【作者】 刘洋；

【导师】 王甦民； 傅瑜； 张宗德；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 免疫学， 2008， 博士

【摘要】 研究背景:结核病仍然是世界所面临的主要卫生问题,全球约有二十亿人感染了结核分枝杆菌。我国的结核病疫情在全球属高流行地区,根据2000年全国结核病流行病学抽样调查显示,我国结核病疫情主要有以下特点:1)患病率高,活动性肺结核患病率为367/10万,涂阳患病率为122/10万;2)耐药率高,初始耐药率为18.6%,获得性耐药率为46.5%;3)全人口感染率高,为44.5%,我国约有5.5亿人感染了结核杆菌。结核病的治疗和预防面临严峻的考验。因此,研究新的、有效的防治策略控制结核病迫在眉睫。结核分枝杆菌是典型的胞内寄生菌,结核分枝杆菌感染巨噬细胞后,可在宿主细胞内繁殖,裂解细胞或处于潜伏状态。IFN-γ是机体抵抗细胞内致病菌关键的细胞因子,由CD4+和CD8+的T淋巴细胞及NK细胞产生。IFN-γ是一种重要的巨噬细胞启动因子,IFN-γ活化巨噬细胞,启动巨噬细胞的氧依赖性和氧非依赖性杀菌系统,使巨噬细胞获得杀灭细胞内寄生菌的能力,同时其抗原递呈能力进一步加强,使机体扩大抗结核的免疫效能。IFN-γ基因的转录由IFN-γ近端启动子区(-73 bp ~ -48 bp)调控,特异的蛋白与启动子区结合将调节启动子的活性和IFN-γ的分泌。环磷酸腺苷反应元件结合蛋白(CREB),是一种真核细胞转录因子,与IFN-γ近端启动子区结合,调节IFN-γ基因的转录。CREB蛋白的磷酸化会促进这种结合。目前,针对IFN-γ转录调控机制的研究刚刚起步,尚不清楚CREB蛋白在IFN-γ转录分泌调节中的作用,CREB蛋白在机体对结核菌的保护性免疫中的作用有待进一步阐明。对CREB蛋白在IFN-γ的转录、分泌调节中作用的研究,将为利用CREB蛋白防治结核病提供实验依据。研究目的:通过CREB蛋白与IFN-γ近端启动子区结合的研究,探讨CREB蛋白对IFN-γ转录的调节作用;比较结核病患者和PPD阳性健康对照人群血液中CREB蛋白及磷酸化的CREB蛋白表达水平的差异,探讨CREB蛋白在结核病免疫调节中的作用,明确CREB蛋白作为新药开发和疫苗研制分子靶位的可行性。研究方法:第一部分:选取25例初治涂阳肺结核患者和18例PPD阳性健康人作为研究对象,留取静脉血,分离外周血单个核细胞(PBMC),利用磁性激活细胞分离技术(MACS)阳性分选得到CD3+T细胞,流式细胞仪检测CD3+T细胞的纯度;从CD3+T细胞提取核蛋白后,采用凝胶电泳迁移率变化分析(EMSA)研究CREB蛋白能否和IFN-γ的近端启动子结合;采用超迁移率(supershift)变化分析实验验证低迁移率条带的DNA-蛋白质复合物的特异性。Western blotting检测结核病患者和PPD阳性健康对照者CREB蛋白表达水平的差异。第二部分:选取25例初治肺结核患者和18例PPD阳性健康人作为研究对象,留取血标本,分离外周血单个核细胞(PBMC);利用磁性激活细胞分离技术(MACS)阳性分选得到CD3+T细胞,流式细胞仪检测CD3+T细胞的纯度;加入通过热灭活方法制备的结核分枝杆菌抗原, RPMI1640培养24小时;利用染色质免疫共沉淀(CHIP)技术研究在体内状态下,结核分枝杆菌抗原刺激CD3+T细胞能否诱导CREB蛋白产生并和IFN-γ近端启动子的结合;并采用Western blotting研究结核分枝杆菌抗原能否诱导CREB蛋白磷酸化。研究结果第一部分:在25例初治肺结核患者中有18例的EMSA结果相比于18例PPD阳性健康人的结果缺失一条低迁移率的条带,说明结核患者缺少和IFN-γ的近端启动子结合的蛋白;超迁移率变化分析实验证明蛋白质-DNA复合物的特异性,结果证明核蛋白特异性的与IFN-γ近端启动子结合,并且蛋白质内含有CREB蛋白;Western blotting结果显示结核患者的CREB蛋白表达水平与PPD阳性健康对照者相比显著降低。第二部分:10例PPD阳性健康对照者ChIP实验PCR产物电泳图都有一条204bp条带,证实PPD阳性健康人活体T细胞在结核分枝杆菌抗原刺激时诱导产生CREB蛋白并与IFN-γ的近端启动子结合。12例结核病患者的电泳图204bp条带缺失,显示缺少与IFN-γ的近端启动子结合的CREB蛋白,表明在体内条件下肺结核患者CD3+T细胞产生CREB蛋白的能力下降;而且,Western blotting结果显示肺结核患者CD3+T细胞在结核分枝杆菌抗原刺激时不能诱导磷酸化CREB蛋白的产生。研究结论:1. CREB蛋白和IFN-γ的近端启动子结合,调控IFN-γ的转录、分泌。2.结核病患者的CREB蛋白表达水平降低,并且活体T细胞在结核分枝杆菌抗原刺激时不能诱导产生磷酸化的CREB蛋白,导致与IFN-γ的近端启动子结合的CREB蛋白减少, IFN-γ的近端启动子的活性下降,限制IFN-γ的转录,下调IFN-γ的分泌。3. CREB蛋白是一种保护性蛋白,促进IFN-γ的转录和分泌,有助于机体对结核分枝杆菌的保护性免疫反应。更多还原

【Abstract】 BackgroundTuberculosis remains a major health problem worldwide. Two billion people are infected with the pathogenic agent, Mycobacterium tuberculosis. Approximately 8-10 million people are infected with this pathogen every year. China was one of the highest TB epidemic countries, according to the results of the national epidemiological sampling surveys on TB conducted in 2000, it’s characterized by higher TB prevalence (the prevalence of pulmonary tuberculosis was 367/100,000 and smear-positive prevalence was 122/100,000) and higher drug resistance rate(the primary resistance rate was 18.6% ). Development of multi-drug