结核不同感染状态下宿主对结核特异性抗原获得性免疫应答的差异及相关分子标识筛选

发布时间：2018-01-14 05:35

本文关键词：结核不同感染状态下宿主对结核特异性抗原获得性免疫应答的差异及相关分子标识筛选　出处：《复旦大学》2011年博士论文　论文类型：学位论文

【摘要】：结核病是由结核分枝杆菌所致的以呼吸系统感染为主的慢性传染病。近年来,结核病疫情越来越严重,其感染后引起患者的死亡率位居我国传染病的首位。目前全球约有1/3的人口感染结核杆菌,每年新发的活动性结核病人有900多万人,并且每年有近200万人死于结核病。大多数人感染结核菌后并无临床症状,称为潜伏感染。但是有10%的潜伏感染者将在其一生的某个阶段发展为活动性结核。潜伏感染不断转化为活动性结核,是造成结核病发病率居高不下的重要原因。因此,潜伏感染者为结核病的控制和诊断带来了巨大的挑战。然而,对于机体如何控制潜伏感染,以避免发展为活动性结核的关键免疫机制仍知之甚少,同时临床亦缺乏鉴别潜伏感染与活动性感染的生物靶标。因此,全面的研究活动性结核与潜伏感染的免疫应答差异对进一步揭开抗结核免疫的机制和发展新的结核诊断方法具有重要意义,也为提出新型的结核病控制策略奠定基础。本研究首先用人全基因组表达谱芯片的方法,寻找结核不同感染状态者的获得性免疫在应答PPD刺激时的差异。我们入选了4名活动性结核患者(TB),4名潜伏感染者(LTBI)及4名未感染结核菌的健康对照者(HC)。采其外周血分离外周血单个核细胞(PBMCs),用结核特异性抗原PPD刺激培养4小时,设置未刺激的PBMCs作为自身对照,消除本底差异。用双荧光标记杂交的方法,将来自于同一人的两份RNA在同一张芯片上进行杂交。对芯片结果的统计分析显示,TB组和LTBI组的PBMCs对PPD刺激的应答差异最为明显,我们从中筛选到了286个差异表达基因;TB组与HC组的差异基因数目与上述两组相当,差异表达基因为239个；LTBI组与HC组的差异最小,差异表达基因为94个。最终,我们通过维恩分析得到了506个在三组组间比较中差异表达的基因群。随后用生物信息学的方法,我们又对差异表达的基因的功能进行了分析。Gene Ontology (GO)分析发现,胞外组分及应对外来刺激的基因簇在LTBI组与HC组的差异表达基因中被显著富集;与应对外来刺激相关的基因簇也在TB组与LTBI组间存在差异；TB组与HC组的差异基因簇最多,主要集中在受体结合、信号转导、胞外组分、应对刺激、免疫系统调节和细胞通信等几个方面。随后,我们又对结核感染者及活动性结核患者的PBMCs在PPD刺激时的特有免疫应答进行研究。我们比较了(1)结核感染者(TB组和LTBI组)与健康对照者(HC组)的PBMCs在应答PPD刺激的差异；(2)活动性结核患者(TB组)与无临床症状者(LTBI组和HC组)的PBMCs在应答PPD刺激的差异。最终我们分别得到了由55个基因所组成的结核感染者的特有的基因表达谱,及由229个基因所组成的活动性结核特有的基因表达谱。GO分析发现,结核感染表达谱的差异基因在受体结合、应对胞外刺激和细胞增殖调控几个方面被显著富集；活动性结核基因表达谱中的229个基因在受体结合、胞外成分、应对胞外刺激、信号转导、细胞膜结构、慢性炎症调控和细胞增殖调控几个方面被显著富集。为了验证表达谱芯片的结果,我们进行了实时定量PCR (qPCR)验证实验。为此,我们另外招募了83名参与者,其中TB组为25名,LTBI组为36名,HC组为22名。结果显示,81%的qPCR结果与芯片试验结果一致,证明了芯片实验数据可靠。同时,我们在该扩大样本的验证中,也得到了一系列在相应组间比较中存在差异表达的基因。基于上述芯片实验筛选及后续qPCR扩大样本验证中得到的差异表达基因的mRNA表达水平,我们筛选到了新的活动性结核患者及结核感染者的分子标识。ROC及决策树分析发现,CXCL10, ATP10A和TLR6三个基因表达水平的联合对于区分TB组和LTBI组的敏感性为80%,特异性为89%。在区分TB组与无结核临床症状组(]LTBIHC组)方面,我们发现CXCL10, ATP10A, TLR6, RAB20和IL2几个基因表达水平的不同的联合可以对二者进行较好的区分。CXCL10, RAB20和IL2三个基因表达水平的联合对于区分TB组和LTBIHC组的阳性预测值最高,为90%,敏感性和特异性分别为72%和97%；另外,CXCL10, RAB20和ATP10A区分这两组的敏感性和特异性为84%和91%；CXCL10, TLR6和ATP10A三个基因联合对于区分TB组和LTBIHC组的敏感性和特异性为68%和97%。在区分结核感染者(TBLTBI组)与HC组方面,IFN-γ, CXCL10, CXCL9及IL2RA四个基因联合的效果良好,敏感性为98%,特异性为59%。综上所述,通过本研究筛选出的CXCL10, ATP10A, TLR6, RAB20, IL2, EFN-γ, CXCL9及IL2RA等基因的不用组合,可用于评价结核感染者的不同疾病状态。
[Abstract]:Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis infection mainly caused by respiratory system. In recent years, the epidemic situation of tuberculosis is more and more serious, the infection causes mortality among infectious diseases in China in the first place. At about 1/3 of the world's population is infected with Mycobacterium tuberculosis, every year new cases of active tuberculosis and about 9000000 people, and every year there are nearly 2 million people died of tuberculosis. The clinical symptoms of tuberculosis after not the majority of people infected, called latent infection. But there are 10% latent infection will at some stage in the development of their life for active tuberculosis. Latent infection into active tuberculosis, which is an important reason for the high incidence of tuberculosis therefore, it brings great challenges for the control of latent infection and diagnosis of tuberculosis. However, for the body to control the latent infection, to avoid developing active tuberculosis The key immune mechanism is still poorly understood, but also the lack of clinical identification of latent infection and active infection of biological targets. Therefore, tuberculosis activity of comprehensive and latent infection of immune response differences has important significance to further uncover the tuberculosis diagnosis method of anti tuberculosis immune mechanism and the development of new, also laid the foundation for the new the tuberculosis control strategy.
This research first used the expression method of human whole genome microarray, differences in immunity in response to PPD stimulation in the search for tuberculosis infection status was different. We selected 4 patients with active tuberculosis (TB), 4 (LTBI) latent infection and 4 uninfected healthy controls (TB HC). Collected peripheral blood from peripheral blood mononuclear cells (PBMCs), with the specific antigens of Mycobacterium tuberculosis PPD cultured for 4 hours, set the untreated PBMCs as control group, the elimination of the background difference. With double fluorescent hybridization, the future two RNA from the same person in the same a chip on chip