人结核病患者血清特异性抗体与CD14基因多态性相关性的初步研究

发布时间：2018-06-15 23:21

本文选题：结核病 + 单核苷酸多态性　；参考：《吉林农业大学》2013年硕士论文

【摘要】：背景：结核病是由结核分枝杆菌引起的一种慢性消耗性传染病。引起该病的结核分枝杆菌作为一种细胞内病原体,主要通过感染肺脏中具有吞噬功能的抗原递呈细胞进而侵入机体,虽然全球结核杆菌的携带者众多,但是感染发病并发展成活动性结核病的仅仅占携带者的5%-10%。个体的遗传因素和差异是影响和增加结核病发生以及发展的重要因素,同时也与结核病的易感性显著相关。CD14,即脂多糖的受体,能通过脂多糖结合蛋白与脂多糖产生特异性结合,同时通过Toll样受体4以及髓样分化蛋白2向下游递呈活化信号,通过释放TNF-α、IL-1和IL-6等多种炎症细胞因子,引发炎症反应的发生,在介导炎症反应过程和初次免疫应答中起到重要的作用。目前研究发现CD14基因多态性位点与结核病易感性有关,通过分析CD14基因多态性及其与患者血清中特异性抗体含量高低的相关性对于深入了解结核病发病机制、寻找新的检测手段有着重要的意义。目的：CD14基因启动子区的多态性与结核病的发生有一定的联系,但CD14基因多态性与患者血清中特异性抗体水平之间是否存在相关性,迄今尚无报道。本研究选择CD14基因中的两个多态性位点,采用直接测序的方法筛选出突变位点并对其多态性进行分析,确定两者之间是否存在相关性及与结核病易感性的联系,为结核病的早期预防、诊断以及治疗方面提供了一定的依据,也为如何区分一些结核高风险人群提供新的思路。方法：运用ELISA的方法筛选出阳性和阴性血液样本。按其数值高低各选取10个血液样本提取基因组DNA；通过直接测序的方法筛选出C-159T和G-1145A的突变位点,分析其突变位点及其频率与结核病易感性是否相关；通过分离血液样本中的单个核细胞,并对其进行免疫荧光染色,观察细胞表面CD14抗原的表达量是否与结核病特异性抗体检测结果之间的联系。结果：成功检测出阳性血清样本132个,阴性血清样本250个；测序结果在C-159T基因中共发现10个突变位点,阳性血液样本中出现了3个突变位点,突变频率分别为12.5%、12.5%、75%,阴性血液样本中出现了8个突变位点,突变频率除一个突变位点为80%外,其他突变位点均为10%；在G-1145A基因中共发现8个突变位点,其中阳性血液样本中出现了6个突变位点,突变频率其中4个位点为11.1%,另外两个位点为66.7%和88.9%,阴性血液样本中出现了4个突变位点,突变频率其中两个位点为11.1%,另外两个位点为66.7%和77.8%；成功分离得到单个核细胞,免疫荧光染色结果阴性血液样本的细胞表面CD14抗原的表达量高于阳性血液样本。结论：发现CD14基因中的两个多态性位点C-159T和G-1145A在阴性和阳性血液样本中存在相同的突变位点,其突变频率分别为77.8%和83.3%,而这两个突变位点和报道过与结核病易感基因相关的突变位点相符,表明CD14基因的多态性与血清中PPD抗体的高低之间无明显的相关性,说明CD14基因C-159T和G-1145A两个多态性位点只能代表机体易感染结核病,但并不代表机体一定会发病。在G-1145A基因的突变位点中,第44个碱基位置在阳性血液样本和阴性血液样本中均出现了碱基缺失的突变现象,这个位点的碱基缺失目前还没有报道,可能是一个新的潜在的多态性位点；在细胞表面CD14抗原的表达量上与PPD抗体量高低存在着负相关性,这说明外周血单个核细胞表面CD14抗原表达量与PPD抗体量高低存在着负相关性。结核病患者CD14的表达量会降低。
[Abstract]:Background : Tuberculosis is a kind of chronic consumptive infectious disease caused by Mycobacterium tuberculosis . Mycobacterium tuberculosis is a kind of intracellular pathogen .

Objective : The polymorphism of CD14 gene promoter region is associated with the occurrence of tuberculosis , but there is no correlation between CD14 gene polymorphism and the level of specific antibody in serum .

Methods : Positive and negative blood samples were screened by ELISA . Genomic DNA was extracted from 10 blood samples .
The mutation sites of C - 159T and G - 1145A were screened by direct sequencing .
By separating individual nuclear cells in the blood sample and performing immunofluorescence staining on it , it was observed whether the expression amount of the CD14 antigen on the surface of the cell was related to the detection result of the tuberculosis specific antibody .

Results : 132 positive serum samples and 250 negative serum samples were successfully detected .
The results showed that there were 10 mutation sites in C - 159T gene , 3 mutation sites appeared in positive blood samples , 12.5 % , 12.5 % , 75 % respectively , 8 mutation sites appeared in negative blood samples , and the mutation frequency was 80 % except for one mutation site and 10 % for other mutation sites .
Eight mutation sites were found in the G - 1145A gene , 6 mutation sites appeared in the positive blood samples , the mutation frequency was 11.1 % , the other two sites were 66.7 % and 89.9 % , 4 mutation sites appeared in the negative blood samples , the mutation frequencies were 11.1 % , and the other two sites were 66.7 % and 77.8 % ;
The expression of CD14 antigen in the cell surface of the negative blood sample was higher than that of the positive blood sample .

Conclusion : Two polymorphic sites C - 159T and G - 1145A in CD14 gene were found to have the same mutation sites in negative and positive blood samples . The mutation frequencies were 77.8 % and 83.3 % , respectively .
There was a negative correlation between the expression level of CD14 antigen and the amount of PPD antibody on the surface CD14 antigen in peripheral blood , suggesting a negative correlation between the expression of CD14 antigen and the amount of PPD antibody in peripheral blood mononuclear cells .
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R378.911

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