【作者】 任涛；

【导师】 蔡映云；

【作者基本信息】 复旦大学 ， 内科学， 2004， 博士

【摘要】 研究背景 结核病仍严重威胁人类健康，是单种致病菌死亡率最高的疾病。当前全球1／3的人口已感染结核菌。免疫抑制宿主的增多增加了结核病的发病率。耐药结核菌的流行以及抗结核药物的相对落后对现有的治疗措施提出了挑战。因此，需要新的防治策略控制结核病。 结核病机体免疫学特点为Th1型免疫反应受抑而Th2型免疫反应亢进。通过激活Th1型免疫反应抑制Th2型免疫反应有助于机体清除结核菌。 CpG ODN含有非甲基化的CpG基序，可刺激脊椎动物的免疫系统，促进机体建立针对病原体的保护性免疫反应。CpG基序可刺激B细胞、NK细胞、T细胞和巨噬细胞等免疫细胞产生IFN-γ，IL-12和IL-2等Th1类细胞因子，建立Th1型免疫反应。并且通过诱导Th1型免疫反应增强小鼠抵抗李斯特杆菌和利什曼原虫感染。CpG ODN在结核病免疫治疗中的研究刚刚起步，目前尚不清楚CoG ODN对结核病人Th1／Th2型免疫反应的影响，并且CpG ODN诱导结核病机体产生免疫保护作用的分子机制有待进一步阐明。 研究目的 观察肺结核病人外周血单个核细胞Th1／Th2型免疫反应变化以及CpG ODN对Th1／Th2型免疫反应的影响。研究CpG ODN对结核病小鼠Th1／Th2型免疫反应的调节作用，明确CpG ODN对结核病的预防和免疫治疗作用。 研究方法 第一部分 选取20例初发活动性肺结核病人和20例健康人为研究对象。留取血标本并分离外周血单个核细胞(PBMC)。分别用RPMI 1640和RPMI 1640+PPD(10μg／ml)培养48小时后，用RT-PCR测定PBMC的FN-γ mRNA和IL-10mRNA表达。同时对PBMC进行CD3／CD8表面抗原染色和胞内抗IFN-γ和IL-4博士学位论文中文摘要染色，用流式细胞仪检测T细胞亚群比例。 第二部分加例初发活动性肺结核病人和20例健康人PBMC各分为4份，分别用RP呱1640，ppD，CpG ODN，Cp(}ODN+PPo培养48小时。用real一timePCR检测PBMC的任N一丫mRN.A、几一lolllRNA和几一12mRNA表达。 第三部分%只雌性BALB/c小鼠随机分成4组(n=24)，分别为cpG ODN预防组(A)，对照ODN预防组(B)，感染对照组(C)和健康对照组(D)。A组和B组小鼠于攻毒前2周分别腹腔注射CpG ODN和对照ODN(30协留只)。随后A组，B组和C组小鼠尾静脉内注射结核菌(l X 106/只)。于感染后3周每组分别处死小鼠12只，称取小鼠体重和)沛脾湿重。肺和脾置10%中性甲醛固定，制作病理切片和抗酸染色。肺组织和脾组织进行结核菌培养和菌落计数。用盯一PeR测定小鼠肺脾组织TL”(toll一like reeeptorg)mRNA表达。realtime一PCR检测小鼠肺脾组织正N一Y n1RN人IL一10nu又NA，几一4mRNA和几-6mRNA的表达。每组剩余12只小鼠，观察60天生存率。 第四部分72只雌性BALB/c小鼠随机分成3组(n=24)，分别为CpG ODN治疗组(A)，感染对照组(C)和健康对照组(C)。A组和B组小鼠尾静脉内注射结核菌(l x 106/只)制作结核病小鼠模型。A组小鼠于攻毒后1周单次腹腔注射CpG ODN(3op岁只)，B组小鼠腹腔注射生理盐水。于CpG ODN治疗后3周每组分别处死小鼠12只，称取小鼠体重和肺脾湿重。肺和脾置10%中性甲醛固定，制作病理切片和抗酸染色。肺组织和脾组织进行结核菌培养和菌落计数。real time一PCR检测小鼠肺脾组织任N一Y n1RNA，几一12p40mRNA，IL-101llRNA，几一4mRNA和几一6mRNA的表达。每组剩余12只小鼠，观察60天生存率。 研究结果 第一部分肺结核病人PBMC的用平Y mRNA的表达低于健康人。肺结核病人PBMC经PPD刺激后IL一10n1RNA的表达增加，并高于健康人。健康人PBMC经PPD刺激后几一10IllRNA的表达不增加。肺结核病人CD4+T细胞与健康人相似，正N一Y+CD4+T细胞百分率低于健康人，IL一4+CD4+T细胞百分率与健康人相似。病人CDS十T的百分率与健康人相似，正N一Y十CDS+T细胞百分率低于健康人，几一4+CDS‘T细胞百分率高于健康人。 第二部分CpG ODN促进肺结核病人PBMC的正N一Y mRNA和几一12mRNA的表达，但不增加几一10 mRNA的表达，CpG ODN与PPD联合应用可下调PPD诱导的几一10 mRNA高表达，并明显增强几一12 n1RNA的表达。 第三部分结核菌感染前使用CpG ODN可减少结核菌感染鼠体重下降，博士学位论文中文摘要减轻感染鼠肺部病理变化和减少肺湿重增加，减少感染鼠肺脾组织结核菌落计数并提高感染鼠的生存率。cpG oDN可增强结核菌感染鼠肺脾组织IFN一YmRNA和几一6 mRNA表达，抑制肺脾组织几一4 mRNA和脾组织几一10 n1RNA的表达。结核菌感染鼠肺脾组织均有TLRgl五RNA表达，并高于健康对照组。 第四部分用cpG ODN免疫治疗结核病小鼠，可见结核病小鼠体重下降减少，肺部病变减轻，肺脾组织结核菌培养菌落数减少，生存率提高。cpG oDN免疫治疗鼠肺脾组织IFN一YmRNA、几一12P4omRNA和几一6mRNA的表达增加，IL一4mRNA和几一101llRN.A的表达降低。 研究结论 1.活动性肺结核病人PBMC Thl/ThZ型免疫紊乱，表现为Thl型免疫反应低下，ThZ型免疫反应增强。 2.CpG ODN可增强结核病人低下的Thl型免疫反应并抑制增强的ThZ型免疫反应。 3小鼠感染结核菌后，肺脾组织TLRg表达上调，可能有利于C更多还原