hybridization. Statistical analysis of the results showed that, TB group and LTBI group of PBMCs responses induced by PPD was the most obvious, we screened 286 differentially expressed genes; the difference between TB group and HC group and the two group is the number of genes, 239 differentially expressed genes A; the difference between LTBI group and HC group is minimum, 94 genes differentially expressed. Finally, we obtained 506 Wayne expression differences in the comparison between the three groups of genes. Then using the method of bioinformatics, we differentially expressed genes of.Gene Ontology (GO) analysis found that extracellular gene cluster and response to external stimuli in the difference between group LTBI and group HC were significantly enriched in gene expression; gene cluster associated with response to external stimulation are also different in TB group and LTBI group; the difference between TB group and HC group gene cluster most concentrated in receptor binding, signal transduction, extracellular components, response to stimulation, several immune system regulation and cell communication.
Then, we study on tuberculosis infection and active tuberculosis patients with PBMCs under the stimulation of PPD specific immune response. We compare (1) tuberculosis infection (TB group and LTBI group) and healthy controls (group HC) the difference of PBMCs in response to PPD stimulation; (2) activity tuberculosis patients (group TB) and clinical symptoms (LTBI group and HC group) the difference of PBMCs in response to PPD stimulation. Finally we obtain the expression consists of 55 genes of tuberculosis infected specific gene,.GO spectrum analysis showed that the expression of genes and specific active tuberculosis consists of 229 a gene, gene expression with tuberculosis infection differentially in response to extracellular stimuli and receptors, cell proliferation regulation aspects was significantly enriched; active tuberculosis combined with gene expression of 229 genes in the spectrum in the receptor extracellular components respond to extracellular stimulation, signal transduction, fine The membrane structure, the regulation of chronic inflammation and the regulation of cell proliferation are significantly enriched.
In order to verify the microarray results, we performed quantitative real-time PCR (qPCR) experiment. Therefore, we also recruited 83 participants, including 25 in TB group, LTBI group 36, HC group was 22. The results showed that 81% qPCR chip results and experimental results agree that. Chip reliable experimental data. At the same time, we expand the sample in the test, get a series of corresponding groups in the differentially expressed genes.
The expression level of the chip screening experiments and subsequent qPCR samples that have been verified in expanding the difference of gene expression based on mRNA, we screened the molecular marker.ROC and the decision tree of active tuberculosis and tuberculosis infection of the new analysis found that CXCL10, ATP10A and TLR6 three gene expression level combined with sensitivity to distinguish between group TB and LTBI group was 80%, the specificity of 89%. in distinguishing between TB group and non tuberculosis clinical symptom group (]LTBIHC group), we found that CXCL10, ATP10A, TLR6, combined with different expression levels of RAB20 and IL2 genes can be used to distinguish the.CXCL10 of two, RAB20 and IL2 three gene expression level the joint for the distinction between TB group and LTBIHC group the positive predictive value was 90%, the highest sensitivity and specificity were 72% and 97%; in addition, CXCL10, RAB20 and ATP10A between the two groups with 84% sensitivity and specificity And 91%; CXCL10, TLR6 and ATP10A three genes combined to distinguish between TB group and LTBIHC group, the sensitivity and specificity of 68% and 97%. in differentiating tuberculosis infection (TBLTBI group) and HC group, IFN- CXCL10, CXCL9 gamma, and IL2RA four gene combined with the good, the sensitivity was 98%, specificity 59%.
In conclusion, the combination of CXCL10, ATP10A, TLR6, RAB20, IL2, EFN- gamma, CXCL9 and IL2RA can be used to evaluate the different disease states of TB infected patients.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378.91

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