【作者】 刘洋； 张继增； 张宗德； 王甦民； 傅瑜；

【Author】 * Department of Molecular Biology,Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149,China LIU Yang,ZHANG Ji-zeng,WANG Su-min,FU Yu,ZHANG Zong-de.

【机构】 北京市结核病胸部肿瘤研究所结核病分子生物学研究室； 北京结核病控制研究所； 中国疾病预防控制中心结核病防治临床中心；

【摘要】 目的研究活动性肺结核患者环腺苷酸反应元件结合蛋白（cyclic adenosine monophosphateresponse element-binding protein,CREB）与γ-干扰素基因近端启动子的关系。方法 2007年1—12月北京胸科医院结核科收治的25例肺结核患者（肺结核组）和18例PPD阳性健康人（对照组）为研究对象。分离外周血中CD₃⁺T细胞,采用凝胶电泳迁移率变化（EMSA）和竞争性EMSA分析CREB及γ-干扰素基因近端启动子结合情况,染色质免疫共沉淀（ChIP）技术研究MTB抗原在体内状态下能否诱导CREB产生并与γ-干扰素基因近端启动子结合。Western blot法检测CREB表达水平及MTB抗原诱导磷酸化CREB蛋白的表达。结果肺结核组25例中有18例缺失低迁移率条带,说明其缺少与γ-干扰素基因近端启动子结合的蛋白,竞争性EMSA试验结果证实该蛋白复合体中含CREB;ChIP试验对照组中10例有204 bp的PCR产物,肺结核组中有12例缺失该产物,提示其缺少与γ-干扰素基因近端启动子结合的CREB;westernblot证实肺结核组中20例未见CREB表达,并且所有25例肺结核组CD3+T细胞在MTB抗原刺激时不能诱导磷酸化CREB蛋白的产生。结论 CREB蛋白可与γ-干扰素基因近端启动子区结合,肺结核患者缺少与γ-干扰素基因近端启动子结合的CREB蛋白。更多还原

【Abstract】 Objective To study the relationship between cAMP response element binding protein（CREB） and the IFN-γproximal promoter.Methods CD₃⁺ T cells were isolated from 25 pulmonary tuberculosis patients, who had been treated in Beijing Chest Hospital from January to December 2007,and18 PPD-positive healthy donors.After extraction of nuclear proteins,electrophoretic mobility shift assay（EMSA） was performed to determine nuclear proteins binding to the IFN-γproximal promoter in vitro,and the specificity of binding complex was tested by competitive EMSA.Chromatin immunoprecipitation（ChIP） with anti-CREB Ab was used to determine whether CREB bind to the IFN-γproximal promoter in vivo in live T cells exposed to microbial Ags. Western blotting with anti-CREB Ab was performed to compare the expression level of CREB in tuberculosis patients and PPD-positive healthy donors.Western blotting with Abs specific for serine 133-phosphorylated CREB was performed to determine whether M.tuberculosis Ags elicited phosphorylation of CREB.Results The results of EMSA showed a low-mobility complex binding to the IFN-γpromoter,the binding pattern observed was similar for T cell from all 18 PPD-posotive healthy donors.However,for T cells from 18 of 25 tuberculosis patients,the low-mobility complex was absent.It suggested that DNA-binding proteins to the IFN-γpromoter reduced in tuberculosis patients.The results of competitive EMSA suggested these nuclear proteins specifically bound to the IFN-γpromoter region and contained CREB.The results of ChIP showed a 204bp band yielded in CD₃⁺ T cells from 10 PPD-positive healthy donors,but 12 tuberculosis patients didn’t yield the band.CREB expression markedly decreased in tuberculosis patients compared with healthy donors detected by western blotting.Furthermore,M.tuberculosis Ags also elicited phosphorylation of CREB in CD₃⁺ T cells from PPD-positive healthy donors,but not in CD₃⁺ T cells from tuberculosis patients.Conclusion CREB protein binding to IFN-γproximal promoter was reduced in tuberculosis patients compared with healthy donors. Tuberculosis patients had diminished CREB protein levels,reduced ability of binding to the IFN-γpromoter.更多还原