【Abstract】 Tuberculosis(TB) is one of greatest public health challenges in the world and remains the leading cause of death among infectious diseases. One third of the world’s population is infected with it. The rise in the number of individuals with compromised immune systems is one of the major reasons that increase the rate of reactivation of TB. As a result ,the percentage that progresses to disease is increasing. In addition to the increasing incidence of TB, there have been a global emergence of drug-resistant strains and a long stagnant period in tuberculosis drug development, posing a threat to existing therapeutic possibilities . Hence, new prophylactic and therapeutic strategies are required for the control of TB.The patients with TB frequently fall into imbanlance between Thl/Th2-like cytokines due to a shift towards a Th2-dominant condition. Such a pathological condition could depress the cleaning of pathogen .And now , it is well known that a Thl immune response conveys protection against infection with Mycobacterium tuberculosis ,while Th2 immune response is associated with impaired immunological function.Unmethylated CpG motifs within synthetic oligodeoxynucleotides (ODN) can activate vertebrate immune system contributing to host defense mechanisms that could recognize invading microbial agents .CpG motifs can stimulate B cells, NK cells, T cells, and macrophages to secrete Thl-like cytokines ,e.g Interferon(EFN)-gamma ,interleukin(IL)-12,IL-2 . A number of studies indicated that CpG could switch on T helper 1 (Thl) immunity and a Thl-dominated cytokine profile .Indeed, protective Thl-biased immune responses could be induced by the administration of CpG ODN in animal models of Listeria and Leishmania infections .At present ,the useof CpG ODN in imunotherapy for tuberculosis is in an infancy stage .And some questions are to be elicidated such as the effect of CpG ODN on Thl/Th2 immune response in peripheral blood mononuclear cells (PBMC) from patients with tuberculosis, the molecular mechanism of immune protection induced by CpG ODN .Purpose1.Observe the imbanlance of Thl/Th2 immunity and the regulation of CpG ODN on Thl/Th2 immunity in PBMC from patients with pulmonary TB.2.Investigate the protective effect of CpG ODN in mice infected with mycobacterium tuberculosis.3.Detect the regulatory effects of CpG ODN on Thl/Th2 immunity in mice infected with mycobacterium tuberculosis.MethodsPart one Venous blood samples were obtained from 20 healthy donors and from 20 HIV-seronegative patients with pulmonary tuberculosis prior to receiving antituberculosis therapy . PBMC were isolated from blood samples by differential centrifugation over Ficoll-Paque . PBMC were suspended in medium (RPMI 1640 ) with or without lOug of tuberculin purified protein derivative (PPD). PBMC were harvested after a 48h incubation at 37癈 in 5% C02, and the expression of EFN-gamma mRNA and IL-lOmRNA were analyzed by reverse transcription-polymerase chain reaction(RT-PCR), Meanwhile the proportions of CD4+/CD8+T lymphocytes producing EFN-gamma or EL-4 in PBMC was assessed by flow cytometry.Part two The every specimens of PBMC from 20 healthy donors and from 20 HIV-seronegative patients with pulmonary tuberculosis were divided into 4 parts , and cultured with none stimulator, PPD, CpG ODNs , CpG ODNs plus PPD separatively. The PBMC were harvested after a 48h incubation at 37癈 in 5% CO2, and the expression of IFN-gamma mRNA , EL-10 mRNA and EL-12 mRNA were analyzed with a real-time PCR techniquePart three 96 female BALB/c mice were randomized into 4 groups, namely CpG ODN treatment (A group),control ODN treatment (B group), infectious control (C group) and healthy control (D group) (n=24). A group and B group were treated once with CpG ODN or control ODN (30 u g/mouse) intraperitoneally 2 weeks before infection with mycobacterium tuberculosis. 1 X 106 bacili in a volume of 300 u 1 saline were injected into the tail vein of mice from A group ,B group and C group.The 12 mice from each group were sacrificed at 3 weeks postin更多还原