發(fā)布時間：2018-06-18 18:41

  本文選題：SLE + 外周血單個核細胞�。� 參考：《安徽醫(yī)科大學》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締組織病,以多種自身抗體的產生和循環(huán)免疫復合物清除障礙為特征,患者臨床表現的輕重緩急存在較大的個體差異,預后也不盡相同。目前大多認為其發(fā)病與遺傳背景、個體生存環(huán)境、體內的雌激素水平等相關,其中遺傳基礎的影響顯著。隨著人類基因組學、遺傳學研究方法的進步,對SLE的相關遺傳學發(fā)生機制的研究越來越深入。不同地區(qū)的多個研究團隊對不同人群進行研究,目前已發(fā)現超過60個SLE的易感基因或位點。2013年楊萬嶺等對多個人群的研究結果進行Meta分析發(fā)現,染色體12p13.1區(qū)域三個位置相鄰的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對應的單個核苷酸多態(tài)性(single nucleotide polymorphism,SNP)rs12822507,rs10845606,rs34330與SLE之間的相關性均達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結合,參與調節(jié)細胞周期的進程。GPR19(G protein-coupled receptor 19)在人體胚胎干細胞中高表達,編碼孤兒受體蛋白,有GPCRs(G protein-coupled receptors)家族的7個跨膜結構域,其與多巴胺D2受體家族、腎上腺素受體、神經肽Y受體之間結構非常相似。CDKN1B(cyclin-dependent kinase inhibitor 1B)編碼細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)的進程,負調控細胞循環(huán),阻礙細胞分化;同時,參與調控T細胞免疫耐受,以及CD4+T和CD8+T之間的動態(tài)平衡。既往已發(fā)現12p13區(qū)域多個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CREBL2,CDKN1B,GPR19與SLE的關聯研究尚未深入。本實驗利用基因分析的實時熒光定量PCR技術(Real-time Quantitative polymerase chain reaction,q PCR),檢測CREBL2,CDKN1B,GPR19在SLE患者和健康對照個體中的相對表達量,進一步探索CREBL2,CDKN1B,GPR19與SLE發(fā)病的關聯及可能的作用機制。目的研究SLE患者和正常對照個體的外周血單個核細胞(PBMCs)中CREBL2,CDKN1B,GPR19基因的相對表達量和組間的表達水平差異,探索基因的表達水平與SNP點rs12822507,rs10845606,rs34330之間的e QTL效應,以及基因表達水平與SLE臨床表型、SLEDAI評分之間的相關性。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并經逆轉錄反應獲得模板c DNA(complementary DNA),加入熒光染料試劑和基因CREBL2,CDKN1B,GPR19及內參基因GAPDH對應的引物,通過ABI 7500HT測定PBMC中CREBL2,CDKN1B,GPR19的相對表達量;應用ABI 3730XL測序儀對所有研究對象的SNP點rs12822507,rs10845606,rs34330進行基因分型;收集整理基因表達、基因分型結果以及臨床資料信息,用SPSS17.0軟件做統(tǒng)計學分析。結果SLE病例組中CREBL2和CDKN1B的相對表達量明顯低于對照組,且差異具有統(tǒng)計學意義(P0.001)。未發(fā)現SNP點rs12822507,rs34330,rs10845606與基因CREBL2,CDKN1B,GPR19表達量之間存在e QTL作用,差異無統(tǒng)計學意義(P0.05)。CREBL2在CRP升高,或抗SSA/Ro抗體陽性,或光敏感陰性的SLE患者中表達水平降低,CDKN1B在C3正�；蛏�,或抗SSA/Ro抗體陽性,或SLEDAI10的SLE患者中表達水平降低(P0.05)。未發(fā)現基因表達量與SLEDAI之間存在線性相關(P0.05)。結論CREBL2,CDKN1B與SLE患病存在相關性,且CREBL2,CDKN1B與SLE的臨床表型存在相關性,暫未發(fā)現SNP rs12822507,rs10845606,rs34330與三個基因表達之間存在e QTL效應。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R593.241

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