IL-10基因启动子区含热休克元件（HSE）位点的寡核苷酸探针进行凝胶电泳迁移率实验（EMSA），分析HSF1与IL-10基因启动子区的HSE的结合情况，并构建IL-10基因启动子的萤光素酶报告基因质粒，与HSF1表达质粒共转染RAW264．7细胞，检测萤光素酶活性，观察HSF1转染对启动子活性的影响。结果生物素标记的HSF1结合片段（-376～-369bp）和核蛋白提取物孵育后能观察到阻滞带，阻滞现象能够被自身非标记探针竞争，但不被非标记的突变探针竞争，加入HSF1单克隆抗体，可以观测到超阻滞带，表明HSF1可以特异性结合于“HSF1识别序列”，HSE核心结合位点突变后，相对萤光素酶活性突变体（34．234-2．14）相对野生型（110．09±5．48）下降3．2倍（P〈0．01），通过EMSA证实了IL-10启动子区域（-688-＋64bp）存在HSF1的结合位点HSE（-376--369bp）；突变体双萤光素酶活性分析发现HSF1的结合位点核心碱基的突变，其转录活性下降。结论HSF1可以特异性结合于IL-10启动子区HSE（-376--369bp），相对萤光素酶活性分析提示HSF1可以转录激活IL-10，上调其表达。

Objective To explore the transcription regulation molecular mechanism of heat shock transcription factor 1 （ HSF-1 ） up-regulating the expression of IL-10 mRNA. Methods The murine IL-10 promoter in RAW264.7 macrophages was analyzed by Matlnspector program to search for heat shock element （HSE）. The murine IL-10 promoter region （-668/＋64bp） was prepared by PCR amplification of RAW264.7 genomic DNA with specific primers, and purified PCR products of IL-10 promoter were end-labeled with biotin. Electrophoretic mobility shift assay （EMSA） was employed to analyze the binding activity of HSF1 and HSE. The luciferase reporter vectors were constructed, and the DNA fragments were inserted into luciferase plasmid pGL-3Basic to form plasmid （HSE-WT）. Mutated murine IL-10 promoter was prepared by PCR amplification of wild-type plasmid DNA with site-directed mutated primers. The mutated DNA fragments were also ligated into pGL-3Basic to form plasmid （HSE-Mut）. The mutation sites were confirmed by DNA sequencing. All of the plasmids for transfection were purified by using the Nucleo Bond endotdxin-free plasmid purification kit. Ceils were transfected with plasmids by lipofection according to the manufacturer＇ s instructions. Luciferase activity was measured by the luciferase assay system. Results EMSA demonstrated an increase in HSF1 binding activity with DNA, which showed the binding of HSFI to the HSE on the promoter of IL-10 gene （ -376- -369 bp）. Luciferase assay system demonstrated that the luciferase activities were decreased in mutated sequence of the IL-10 promoter （34.23±2.14） as com- pared with wild type （ 110.09 ± 5.48）. Conclusion HSF-1 may up-regulate the expression of IL-10 by binding of HSF-1 to the HSEs on the promoter of IL-10 gene and enhance IL-10 transcription activity.