中文摘要：

脓毒症早期内毒素（脂多糖,LPS）可激活丝裂原活化蛋白激酶通路,诱导肿瘤坏死因子α（TNF-α）等多种炎性细胞因子大量生成.TNF-α作为一种重要的早期炎症介质,间接激活Janus激酶/信号转导和转录激活因子（JAK/STAT）通路.STATs活化后直接进入核内参与LPS诱导的基因表达.本研究拟探讨STAT3在TNF-α启动子上的作用位点,为深入认识JAK/STAT信号通路在脓毒症发生中的分子基础及其干预途径提供理论依据.a.将Flag-STAT3与全长TNF-α启动子报告基因共同转染COS-7细胞,观察STAT3作用的剂量-效应.结果证实,STAT3对TNF-α基因的表达不受LPS调节,无论是否有LPS刺激,随着STAT3剂量的增加,TNF-α基因的表达亦随之增加.在将200μg/L浓度的Flag-STAT3质粒分别与不同长度的TNF-α启动子报告基因质粒共同转染COS-7细胞并用LPS刺激,观察不同片段的TNF-α启动子活性.结果发现,95 bp片段的TNF-α缺失突变体作用最为明显,增加了6.9倍.b.分别构建了70 bp、75 bp、80 bp和85 bp片段的TNF-α缺失突变体———pTNF-α（70 bp）、pTNF-α（75 bp）、pTNF-α（80 bp）和pTNF-α（85 bp）,将它们与Flag-STAT3共同转染COS-7细胞并用LPS刺激,观察到85 bp片段的活性与95 bp片段相似,当片段到达80 bp时,活性降低到对照水平.为了进一步了解其精确结合位点,将85 bp片段突变体的81和82位碱基突变,检测其对TNF-α活性影响,发现启动子活性下降.c.为了证明STAT3的潜在结合位点在80 bp到85 bp之间,且81和82位这2个位点的突变确实改变了STAT3对TNF-α启动子活性,进行凝胶迁移阻滞实验,观察到TNF-α启动子62～85 bp的野生型γ-32P标记的探针可以与核蛋白STAT3结合,而突变的62～85 bp标记的探针不能与核蛋白STAT3结合.上述结果表明,STAT3对TNF-α基因表达的调节不依赖LPS的刺激,STAT3在TNF-α启动子的潜在结合位点是在80到85碱基片段之间.

英文摘要：

At the early stage of sepsis,it is clear that tumor necrosis factor alpha（TNF-α） will be excessively produced after mitogen-activated protein kinase is activated by lipopolysaccharide（LPS）.TNF-α,which is an important early inflammatory cytokine,activates indirectly signal transducer and activator of transcription （STAT3） pathways.Activated STATs enter into the nucleus to take part in the gene expression induced by LPS. The present study was performed to explore the potential sites of STAT3 binding to TNF-α promoter.The purpose is to provide the theory basis of understanding molecular mechanism and intervention pathway of JAK/STAT signal pathway in the development of sepsis.In the present study,dose-dependent response of STAT3 on TNF-α expression was observed after Flag-STAT3 and full length TNF-α promoter reporter gene were co-transfected into COS-7 cells.The results indicated that TNF-α gene expression was enhanced along with increased doses of STAT3 with or without LPS.Flag-STAT3（200 μg/L） and TNF-α promoter reporter gene of different length were co-transfected into COS-7 cells respectively,and LPS was added into cells 4 hours later.Compared to the controls, fold activity value of pTNF-α（95 bp） was found to be the raised 6.9-fold.Then,pTNF-α（70 bp）,pTNF-α（75 bp）, pTNF-α（80 bp） and pTNF-α（85 bp） deletion mutants were constructed and co-transfected with Flag-STAT3 （200 μg/L） to COS-7 cells induced by LPS.Data showed that fold activity value of pTNF-α（85 bp） was similar to that of pTNF-α（95 bp）,and fold activity value of pTNF-α（80 bp） decreased to the control levels.81 base and 82 base in the TNF-α promoter were mutated and then the activity results proved that the two sites were important to the TNF-α gene expression induced by STAT3.The potential binding site of STAT3 on TNF-α promoter was identified to be between 80 bp and 85 bp.Furthermore,the results of EMSA showed that it was wild probe tagged with γ-32P of the TNF-α promoter 62～85 b