HMGB1及其基因多态性与山东鲁西南地区儿童过敏性紫癜的关联性研究

发布时间：2018-05-03 15:37

本文选题：HMGB1 + 基因多态性　；参考：《安徽医科大学》2017年硕士论文

【摘要】：背景与目的:过敏性紫癜(Henoch-Schonlein purpura,HSP)是儿童最常见的且有一定自限性的毛细血管和细小血管炎,除皮肤紫癜外,还可引起腹痛、关节炎甚至肾脏的损害,其中过敏性紫癜性肾炎(Henoch-Schonlein purpura nephritis,HSPN)是最严重的并发症,与过敏性紫癜病情的进展及预后关系最为密切。该病病因尚未完全明确,认为与遗传、微生物感染、药物、食物等有关,随着对人类基因学研究的不断探索,目前已发现多种基因的遗传多态性与HSP相关。HSP的发病近年呈上升趋势,影响患者的健康和生活质量,有必要对其病因及发病机制进行深入研究。高迁移率族蛋白(High mobility group protein,HMGB1)被认为是晚期重要的炎症介质,具有广泛的免疫调节作用,并与IL-6、TNF-a等炎症因子相关作用参与炎症的级联放大反应,其基因多态性与多种自身免疫性炎症疾病相关,但在HSP中尚未见报道。本研究旨在探讨HMGB1基因多态性与儿童HSP易感性的关系,筛查与疾病相关的SNP位点,并通过ELISA实验进一步从分子水平研究HMGB1在发病机制中起到的作用,为深入理解HSP的遗传易感性、阐明HSP的发病机制、疾病的风险预测提供理论依据,为临床诊疗提供新的思路。方法与资料:1、外周血样本选择。采用病例-对照研究设计,自2015年3月至2016年8月间,在济宁市第一人民医院病房及门诊确诊为HSP的160例患儿为HSP组,同期我院体检的82例健康儿童为对照组。2、标签单核苷酸多态性的筛选。HMGB1基因的转录起始位点上下游3-10kb范围内,通过HMGB1基因的连锁不平衡结构图,以及查阅前期文献,最终选择标签SNP(tagging SNP,tag SNP):rs2249825,rs1045411,rs1412125。3、引物设计及测序。测序的范围包括4个外显子以及包含在内含子中的两个标签SNP,共设计6对引物。应用ABI 3730测序仪对HMGB1基因的tag SNP进行测序分型,并根据每个碱基的高峰进行基因型的自行判读。4、统计分析。对标签SNP及筛查到的有意义的SNP的基因型频率、等位基因频率进行H-W平衡检验及统计分析,筛查与HSP/HSPN相关的SNP。5、血清样本选择。从160例HSP患儿中随机选择60例为HSP组,同期我院体检的59例健康儿童为对照组。6、血浆HMGB1、IL-6、INF-a的测定。取采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测两组HMGB1、IL-6、INF-a的水平。7、统计分析。T检验分析HMGB1、IL-6、INF-a在两组间的差异,并做HMGB1与IL-6、INF-a间的pearson相关性分析。结果:1、HMGB1基因的rs3742305位点,CC基因型在HSP组低于对照组,差异有统计学意(P=0.0140.05),G→C突变与HSP相关,C等位基因频率在两组间无显著性差异(P0.05),且在HSPN组与对照组中亦未发现基因型与等位基因频率存在统计学差异(P0.05),该位点多态性可能与HSPN的发生无关;rs2249825,rs1045411,rs1412125位点基因型和等位基因频率差异无统计学意义(P0.05),与HSP/HSPN的发生无相关性。2、HSP组血浆HMGB1、IL-6及INF-a浓度高于对照组并有统计学差异(P0.05),HMGB1与IL-6的表达、HMGB1与INF-a的表达均呈正相关。结论:1、本研究结果表明,在山东鲁西南地区患儿中,HMGB1基因rs3742305位点CC基因型与HSP相关,可能是其发病的保护因素,而该位点的基因多态性与HSPN的发生可能无关。但未发现rs2249825、rs1045411和rs1412125的基因多态性与HSP/HSPN的相关性。2、HMGB1在HSP疾病的炎症反应过程中起到重要作用,它可上调炎症因子IL-6、TNF-a的表达,可能促进了疾病的发生发展。
[Abstract]:Background and purpose: Henoch-Schonlein purpura (HSP) is the most common and self limiting capillary and small vasculitis in children. Besides skin purpura, it can also cause abdominal pain, arthritis and even renal damage. The most serious complication is allergic purpura nephritis (Henoch-Schonlein purpura nephritis, HSPN). The disease is most closely related to the progress and prognosis of Henoch Schonlein purpura. The cause of the disease is not completely clear. It is considered to be related to heredity, microbial infection, drugs, food and so on. With the continuous exploration of human genetics research, the genetic polymorphism of various genes and the incidence of HSP related.HSP have been on the rise in recent years. High mobility group protein (HMGB1) is considered to be an important late inflammatory mediator and has extensive immunomodulatory effects and is involved in the cascade amplification of inflammation, and its genes are associated with IL-6, TNF-a and other inflammatory factors. Polymorphism is associated with a variety of autoimmune inflammatory diseases, but it has not been reported in HSP. The purpose of this study was to explore the relationship between HMGB1 gene polymorphism and HSP susceptibility in children, to screen the SNP loci associated with disease, and to further study the role of HMGB1 in the pathogenesis of the disease from the molecular level by ELISA experiment in order to understand the remains of HSP in depth. Transmission of susceptibility, elucidate the pathogenesis of HSP, provide a theoretical basis for the prediction of the risk of disease, and provide new ideas for clinical diagnosis and treatment. 