本文關(guān)鍵詞：結(jié)核不同感染狀態(tài)下宿主對(duì)結(jié)核特異性抗原獲得性免疫應(yīng)答的差異及相關(guān)分子標(biāo)識(shí)篩選　出處：《復(fù)旦大學(xué)》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 活動(dòng)性結(jié)核 潛伏感染 表達(dá)譜 PBMCs PPD 分子標(biāo)識(shí) qPCR 決策樹(shù)

【摘要】：結(jié)核病是由結(jié)核分枝桿菌所致的以呼吸系統(tǒng)感染為主的慢性傳染病。近年來(lái),結(jié)核病疫情越來(lái)越嚴(yán)重,其感染后引起患者的死亡率位居我國(guó)傳染病的首位。目前全球約有1/3的人口感染結(jié)核桿菌,每年新發(fā)的活動(dòng)性結(jié)核病人有900多萬(wàn)人,并且每年有近200萬(wàn)人死于結(jié)核病。大多數(shù)人感染結(jié)核菌后并無(wú)臨床癥狀,稱為潛伏感染。但是有10%的潛伏感染者將在其一生的某個(gè)階段發(fā)展為活動(dòng)性結(jié)核。潛伏感染不斷轉(zhuǎn)化為活動(dòng)性結(jié)核,是造成結(jié)核病發(fā)病率居高不下的重要原因。因此,潛伏感染者為結(jié)核病的控制和診斷帶來(lái)了巨大的挑戰(zhàn)。然而,對(duì)于機(jī)體如何控制潛伏感染,以避免發(fā)展為活動(dòng)性結(jié)核的關(guān)鍵免疫機(jī)制仍知之甚少,同時(shí)臨床亦缺乏鑒別潛伏感染與活動(dòng)性感染的生物靶標(biāo)。因此,全面的研究活動(dòng)性結(jié)核與潛伏感染的免疫應(yīng)答差異對(duì)進(jìn)一步揭開(kāi)抗結(jié)核免疫的機(jī)制和發(fā)展新的結(jié)核診斷方法具有重要意義,也為提出新型的結(jié)核病控制策略奠定基礎(chǔ)。 本研究首先用人全基因組表達(dá)譜芯片的方法,尋找結(jié)核不同感染狀態(tài)者的獲得性免疫在應(yīng)答PPD刺激時(shí)的差異。我們?nèi)脒x了4名活動(dòng)性結(jié)核患者(TB),4名潛伏感染者(LTBI)及4名未感染結(jié)核菌的健康對(duì)照者(HC)。采其外周血分離外周血單個(gè)核細(xì)胞(PBMCs),用結(jié)核特異性抗原PPD刺激培養(yǎng)4小時(shí),設(shè)置未刺激的PBMCs作為自身對(duì)照,消除本底差異。用雙熒光標(biāo)記雜交的方法,將來(lái)自于同一人的兩份RNA在同一張芯片上進(jìn)行雜交。對(duì)芯片結(jié)果的統(tǒng)計(jì)分析顯示,TB組和LTBI組的PBMCs對(duì)PPD刺激的應(yīng)答差異最為明顯,我們從中篩選到了286個(gè)差異表達(dá)基因;TB組與HC組的差異基因數(shù)目與上述兩組相當(dāng),差異表達(dá)基因?yàn)?39個(gè)；LTBI組與HC組的差異最小,差異表達(dá)基因?yàn)?4個(gè)。最終,我們通過(guò)維恩分析得到了506個(gè)在三組組間比較中差異表達(dá)的基因群。隨后用生物信息學(xué)的方法,我們又對(duì)差異表達(dá)的基因的功能進(jìn)行了分析。Gene Ontology (GO)分析發(fā)現(xiàn),胞外組分及應(yīng)對(duì)外來(lái)刺激的基因簇在LTBI組與HC組的差異表達(dá)基因中被顯著富集;與應(yīng)對(duì)外來(lái)刺激相關(guān)的基因簇也在TB組與LTBI組間存在差異；TB組與HC組的差異基因簇最多,主要集中在受體結(jié)合、信號(hào)轉(zhuǎn)導(dǎo)、胞外組分、應(yīng)對(duì)刺激、免疫系統(tǒng)調(diào)節(jié)和細(xì)胞通信等幾個(gè)方面。 隨后,我們又對(duì)結(jié)核感染者及活動(dòng)性結(jié)核患者的PBMCs在PPD刺激時(shí)的特有免疫應(yīng)答進(jìn)行研究。我們比較了(1)結(jié)核感染者(TB組和LTBI組)與健康對(duì)照者(HC組)的PBMCs在應(yīng)答PPD刺激的差異；(2)活動(dòng)性結(jié)核患者(TB組)與無(wú)臨床癥狀者(LTBI組和HC組)的PBMCs在應(yīng)答PPD刺激的差異。