汉族人群TLRs基因变异与侵袭性曲霉病易感性关系的研究

发布时间：2018-03-12 14:22

本文选题：侵袭性曲霉病　切入点：TLRa　出处：《南京大学》2013年硕士论文　论文类型：学位论文

【摘要】：侵袭性曲霉病(Invasive Aspergillosis, IA)不仅多发于免疫抑制患者,也可见于无基础疾病的健康宿主。研究表明,TLRs参与宿主对曲霉的识别、信号转导和促炎症因子释放,并调节免疫平衡,在宿主抵抗曲霉感染中起到重要作用。近几年,国外已有多篇临床研究报告发现TLR4基因多态性位点1063AG与1363CT与曲霉易感性相关,但均以骨髓造血干细胞移植的病例为研究对象,尚无对无基础疾病的IA患者的研究。我们在临床上发现许多IA患者并无基础疾病,且不存在其它IA的易感因素,因此以这类患者作为研究对象更有意义。且我国汉族人种与白色人种存在一定的基因差异,国内至今尚无汉族人群IA患者基因变异的文献报道,故有待进一步研究。目的：研究我国汉族人群TLR2、TLR4编码区基因变异情况,旨在探讨汉族人群TLRs基因变异与IA易感性的关系。方法：收集自2011.06至2012.12期间在我科确诊及临床诊断的侵袭性曲霉病25例,其中免疫受损者12例,无基础疾病的健康宿主13例；有基础疾病非IA病例48例,健康志愿者80例。所有研究对象均为我国汉族人种。采取患者及志愿者外周血,提取外周血有核粒细胞中的DNA；通过NCBI查找TLR2、TLR4基因信息,设计引物,采用聚合酶链式反应(PCR)扩增TLR2、TLR4基因编码区序列后测序,比对序列,比较IA组与非IA组基因变异的差异,采用χ2检验进行统计学分析。同时分析突变点氨基酸改变,并通过SMART在线工具(http://amart.embl-heidelberg.de)分析突变位点是否造成TLRs蛋白质结构域改变。结果：1、TLR2基因变异情况：测序发现2个TLR2的变异位点597TC,1350TC,均为同义突变,不引起氨基酸改变,两个位点在对照组的发生率1%,故均为单核苷酸基因多态性位点,与对照组相比差异无统计学意义。2、TLR4基因变异情况：测序发现2个TLR4的变异位点219C＞G,1420GA。219C＞G导致第73位丝氨酸变为精氨酸；1420GA导致第474位谷氨酸变为赖氨酸,对照组中的发生率1%,故为突变且国内外未见报道。蛋白结构预测发现TLR4219CG改变导致TLR4s胞外区缺失一个富含亮氨酸重复序列的结构域,1420GA经预测未发现蛋白结构改变。3、未发现国外报道与IA易感性相关的SNPs位点。结论：本组汉族人群中未发现与IA易感相关的TLR2编码区基因变异。首次发现TLR4突变位点219CG,1420GA,其中TLR4219C＞G可影响蛋白质结构,推测可能导致TLR4识别病原体功能的异常,从而影响宿主对曲霉的抵抗力,故TLR4编码区基因变异可能与IA易感相关。
[Abstract]:Invasive Aspergillosis (IAA) is found not only in immunosuppressive patients, but also in healthy hosts without underlying diseases. Studies have shown that TLRs are involved in the recognition of Aspergillus sp., signal transduction and inflammatory factor release, and in regulating immune balance. In recent years, many clinical studies have found that the polymorphic sites of TLR4 gene 1063 AG and 1363 CT are associated with susceptibility to Aspergillus, but all cases of bone marrow hematopoietic stem cell transplantation were studied. No research has been done on IA patients without underlying diseases. We have found that many IA patients do not have underlying diseases and there are no predisposing factors for other IA patients. Therefore, it is more meaningful to study this kind of patients, and there is a certain genetic difference between Han and white race in China. There is no literature report on the gene variation of IA patients in China, so it needs further study. Objective: to study the variation of TLR2TLR4 gene in Chinese Han population, and to explore the relationship between the mutation of TLRs gene and the susceptibility of IA. Methods: a total of 25 cases of invasive aspergillosis diagnosed and clinically diagnosed in our department from June 6 to December 2012 were collected, including 12 cases of immune impairment, 13 cases of healthy host without underlying diseases, 48 cases of non-IA patients with underlying diseases, 80 healthy volunteers. All the subjects were Chinese Han race. The peripheral blood of patients and volunteers were used to extract the DNA from the nucleated granulocytes of peripheral blood, and the information of TLR2TLR4 gene was searched by NCBI, and primers were designed. The coding region of TLR2nTLR4 gene was amplified by polymerase chain reaction (PCR) and sequenced. The sequence was compared between IA group and non-IA group. The difference between IA group and non-IA group was analyzed statistically by 蠂 2 test. The SMART online tool http: / / amart.embl-heidelberg.de) was used to analyze whether the mutation site caused the change of TLRs protein domain. Results the mutation of TLR2 gene in 1: 1: two mutation sites of TLR2, 597TCn1350TC, were found to be synonymous mutations, which did not cause amino acid changes. The incidence of the two loci in the control group was 1%, so they were all single nucleotide polymorphisms. There was no significant difference in the variation of TLR4 gene between the control group and the control group. The results showed that the mutation sites of 2 TLR4 were 219C > 1420GA.219C > G, which led to the transformation of 73rd position serine to arginine 1420GA, resulting in the conversion of glutamate to lysine at 474 position. In the control group, the rate of mutation was 1%, which was not reported at home and abroad. The prediction of protein structure showed that the change of TLR4219CG resulted in the deletion of a leucine-rich repeats in the extracellular domain of TLR4s. No SNPs loci associated with IA susceptibility were found abroad. Conclusion: no mutation of TLR2 coding region associated with IA susceptibility was found in this group of Han population. TLR4 mutation site 219CGN 1420GA was first found, in which TLR4219C > G may affect protein structure, and may lead to abnormal function of TLR4 to recognize pathogens. Thus, the host resistance to Aspergillus sp., so the mutation of TLR4 coding region may be related to IA susceptibility.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R519.8

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