CAJ | 학술논문

目的探讨热休克因子1(HSFl)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HSFl结合片段(-376～- 369 bp)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HSFl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"HSFI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(34.23±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～+64 bp)存在HSF1的结合位点HSE(-376～-369 bp);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～-369 bp),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.
목적 탐토열휴극인자1(HSFl)대백세포개소(IL)-10적전록조공작용급기궤제.방법 합성IL-10기인계동자구함열휴극원건(HSE)위점적과핵감산탐침진행응효전영천이솔실험(EMSA),분석HSF1여IL-10기인계동자구적HSE적결합정황,병구건IL-10기인계동자적형광소매보고기인질립,여HSF1표체질립공전염RAW264.7세포,검측형광소매활성,관찰HSF1전염대계동자활성적영향.결과 생물소표기적HSFl결합편단(-376～- 369 bp)화핵단백제취물부육후능관찰도조체대,조체현상능구피자신비표기탐침경쟁,단불피비표기적돌변탐침경쟁,가입HSFl단극륭항체,가이관측도초조체대,표명HSF1가이특이성결합우"HSFI식별서렬",HSE핵심결합위점돌변후,상대형광소매활성돌변체(34.23±2.14)상대야생형(110.09±5.48)하강3.2배(P<0.01),통과EMSA증실료IL-10계동자구역(-688～+64 bp)존재HSF1적결합위점HSE(-376～-369 bp);돌변체쌍형광소매활성분석발현HSF1적결합위점핵심감기적돌변,기전록활성하강.결론 HSF1가이특이성결합우IL-10계동자구HSE(-376～-369 bp),상대형광소매활성분석제시HSF1가이전록격활IL-10,상조기표체.
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 MatInspector program to search for heat shock ele-ment (HSE).The murine IL-IO 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-la-beled with biotin.Electrophoretic mobility shift assay (EMSA) was employed to analyze the binding activi-ty of HSF1 and HSE.The luciferase reporter vectors were constructed, and the DNA fragments were insert-ed 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.Cells 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 HSFI binding activity with DNA,which showed the binding of HSF1 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-IO 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.