1, the selection of peripheral blood samples. A case control study was designed, and from March 2015 to August 2016, 160 cases of HSP in the ward and outpatient department of the first people's Hospital in Jining were group HSP, In the same period, 82 healthy children were selected as the control group.2. The label single nucleotide polymorphisms were used to screen the transcriptional initiation site of the.HMGB1 gene in the lower 3-10kb range, the linkage disequilibrium structure map of the HMGB1 gene, and the previous literature, and the final selection of the label SNP (tagging SNP, tag SNP): rs2249825, rs1045411, rs1412125.3, primers. Design and sequencing. The sequence includes 4 exons and two tags SNP contained in the intron. A total of 6 pairs of primers are designed. The tag SNP of the HMGB1 gene is sequenced by the ABI 3730 sequencer, and according to the peak of each base, a self-judged.4, a series analysis, and a meaningful SN for tagging SNP and screening are made. The genotype frequency of P, the allele frequency of H-W balance test and statistical analysis, the screening of SNP.5 related to HSP/HSPN and the selection of serum samples. From 160 children with HSP, 60 cases were randomly selected as group HSP, 59 healthy children in the same period of our hospital were the control group.6, the plasma HMGB1, IL-6, INF-a were measured. The enzyme linked immunosorbent assay (enzyme) was taken. Linked immunosorbent assay, ELISA) was used to detect the level.7 of two groups of HMGB1, IL-6 and INF-a. Statistical analysis of.T test was used to analyze the differences between HMGB1, IL-6, INF-a in the two groups. The mutation of G to C was associated with HSP, and there was no significant difference in the frequency of C allele between the two groups (P0.05), and there was no statistical difference between the genotype and allele frequencies in the HSPN group and the control group (P0.05). The polymorphism of the loci was not related to the occurrence of HSPN; rs2249825, rs1045411, rs1412125 loci, and allele frequency differences were not related to the polymorphism of the allele. There was no statistical significance (P0.05) and no correlation with HSP/HSPN, and the plasma HMGB1, IL-6 and INF-a concentrations in group HSP were higher than those of the control group (P0.05). The expression of HMGB1 and IL-6 was positively correlated with the expression of HMGB1 and INF-a. Conclusion: 1. Genotype is associated with HSP and may be a protective factor for its pathogenesis, and the genetic polymorphism of this loci may not be related to the occurrence of HSPN. But rs2249825, rs1045411 and rs1412125 gene polymorphisms are not found to be associated with HSP/HSPN.2. HMGB1 plays an important role in the inflammatory reaction of the HSP disease. It can up regulate the inflammatory factors IL-6, TNF-a. Expression may promote the development of the disease.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.5

【参考文献】