本文選題：SLE + 外周血單個(gè)核細(xì)胞��； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景系統(tǒng)性紅斑狼瘡(SLE)是一種典型的較常見的自身免疫性結(jié)締組織病,以多種自身抗體的產(chǎn)生和循環(huán)免疫復(fù)合物清除障礙為特征,患者臨床表現(xiàn)的輕重緩急存在較大的個(gè)體差異,預(yù)后也不盡相同。目前大多認(rèn)為其發(fā)病與遺傳背景、個(gè)體生存環(huán)境、體內(nèi)的雌激素水平等相關(guān),其中遺傳基礎(chǔ)的影響顯著。隨著人類基因組學(xué)、遺傳學(xué)研究方法的進(jìn)步,對SLE的相關(guān)遺傳學(xué)發(fā)生機(jī)制的研究越來越深入。不同地區(qū)的多個(gè)研究團(tuán)隊(duì)對不同人群進(jìn)行研究,目前已發(fā)現(xiàn)超過60個(gè)SLE的易感基因或位點(diǎn)。2013年楊萬嶺等對多個(gè)人群的研究結(jié)果進(jìn)行Meta分析發(fā)現(xiàn),染色體12p13.1區(qū)域三個(gè)位置相鄰的SLE易感基因基因CREBL2、CDKN1B、GPR19,且三者之間存在最小的有用連鎖不平衡(minimum linkage disequilibrium,LD)。其中CREBL2,CDKN1B,GPR19對應(yīng)的單個(gè)核苷酸多態(tài)性(single nucleotide polymorphism,SNP)rs12822507,rs10845606,rs34330與SLE之間的相關(guān)性均達(dá)到基因組水平的意義(3.8 3×10-17≤Pcombined≤2.2 3×10-8)。CREBL2(cAMP responsive element binding protein like 2)有48個(gè)堿基片段與CREB基因相同,該片段編碼CREB蛋白的b ZIP區(qū)域,可與DNA結(jié)合,參與調(diào)節(jié)細(xì)胞周期的進(jìn)程。GPR19(G protein-coupled receptor 19)在人體胚胎干細(xì)胞中高表達(dá),編碼孤兒受體蛋白,有GPCRs(G protein-coupled receptors)家族的7個(gè)跨膜結(jié)構(gòu)域,其與多巴胺D2受體家族、腎上腺素受體、神經(jīng)肽Y受體之間結(jié)構(gòu)非常相似。CDKN1B(cyclin-dependent kinase inhibitor 1B)編碼細(xì)胞周期蛋白依賴性激酶抑制劑,抑制DNA合成前期(G1期)到DNA合成期(S期)的進(jìn)程,負(fù)調(diào)控細(xì)胞循環(huán),阻礙細(xì)胞分化;同時(shí),參與調(diào)控T細(xì)胞免疫耐受,以及CD4+T和CD8+T之間的動(dòng)態(tài)平衡。既往已發(fā)現(xiàn)12p13區(qū)域多個(gè)基因參與自身免疫性疾病的發(fā)生,如NKG2,CD4,C1s/r,CD163,AID等,有關(guān)CREBL2,CDKN1B,GPR19與SLE的關(guān)聯(lián)研究尚未深入。本實(shí)驗(yàn)利用基因分析的實(shí)時(shí)熒光定量PCR技術(shù)(Real-time Quantitative polymerase chain reaction,q PCR),檢測CREBL2,CDKN1B,GPR19在SLE患者和健康對照個(gè)體中的相對表達(dá)量,進(jìn)一步探索CREBL2,CDKN1B,GPR19與SLE發(fā)病的關(guān)聯(lián)及可能的作用機(jī)制。目的研究SLE患者和正常對照個(gè)體的外周血單個(gè)核細(xì)胞(PBMCs)中CREBL2,CDKN1B,GPR19基因的相對表達(dá)量和組間的表達(dá)水平差異,探索基因的表達(dá)水平與SNP點(diǎn)rs12822507,rs10845606,rs34330之間的e QTL效應(yīng),以及基因表達(dá)水平與SLE臨床表型、SLEDAI評分之間的相關(guān)性。方法收集SLE女性患者119例和女性正常對照129例的外周靜脈血樣本及臨床病歷信息,從PBMC中提取出RNA并經(jīng)逆轉(zhuǎn)錄反應(yīng)獲得模板c DNA(complementary DNA),加入熒光染料試劑和基因CREBL2,CDKN1B,GPR19及內(nèi)參基因GAPDH對應(yīng)的引物,通過ABI 7500HT測定PBMC中CREBL2,CDKN1B,GPR19的相對表達(dá)量;應(yīng)用ABI 3730XL測序儀對所有研究對象的SNP點(diǎn)rs12822507,rs10845606,rs34330進(jìn)行基因分型;收集整理基因表達(dá)、基因分型結(jié)果以及臨床資料信息,用SPSS17.0軟件做統(tǒng)計(jì)學(xué)分析。結(jié)果SLE病例組中CREBL2和CDKN1B的相對表達(dá)量明顯低于對照組,且差異具有統(tǒng)計(jì)學(xué)意義(P0.001)。未發(fā)現(xiàn)SNP點(diǎn)rs12822507,rs34330,rs10845606與基因CREBL2,CDKN1B,GPR19表達(dá)量之間存在e QTL作用,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。CREBL2在CRP升高,或抗SSA/Ro抗體陽性,或光敏感陰性的SLE患者中表達(dá)水平降低,CDKN1B在C3正常或升高,或抗SSA/Ro抗體陽性,或SLEDAI10的SLE患者中表達(dá)水平降低(P0.05)。未發(fā)現(xiàn)基因表達(dá)量與SLEDAI之間存在線性相關(guān)(P0.05)。結(jié)論CREBL2,CDKN1B與SLE患病存在相關(guān)性,且CREBL2,CDKN1B與SLE的臨床表型存在相關(guān)性,暫未發(fā)現(xiàn)SNP rs12822507,rs10845606,rs34330與三個(gè)基因表達(dá)之間存在e QTL效應(yīng)。
[Abstract]:Background systemic lupus erythematosus (SLE) is a typical and common autoimmune connective tissue disease. It is characterized by a variety of autoantibodies and scavenging disorders of circulating immune complex. There are large individual differences in the patient's clinical manifestation and the prognosis is different. Landscape, individual living environment, and estrogen levels in the body are related, in which the genetic basis has a significant impact. With the progress of human genomics and genetic research methods, the research on the genetic mechanism of SLE is becoming more and more in-depth. Several research teams in different regions have studied different populations, and more than 60 SLE have been found. Meta analysis of the research results of multiple populations, such as the susceptibility gene or.2013 Yang Wanling, found that the SLE susceptible gene gene CREBL2, CDKN1B, GPR19, and the minimum useful linkage