结核分枝杆菌分泌蛋白ESAT-6、Ag85B对外周血FoxP3mRNA的诱导作用

发布时间：2018-06-18 08:31

本文选题：结核分枝杆菌 + CD4+CD25+调节性T细胞　；参考：《汕头大学》2011年硕士论文

【摘要】：背景和目的:结核病是我国重大传染病之一,是严重危害我国人群健康的呼吸道传染病。本课题组前期研究发现,耐药性空洞型肺结核病人经有效手术治疗后,CD4+CD25+调节性T细胞(Treg)细胞水平明显下降。CD4+CD25+Treg细胞水平上调与结核分枝杆菌感染存在高度相关性,但其产生机制并不清楚。因此我们选取结核菌分泌蛋白ESAT-6和Ag85B对人外周血进行刺激,运用流式细胞术检测外周血中CD4+CD25+Treg细胞(%PBMC),证明经蛋白刺激后CD4+CD25+Treg细胞明显升高。由于CD25在活化的T细胞(包括CD4+ CD25-T细胞)中表达,而叉头样转录因子FoxP3在激活的CD4+CD25-T细胞上并不表达,是CD4+CD25+Treg细胞的特异性生物标志,其表达水平与CD4+CD25+Treg细胞数量成正相关。因CD25作为明确的Treg的生物标志受限,为了更精确的检测Treg水平,应用RT-PCR技术检测FoxP3mRNA表达水平。本论文主要通过检测FoxP3 mRNA表达水平,从基因水平上观察经ESAT-6和Ag85B刺激后的外周血CD4+CD25+FoxP3+Treg细胞改变,为研究结核分枝杆菌诱导CD4+CD25+ FoxP3+调节性T细胞上调产生免疫逃逸机制提供新的依据。材料和方法: 1.对汕头市第三人民院进行结核病流行病学调查,获取痰培养和药敏试验及病人的一般信息,描述当地结核病分布情况。 2.从健康人群、潜伏期感染者、耐药性结核病病人中随机选取志愿者,对其空腹静脉取血。 3.采用不同浓度(0μg/ml、1μg/ml、2μg/ml、4μg/ml)的ESAT-6、Ag85B、ESAT-6+Ag85B、BCG处理血液,培养24h、48h、72h后,实时荧光定量PCR检测FoxP3 mRNA表达水平。结果: 1.结核病流行病学调查发现,痰培养阳性者中男性多于女性,且男性年龄高于女性。而药敏试验中耐药性结核病所占比例偏高。 2.不同浓度ESAT-6,Ag85B刺激外周血24h、48h、72h后发现随浓度的增加,FoxP3mRNA有升高趋势,培养24小时FoxP3mRNA表达水平最高,后随时间延长有下降趋势,但均无统计学意义。 3.在未经分泌蛋白刺激前,三组(健康对照组、潜伏期感染组、病例组)中FoxP3 mRNA表达水平比较,病例组与潜伏期感染组高于对照组,但病例组和潜伏期感染组间比较差别无统计学意义。 4.分泌蛋白处理前后比较,经ESAT-6、Ag85B、ESAT-6+Ag85B处理后FoxP3mRNA水平高于处理前水平(P0.05),但BCG处理后FoxP3mRNA略有升高,但差别无统计学意义。 5.不同蛋白处理后三组间比较发现,潜伏期感染组经抗原刺激后,FoxP3mRNA水平高于病例组和健康对照组。病例组低于对照组,但无统计意义。结论:目前耐药性结核病流行,迫切需要了解结核菌的免疫逃逸机制以寻找治疗办法。经体外抗原刺激外周血培养实验发现,外周血FoxP3 mRNA表达水平与ESAT-6,Ag85B存在剂量-反应关系。ESAT-6、Ag85B作为结核菌的主要分泌蛋白,意味着菌量负荷增加后可诱导FoxP3 mRNA表达水平上调。CD4+CD25+ FoxP3+Treg细胞是结核分枝杆菌逃避机体免疫清除过程中不可或缺的因素,故认为ESAT-6、Ag85B两种抗原可能与结核菌的免疫逃逸有关。
[Abstract]:Background and objective: tuberculosis is one of the major infectious diseases in China and is a respiratory infectious disease which seriously endangers the healthy population of our country. In our previous study, we found that after effective surgical treatment, the level of CD4+CD25+ regulatory T cell (Treg) cells decreased significantly and the level of.CD4+CD25+Treg cells was up to the tuberculosis score. There is a high correlation between Mycobacterium infection, but the mechanism of its production is not clear. Therefore, we select ESAT-6 and Ag85B to stimulate human peripheral blood, and use flow cytometry to detect CD4+CD25+Treg cells (%PBMC) in peripheral blood. It is proved that CD4+ CD25+Treg cells are significantly increased after the protein stimulation. Because CD25 is activated T The cell (including CD4+ CD25-T cells) is expressed, and the cross head like transcription factor FoxP3 is not expressed on activated CD4+CD25-T cells. It is a specific biomarker of CD4+CD25+Treg cells, and its expression level is positively related to the number of CD4+CD25+Treg cells. The biological marker of CD25 as a clear Treg is limited, in order to detect the Treg level more accurately. In this paper, the expression level of FoxP3mRNA was detected by RT-PCR technique. By detecting the expression level of FoxP3 mRNA, this paper observed the changes in the peripheral blood CD4+CD25+FoxP3+Treg cells from the ESAT-6 and Ag85B stimuli at the gene level, providing a new method for the study of the immune escape mechanism induced by Mycobacterium tuberculosis induced FoxP3+ regulated T cells up regulation. Basis.
