本文選題：侵襲性曲霉病　切入點：TLRa　出處：《南京大學》2013年碩士論文　論文類型：學位論文

【摘要】：侵襲性曲霉病(Invasive Aspergillosis, IA)不僅多發(fā)于免疫抑制患者,也可見于無基礎疾病的健康宿主。研究表明,TLRs參與宿主對曲霉的識別、信號轉導和促炎癥因子釋放,并調節(jié)免疫平衡,在宿主抵抗曲霉感染中起到重要作用。近幾年,國外已有多篇臨床研究報告發(fā)現TLR4基因多態(tài)性位點1063AG與1363CT與曲霉易感性相關,但均以骨髓造血干細胞移植的病例為研究對象,尚無對無基礎疾病的IA患者的研究。我們在臨床上發(fā)現許多IA患者并無基礎疾病,且不存在其它IA的易感因素,因此以這類患者作為研究對象更有意義。且我國漢族人種與白色人種存在一定的基因差異,國內至今尚無漢族人群IA患者基因變異的文獻報道,故有待進一步研究。 目的：研究我國漢族人群TLR2、TLR4編碼區(qū)基因變異情況,旨在探討漢族人群TLRs基因變異與IA易感性的關系。 方法：收集自2011.06至2012.12期間在我科確診及臨床診斷的侵襲性曲霉病25例,其中免疫受損者12例,無基礎疾病的健康宿主13例；有基礎疾病非IA病例48例,健康志愿者80例。所有研究對象均為我國漢族人種。采取患者及志愿者外周血,提取外周血有核粒細胞中的DNA；通過NCBI查找TLR2、TLR4基因信息,設計引物,采用聚合酶鏈式反應(PCR)擴增TLR2、TLR4基因編碼區(qū)序列后測序,比對序列,比較IA組與非IA組基因變異的差異,采用χ2檢驗進行統(tǒng)計學分析。同時分析突變點氨基酸改變,并通過SMART在線工具(http://amart.embl-heidelberg.de)分析突變位點是否造成TLRs蛋白質結構域改變。 結果：1、TLR2基因變異情況：測序發(fā)現2個TLR2的變異位點597TC,1350TC,均為同義突變,不引起氨基酸改變,兩個位點在對照組的發(fā)生率1%,故均為單核苷酸基因多態(tài)性位點,與對照組相比差異無統(tǒng)計學意義。2、TLR4基因變異情況：測序發(fā)現2個TLR4的變異位點219C＞G,1420GA。219C＞G導致第73位絲氨酸變?yōu)榫彼幔?420GA導致第474位谷氨酸變?yōu)橘嚢彼?對照組中的發(fā)生率1%,故為突變且國內外未見報道。蛋白結構預測發(fā)現TLR4219CG改變導致TLR4s胞外區(qū)缺失一個富含亮氨酸重復序列的結構域,1420GA經預測未發(fā)現蛋白結構改變。3、未發(fā)現國外報道與IA易感性相關的SNPs位點。 結論：本組漢族人群中未發(fā)現與IA易感相關的TLR2編碼區(qū)基因變異。首次發(fā)現TLR4突變位點219CG,1420GA,其中TLR4219C＞G可影響蛋白質結構,推測可能導致TLR4識別病原體功能的異常,從而影響宿主對曲霉的抵抗力,故TLR4編碼區(qū)基因變異可能與IA易感相關。
[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.
【學位授予單位】：南京大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R519.8

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