最終我們分別得到了由55個(gè)基因所組成的結(jié)核感染者的特有的基因表達(dá)譜,及由229個(gè)基因所組成的活動(dòng)性結(jié)核特有的基因表達(dá)譜。GO分析發(fā)現(xiàn),結(jié)核感染表達(dá)譜的差異基因在受體結(jié)合、應(yīng)對(duì)胞外刺激和細(xì)胞增殖調(diào)控幾個(gè)方面被顯著富集；活動(dòng)性結(jié)核基因表達(dá)譜中的229個(gè)基因在受體結(jié)合、胞外成分、應(yīng)對(duì)胞外刺激、信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞膜結(jié)構(gòu)、慢性炎癥調(diào)控和細(xì)胞增殖調(diào)控幾個(gè)方面被顯著富集。 為了驗(yàn)證表達(dá)譜芯片的結(jié)果,我們進(jìn)行了實(shí)時(shí)定量PCR (qPCR)驗(yàn)證實(shí)驗(yàn)。為此,我們另外招募了83名參與者,其中TB組為25名,LTBI組為36名,HC組為22名。結(jié)果顯示,81%的qPCR結(jié)果與芯片試驗(yàn)結(jié)果一致,證明了芯片實(shí)驗(yàn)數(shù)據(jù)可靠。同時(shí),我們?cè)谠摂U(kuò)大樣本的驗(yàn)證中,也得到了一系列在相應(yīng)組間比較中存在差異表達(dá)的基因。 基于上述芯片實(shí)驗(yàn)篩選及后續(xù)qPCR擴(kuò)大樣本驗(yàn)證中得到的差異表達(dá)基因的mRNA表達(dá)水平,我們篩選到了新的活動(dòng)性結(jié)核患者及結(jié)核感染者的分子標(biāo)識(shí)。ROC及決策樹(shù)分析發(fā)現(xiàn),CXCL10, ATP10A和TLR6三個(gè)基因表達(dá)水平的聯(lián)合對(duì)于區(qū)分TB組和LTBI組的敏感性為80%,特異性為89%。在區(qū)分TB組與無(wú)結(jié)核臨床癥狀組(]LTBIHC組)方面,我們發(fā)現(xiàn)CXCL10, ATP10A, TLR6, RAB20和IL2幾個(gè)基因表達(dá)水平的不同的聯(lián)合可以對(duì)二者進(jìn)行較好的區(qū)分。CXCL10, RAB20和IL2三個(gè)基因表達(dá)水平的聯(lián)合對(duì)于區(qū)分TB組和LTBIHC組的陽(yáng)性預(yù)測(cè)值最高,為90%,敏感性和特異性分別為72%和97%；另外,CXCL10, RAB20和ATP10A區(qū)分這兩組的敏感性和特異性為84%和91%；CXCL10, TLR6和ATP10A三個(gè)基因聯(lián)合對(duì)于區(qū)分TB組和LTBIHC組的敏感性和特異性為68%和97%。在區(qū)分結(jié)核感染者(TBLTBI組)與HC組方面,IFN-γ, CXCL10, CXCL9及IL2RA四個(gè)基因聯(lián)合的效果良好,敏感性為98%,特異性為59%。 綜上所述,通過(guò)本研究篩選出的CXCL10, ATP10A, TLR6, RAB20, IL2, EFN-γ, CXCL9及IL2RA等基因的不用組合,可用于評(píng)價(jià)結(jié)核感染者的不同疾病狀態(tài)。
[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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R378.91

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 趙雁林;尚美;;我國(guó)結(jié)核病實(shí)驗(yàn)室診斷的現(xiàn)狀[J];中華檢驗(yàn)醫(yī)學(xué)雜志;2007年07期

2 高謙;梅建;;早期診斷技術(shù)的突破是當(dāng)前控制結(jié)核病的關(guān)鍵[J];中華檢驗(yàn)醫(yī)學(xué)雜志;2007年07期

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