Materials and methods:
1. the epidemiological investigation of tuberculosis in the third people's Hospital of Shantou was carried out to obtain the phlegm culture and drug sensitivity test and the general information of the patients, and to describe the distribution of local tuberculosis.
2. randomly selected volunteers from healthy populations, latent infections and drug-resistant TB patients, took blood from fasting veins.
3. the blood was treated with ESAT-6, Ag85B, ESAT-6+Ag85B, BCG, ESAT-6, Ag85B, ESAT-6+Ag85B, and BCG, with different concentrations (0 g/ml, 1, g/ml, 2 g/ml, 4 g/ml).
Result:
1. the epidemiological survey of tuberculosis found that there were more males than women in sputum positive, and the male age was higher than that of women. The proportion of drug-resistant tuberculosis was higher in the drug sensitivity test.
2. different concentrations of ESAT-6 and Ag85B stimulated 24h, 48h, and 72h in peripheral blood. After 72h, it was found that FoxP3mRNA increased with the increase of concentration, and the expression level of FoxP3mRNA was highest at 24 hours, and then decreased with time, but no statistical significance was found.
3. before the stimulation of the secretory protein, the level of FoxP3 mRNA expression in the three groups (healthy control, latent infection group, case group) was compared, the case group and the latent period infection group were higher than the control group, but there was no statistical difference between the case group and the latent period infection group.
Before and after treatment of 4. secretory proteins, the level of FoxP3mRNA after treatment with ESAT-6, Ag85B and ESAT-6+Ag85B was higher than that before treatment (P0.05), but FoxP3mRNA increased slightly after BCG treatment, but the difference was not statistically significant.
5. after the three groups of protein treatment, it was found that the level of FoxP3mRNA was higher than that of the case group and the healthy control group after the antigen stimulation in the latent period infection group. The case group was lower than the control group, but no statistical significance was found.
Conclusion: at present, the epidemic of drug-resistant tuberculosis is an urgent need to understand the immune escape mechanism of Mycobacterium tuberculosis in order to find the treatment method. The expression level of FoxP3 mRNA in peripheral blood has a dose response relationship with ESAT-6, Ag85B,.ESAT-6, Ag85B as the main secretory protein of Mycobacterium tuberculosis. The increase of FoxP3 mRNA expression level after the increase of.CD4+CD25+ FoxP3+Treg cells is an indispensable factor in the immune clearance of Mycobacterium tuberculosis. Therefore, it is believed that the two antigens of ESAT-6 and Ag85B may be related to the immune escape of Mycobacterium tuberculosis.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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