中文摘要：

探讨信号转导和转录激活因子3（signal transducer and activator of transcription 3，STAT3）与丝裂原活化蛋白激酶（mitogen—activated protein kinase，MAPK）在体内是否存在相互作用，并观察其作用如何影响肿瘤坏死因子-α（tumor necrosis factor，TNF—α）的转录活性．采用聚合酶链反应技术，从人Flag—p38和Flag-细胞外信号蛋白调节激酶2（extracellular-signal regulated protein kinase2，ERK2）中扩增出p38和ERK2基因，将其插入载体pcDNA3-HA中；用Westernblot方法在293T细胞中检测其表达后应用免疫共沉淀技术检测STAT3蛋白与p38／ERK2蛋白之间是否存在相互作用．然后应用报告基因技术检测这种相互作用如何影响TNF-α的转录表达，并在应用RNA干扰技术将STAT3通路抑制后，观察TNF—α启动子转录活性如何变化．酶切和测序结果表明，扩增的p38和ERK2基因序列正确，大小为1080bp，在293T细胞中正确表达大小约40ku的p38和ERK2蛋白．免疫共沉淀实验结果证实，p38和STAT3蛋白以及ERK2和STAT3蛋白在体内相互作用．下游基因TNF—α荧光素酶活性实验显示，p38和STAT3蛋白以及ERK2和STAT3蛋白复合物协同升高TNF-α的活性，应用STAT3的干扰RNA后其活性则明显下降．该研究表明，STAT3和p38／ERK2蛋白在体内存在相互作用，STAT3与p38、STAT3与ERK2均对TNF-α的表达发挥协同效应，在阻断STAT3通路后，STAT3与p38、STAT3与ERK2对TNF-α表达的协同效应将明显降低．

英文摘要：

In order to investigate if there exists interaction between mitogen-activated protein kinase （MAPK） and signal transducer and activator of transcription 3 （STAT3） protein, and how the interaction regulates tumor necrosis factor-α （TNF-α） transcription activity, the human p38 and extracellular-signal regulated protein kinase 2 （ERK2） genes were amplified from human flag-p38 and flag-ERK2 by polymerase chain reaction （PCR） and cloned into pcDNA3-HA. Protein expression of the plasmids was examined by Western blotting. Co-immunoprecipitation was used to identify if there exists interaction between MAPK and STAT3 proteins. If the interaction was approved to be true, report gene system was applied to find how the interaction affect transcriptional expression of TNF-α. After STAT3 pathway was inhibited by RNA interfering, the action on TNF-α activity was determined. The results of DNA sequencing and enzyme digestion showed that the cloned p38 and ERK2 genes were correct, to be 1 080 bp or so. p38 and ERK2 proteins were expressed in 293T cell to be approximately 40 ku. Co-immunoprecipitation data showed that p38 and ERK2. proteins integrated with STAT3 protein in vivo. TNF-α reporter gene activity results found that protein complex ofp38-STAT3 and ERK2-STAT3 coordinately increased TNF-α activity. After STAT3 was interfered, the TNF-α activity markedly decreased. These data indicated that there exists interaction between p38 and STAT3 protein, ERK2 and STAT3 protein. The complex of the proteins can coordinately regulate TNF-α expression. After interfereing STAT3 pathway, the coordinated action on TNF-α transcription activity might be obviously reduced.