resistant TB (MDR-TB) and HIV-TB coinfection are greatly increased worldwide, posing a threat to the existing therapeutic possibilities. Hence, the reach of new prophylactic and therapeutic strategies are urgent to control TB.TB is a disease caused by Mycobacterium tuberculosis whose interaction with the host may lead to a cell-mediated protective immune response. Interferon- gamma (IFN-γ) is essential to the response. IFN-γ, a key cytokine in control of M. tuberculosis infection is produced by both CD4+ and CD8+ T cells, as well as by NK cells. IFN-γmight augment antigen presentation, leading to recruitment of CD4+ T-lymphocytes and/or cytotoxic T-lymphocytes, which might participate in mycobacterial killing. Although IFN-γproduction alone is insufficient to control M. tuberculosis infection, it is required for the protective response to this pathogen. IFN-γis the major activator of macrophages, such as generation of reactive oxygen intermediates (ROI), reactive nitrogen intermediates (RNI), so it helps macrophages to inhibit the growth of M. tuberculosis.Transcription of the IFN-γgene in activated T cells is controlled by the proximal promoter element (-73 bp to -48 bp). Binding of specific proteins to these regulatory regions markedly affects IFN-γpromoter activity and the production of IFN-γ. Cyclic adenosine monophosphate response element binding protein (CREB), as a transcription factor, binds to the IFN-γproximal promoter to adjust the transcription of the IFN-γ, and the binding is enhanced by phosphorylation of CREB.At present, the study of CREB protein regulatory of IFN-γtranscription is in an infancy stage. Some questions need to be elucidated such as CREB protein is a positive or negative regulator of IFN-γtranscription, the function of CREB in immune protection. Knowledge about the functioning of CREB protein during IFN-γtranscription and production provides the basis for rational tuberculosis treatment and prevention.Purpose1. Study binding of CREB protein to the proximal IFN-γpromoter in vitro and in vivo; discuss the function of CREB protein in IFN-γtranscription.2. Compare the level of CREB protein in tuberculosis patients and PPD-positive healthy donors; investigate the functions of CREB protein in the immunity against tuberculosis.MethodsPart one:Venous blood samples were obtained from 18 PPD-positive healthy donors and from 25 HIV-seronegative patients with smear-positive pulmonary tuberculosis, all of whom had received < 4 weeks of antituberculosis therapy. PBMC were isolated by differential centrifugation over Ficoll-Paque. Freshly isolated PBMC were incubated with magnetic beads conjugated to anti-CD3 and a magnetic cell separator was used to positively select CD3+ cells. The purity of CD3+ cells was measured by flow cytometer. After Extraction of nuclear