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...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 kinase 2,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.展开更多
Objective： To explore the role that ceramide plays in the activation of mitogen-activated protein kinases （MAPKs） during cerebral ischemia and reperfusion. Methods： Rats were subjected to ischemia by the fourvesse...Objective： To explore the role that ceramide plays in the activation of mitogen-activated protein kinases （MAPKs） during cerebral ischemia and reperfusion. Methods： Rats were subjected to ischemia by the fourvessel occlusion （4-VO） method. The sphingomyelinase inhibitor TPCK was administered to the CA1 subregion of the rat hippocampus before inducing ischemia. Western blot was used to examine the activity of extracellular- signal regulated kinase （ERK） and c-Jun N-terminal protein kinase （JNK） using antibodies against ERK, JNK and diphosphorylated ERK and JNK. Results： At lh reperfusion post-ischemia, JNK reached its peak activity while ERK was undergoing a sharp inactivation （P 〈 0.05）. The level of diphosphorylated JNK was significantly reduced but the sharp inactivation of ERK was visibly reversed （P 〈 0.05） by the sphingomyelinase inhibitor. Conclusion： The ceramide signaling pathway is up-regulated through sphingomyelin hydrolysis in brain ischemia, promoting JNK activation and suppressing ERK activation, culminating in the ischemic lesion.展开更多
文摘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 kinase 2,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.
基金supported by grants from the National Natural Science Foundation of China (No.30871200)the Practice and Innovation Training Program for Students in Colleges and Universities of Jiangsu Province (NO.20090370)
文摘Objective： To explore the role that ceramide plays in the activation of mitogen-activated protein kinases （MAPKs） during cerebral ischemia and reperfusion. Methods： Rats were subjected to ischemia by the fourvessel occlusion （4-VO） method. The sphingomyelinase inhibitor TPCK was administered to the CA1 subregion of the rat hippocampus before inducing ischemia. Western blot was used to examine the activity of extracellular- signal regulated kinase （ERK） and c-Jun N-terminal protein kinase （JNK） using antibodies against ERK, JNK and diphosphorylated ERK and JNK. Results： At lh reperfusion post-ischemia, JNK reached its peak activity while ERK was undergoing a sharp inactivation （P 〈 0.05）. The level of diphosphorylated JNK was significantly reduced but the sharp inactivation of ERK was visibly reversed （P 〈 0.05） by the sphingomyelinase inhibitor. Conclusion： The ceramide signaling pathway is up-regulated through sphingomyelin hydrolysis in brain ischemia, promoting JNK activation and suppressing ERK activation, culminating in the ischemic lesion.
