CAJ | 학술논문

目的研究活动性肺结核患者环腺苷酸反应元件结合蛋白(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+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CREB磷酸化.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;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;肺结核组有20例未见CREB表达,且所有病例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.
목적 연구활동성폐결핵환자배선감산반응원건결합단백(CREB)여γ-간우소기인근단계동자적관계.방법 2007년1-12월북경흉과의원결핵과수치적25례폐결핵환자(폐결핵조)화18례PPD양성건강인(대조조)위연구대상.분리외주혈중CD3+세포,채용응효전영천이솔변화(EMSA)화경쟁성EMSA분석CREB급γ-간우소기인근단계동자결합정황,염색질면역공침정(ChIP)기술연구MTB항원재체내상태하능부유도CREB산생병여γ-간우소기인근단계동자결합.Western blot법검측CREB표체수평급MTB항원유도CREB린산화.결과 폐결핵조25례중유18례결실저천이솔조대,설명기결소여γ-간우소기인근단계동자결합적단백,경쟁성EMSA시험결과증실해단백복합체중함CREB;대조조중10례유204 bp적PCR산물,폐결핵조중유12례결실해산물,제시기결소여γ-간우소기인근단계동자결합적CREB;폐결핵조유20례미견CREB표체,차소유병례CD3+T세포재MTB항원자격시불능유도린산화CREB단백적산생.결론 CREB단백가여γ-간우소기인근단계동자구결합,폐결핵환자결소여γ-간우소기인근단계동자결합적CREB단백.
Objective To study the relationship between cAMP response element binding protein (CREB) and the interferon-γ (IFN-γ) proximal promoter in patients with tuberculosis. Methods CD3+ T cells were isolated from 25 pulmonary tuberculosis patients, who had been treated in Beijing Chest Hospital from January to December 2007, and 18 PPD-positive healthy donors. After extraction of nuclear proteins, electrophoretic mobility shift assay (EMSA) was performed to determine nuclear protein binding to the IFN-γ proximal 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 binded to the IFN-γ proximal 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-γ promoter, and the binding pattern observed was similar for T cells from all 18 PPD-positive healthy donors. However, for T cells from 18 of 25 tuberculosis patients, the low-mobility complex was absent The results of competitive EMSA showed that these nuclear proteins specifically bound to the IFN-γ promoter region and contained CREB. The results of ChIP showed a 204 bp 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.Conclusions CREB protein binding to IFN-γ proximal promoter was reduced in tuberculosis patients compared with healthy donors. Tuberculosis patients had diminished CREB protein levels, and reduced ability of binding to the IFN-γ promoter.