發(fā)布時(shí)間：2018-05-03 15:37

  本文選題：HMGB1 + 基因多態(tài)性�。� 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景與目的:過(guò)敏性紫癜(Henoch-Schonlein purpura,HSP)是兒童最常見的且有一定自限性的毛細(xì)血管和細(xì)小血管炎,除皮膚紫癜外,還可引起腹痛、關(guān)節(jié)炎甚至腎臟的損害,其中過(guò)敏性紫癜性腎炎(Henoch-Schonlein purpura nephritis,HSPN)是最嚴(yán)重的并發(fā)癥,與過(guò)敏性紫癜病情的進(jìn)展及預(yù)后關(guān)系最為密切。該病病因尚未完全明確,認(rèn)為與遺傳、微生物感染、藥物、食物等有關(guān),隨著對(duì)人類基因?qū)W研究的不斷探索,目前已發(fā)現(xiàn)多種基因的遺傳多態(tài)性與HSP相關(guān)。HSP的發(fā)病近年呈上升趨勢(shì),影響患者的健康和生活質(zhì)量,有必要對(duì)其病因及發(fā)病機(jī)制進(jìn)行深入研究。高遷移率族蛋白(High mobility group protein,HMGB1)被認(rèn)為是晚期重要的炎癥介質(zhì),具有廣泛的免疫調(diào)節(jié)作用,并與IL-6、TNF-a等炎癥因子相關(guān)作用參與炎癥的級(jí)聯(lián)放大反應(yīng),其基因多態(tài)性與多種自身免疫性炎癥疾病相關(guān),但在HSP中尚未見報(bào)道。本研究旨在探討HMGB1基因多態(tài)性與兒童HSP易感性的關(guān)系,篩查與疾病相關(guān)的SNP位點(diǎn),并通過(guò)ELISA實(shí)驗(yàn)進(jìn)一步從分子水平研究HMGB1在發(fā)病機(jī)制中起到的作用,為深入理解HSP的遺傳易感性、闡明HSP的發(fā)病機(jī)制、疾病的風(fēng)險(xiǎn)預(yù)測(cè)提供理論依據(jù),為臨床診療提供新的思路。方法與資料:1、外周血樣本選擇。采用病例-對(duì)照研究設(shè)計(jì),自2015年3月至2016年8月間,在濟(jì)寧市第一人民醫(yī)院病房及門診確診為HSP的160例患兒為HSP組,同期我院體檢的82例健康兒童為對(duì)照組。2、標(biāo)簽單核苷酸多態(tài)性的篩選。HMGB1基因的轉(zhuǎn)錄起始位點(diǎn)上下游3-10kb范圍內(nèi),通過(guò)HMGB1基因的連鎖不平衡結(jié)構(gòu)圖,以及查閱前期文獻(xiàn),最終選擇標(biāo)簽SNP(tagging SNP,tag SNP):rs2249825,rs1045411,rs1412125。3、引物設(shè)計(jì)及測(cè)序。測(cè)序的范圍包括4個(gè)外顯子以及包含在內(nèi)含子中的兩個(gè)標(biāo)簽SNP,共設(shè)計(jì)6對(duì)引物。應(yīng)用ABI 3730測(cè)序儀對(duì)HMGB1基因的tag SNP進(jìn)行測(cè)序分型,并根據(jù)每個(gè)堿基的高峰進(jìn)行基因型的自行判讀。4、統(tǒng)計(jì)分析。對(duì)標(biāo)簽SNP及篩查到的有意義的SNP的基因型頻率、等位基因頻率進(jìn)行H-W平衡檢驗(yàn)及統(tǒng)計(jì)分析,篩查與HSP/HSPN相關(guān)的SNP。5、血清樣本選擇。從160例HSP患兒中隨機(jī)選擇60例為HSP組,同期我院體檢的59例健康兒童為對(duì)照組。6、血漿HMGB1、IL-6、INF-a的測(cè)定。取采用酶聯(lián)免疫吸附試驗(yàn)(enzyme linked immunosorbent assay,ELISA)檢測(cè)兩組HMGB1、IL-6、INF-a的水平。7、統(tǒng)計(jì)分析。T檢驗(yàn)分析HMGB1、IL-6、INF-a在兩組間的差異,并做HMGB1與IL-6、INF-a間的pearson相關(guān)性分析。結(jié)果:1、HMGB1基因的rs3742305位點(diǎn),CC基因型在HSP組低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意(P=0.0140.05),G→C突變與HSP相關(guān),C等位基因頻率在兩組間無(wú)顯著性差異(P0.05),且在HSPN組與對(duì)照組中亦未發(fā)現(xiàn)基因型與等位基因頻率存在統(tǒng)計(jì)學(xué)差異(P0.05),該位點(diǎn)多態(tài)性可能與HSPN的發(fā)生無(wú)關(guān);rs2249825,rs1045411,rs1412125位點(diǎn)基因型和等位基因頻率差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),與HSP/HSPN的發(fā)生無(wú)相關(guān)性。2、HSP組血漿HMGB1、IL-6及INF-a濃度高于對(duì)照組并有統(tǒng)計(jì)學(xué)差異(P0.05),HMGB1與IL-6的表達(dá)、HMGB1與INF-a的表達(dá)均呈正相關(guān)。結(jié)論:1、本研究結(jié)果表明,在山東魯西南地區(qū)患兒中,HMGB1基因rs3742305位點(diǎn)CC基因型與HSP相關(guān),可能是其發(fā)病的保護(hù)因素,而該位點(diǎn)的基因多態(tài)性與HSPN的發(fā)生可能無(wú)關(guān)。但未發(fā)現(xiàn)rs2249825、rs1045411和rs1412125的基因多態(tài)性與HSP/HSPN的相關(guān)性。2、HMGB1在HSP疾病的炎癥反應(yīng)過(guò)程中起到重要作用,它可上調(diào)炎癥因子IL-6、TNF-a的表達(dá),可能促進(jìn)了疾病的發(fā)生發(fā)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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R725.5

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