proteins, EMSA was performed to determine nuclear proteins binding to the proximal IFN-γpromoter in vitro, and the specificity of binding complex was tested by competitive EMSA. Western blotting was performed to compare the difference of expression of CREB protein in tuberculosis patients and PPD-positive healthy donors.Part two:Venous blood samples were obtained from 18 PPD-positive healthy donors and from 25 HIV-seronegative patients with smear-positive pulmonary tuberculosis. PBMC were isolated by differential centrifugation over Ficoll-Paque,Freshly isolated PBMC were incubated with magnetic beads conjugated to anti-CD3 and a magnetic cell separator was used to positively select CD3+ cells. The purity of CD3+ cells was measured by flow cytometer. CD3+ cells were cultured with heat-killed mycobacterial Ags for 24h. Chromatin immunoprecipitation with anti-CREB Ab was used to determine whether CREB binds to the chromosomal IFN-γproximal promoter in vivo in live T cells exposed to microbial Ags. Western blotting with Abs specific for serine 133-phosphorylated CREB was performed to determine whether M. tubtuberculosis Ags elicited phosphorylation of CREB.ResultsPart one:The results of EMSA showed a low-mobility complex binding to the IFN-γpromoter, the binding pattern observed was similar for T cells from all 18 PPD-positive healthy donors. However, in T cells from 18 of 25 tuberculosis patients, the low-mobility complex binding to the IFN-γpromoter 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. CREB protein expression markedly decreased in tuberculosis patients compared with PPD-positive healthy donors detected by Western blotting.Part two: The results of ChIP showed a 204bp product yielded in CD3+T cell from 10 PPD-positive healthy donors cultured with heat-killed M. tuberculosis for 24h. However, CD3+T cell from 12 tuberculosis patients didn’t yield a 204bp product. The results elicited that CREB protein can bind to the chromosomal IFN-γproximal promoter in vivo in live T cells from PPD-positive healthy donors exposed to microbial Ag, but no binding was seen in live T cells from tuberculosis patients. Furthermore, M. tubtuberculosis Ags also elicited phosphorylation of CREB in CD3~+T cell from PPD-positive healthy donors, but not in CD3+T cell from tuberculosis patients.conclusions1. CREB protein binds to IFN-γproximal promoter to regulate the transcription and production of IFN-γ.2. CREB protein binding to IFN-γproximal promoter reduced in tuberculosis patients compared with PPD-positive healthy donors. Tuberculosis patients had diminished CREB protein levels, reduced ability of binding to the IFN-γpromoter, diminished IFN-γpromoter activity, and low IFN-γproduction.3. CREB positively regulates the production of IFN-γby human T cells in response to M. tuberculosis.更多还原