disequilibrium (minimum linkage disequilibrium, LD) between the three parts of the chromosome 12p13.1 region were found in the chromosome 12p13.1 region. The single nucleotide polymorphisms (single nucleotide polymorphism, SNP) rs12822507, rs10845606, the correlation between rs34330 and SLE reached the significance of the genome level (3.83 * 10-17 < < Pcombined < 2.23 * 10-8).CREBL2 (cAMP responsive) and 48 base fragments were the same as those of the gene. The fragment was encoded. The B ZIP region of CREB protein, which can be combined with DNA, participates in the process of regulating cell cycle,.GPR19 (G protein-coupled receptor 19) is highly expressed in human embryonic stem cells, encoding the orphan receptor protein, and there are 7 trans membrane domains of the GPCRs (G protein-coupled receptors) family, which are associated with the dopamine receptor family, adrenoceptor, neuropeptide. The structure of the receptor is very similar to.CDKN1B (cyclin-dependent kinase inhibitor 1B), which encodes a cyclin dependent kinase inhibitor, which inhibits the process of DNA synthesis (G1 phase) to DNA synthesis phase (S phase), negatively regulates cell cycle and hinders cell differentiation; meanwhile, participates in the regulation of T cell immune tolerance, and the dynamics between CD4+T and CD8+T. In the past, many genes in the 12p13 region have been found to be involved in the occurrence of autoimmune diseases, such as NKG2, CD4, C1s/r, CD163, AID and so on. The correlation of CREBL2, CDKN1B, GPR19 and SLE has not been further studied. The relative expression of CDKN1B, GPR19 in SLE patients and healthy controls to further explore the association of CREBL2, CDKN1B, GPR19 with SLE and the possible mechanism of action. Objective to study the relative expression of CREBL2, CDKN1B, GPR19 genes and the difference of expression levels in peripheral blood mononuclear cells (PBMCs) of patients with SLE and normal controls. To explore the correlation between the expression level of gene and the e QTL effect between SNP point rs12822507, rs10845606, rs34330, and the correlation between the gene expression level and the SLE clinical phenotype and SLEDAI score. Methods the peripheral venous blood samples and the clinical records were collected from 119 cases of SLE female patients and 129 female normal controls, and RNA and inverse were extracted from PBMC. The transcriptional reaction obtained the template C DNA (complementary DNA), added the fluorescent dye reagent and the primers corresponding to the gene CREBL2, CDKN1B, GPR19 and the internal reference gene GAPDH. The expression of gene expression, genotyping and clinical data were collected and analyzed by SPSS17.0 software. Results in SLE case group, the relative expression of CREBL2 and CDKN1B was significantly lower than that of the control group, and the difference was statistically significant (P0.001). No SNP point rs12822507, rs34330, rs10845606 and gene CREBL2, CDKN1B, GPR were not found. There was no significant difference between the 19 expressions of E QTL, the difference was not statistically significant (P0.05).CREBL2 in CRP, or anti SSA/Ro positive, or light sensitive negative SLE, the level of expression decreased, CDKN1B in C3 normal or elevated, or anti SSA/Ro antibody positive, or SLEDAI10 SLE patients. There is a linear correlation between I (P0.05). Conclusion CREBL2, CDKN1B is associated with SLE disease, and CREBL2, there is a correlation between the clinical phenotype of CDKN1B and SLE, and there are no SNP rs12822507, rs10845606, rs34330 and the three gene expressions exist.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R593.241

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