系统性红斑狼疮患者外周血单个核细胞CREBL2，CDKN1B，GPR19基因表达研究

发布时间：2018-06-18 18:41

本文选题：SLE + 外周血单个核细胞　；参考：《安徽医科大学》2017年硕士论文

【摘要】：研究背景系统性红斑狼疮(SLE)是一种典型的较常见的自身免疫性结缔组织病,以多种自身抗体的产生和循环免疫复合物清除障碍为特征,患者临床表现的轻重缓急存在较大的个体差异,预后也不尽相同。目前大多认为其发病与遗传背景、个体生存环境、体内的雌激素水平等相关,其中遗传基础的影响显著。随着人类基因组学、遗传学研究方法的进步,对SLE的相关遗传学发生机制的研究越来越深入。不同地区的多个研究团队对不同人群进行研究,目前已发现超过60个SLE的易感基因或位点。2013年杨万岭等对多个人群的研究结果进行Meta分析发现,染色体12p13.1区域三个位置相邻的SLE易感基因基因CREBL2、CDKN1B、GPR19,且三者之间存在最小的有用连锁不平衡(minimum linkage disequilibrium,LD)。其中CREBL2,CDKN1B,GPR19对应的单个核苷酸多态性(single nucleotide polymorphism,SNP)rs12822507,rs10845606,rs34330与SLE之间的相关性均达到基因组水平的意义(3.8 3×10-17≤Pcombined≤2.2 3×10-8)。CREBL2(cAMP responsive element binding protein like 2)有48个碱基片段与CREB基因相同,该片段编码CREB蛋白的b ZIP区域,可与DNA结合,参与调节细胞周期的进程。GPR19(G protein-coupled receptor 19)在人体胚胎干细胞中高表达,编码孤儿受体蛋白,有GPCRs(G protein-coupled receptors)家族的7个跨膜结构域,其与多巴胺D2受体家族、肾上腺素受体、神经肽Y受体之间结构非常相似。CDKN1B(cyclin-dependent kinase inhibitor 1B)编码细胞周期蛋白依赖性激酶抑制剂,抑制DNA合成前期(G1期)到DNA合成期(S期)的进程,负调控细胞循环,阻碍细胞分化;同时,参与调控T细胞免疫耐受,以及CD4+T和CD8+T之间的动态平衡。既往已发现12p13区域多个基因参与自身免疫性疾病的发生,如NKG2,CD4,C1s/r,CD163,AID等,有关CREBL2,CDKN1B,GPR19与SLE的关联研究尚未深入。本实验利用基因分析的实时荧光定量PCR技术(Real-time Quantitative polymerase chain reaction,q PCR),检测CREBL2,CDKN1B,GPR19在SLE患者和健康对照个体中的相对表达量,进一步探索CREBL2,CDKN1B,GPR19与SLE发病的关联及可能的作用机制。目的研究SLE患者和正常对照个体的外周血单个核细胞(PBMCs)中CREBL2,CDKN1B,GPR19基因的相对表达量和组间的表达水平差异,探索基因的表达水平与SNP点rs12822507,rs10845606,rs34330之间的e QTL效应,以及基因表达水平与SLE临床表型、SLEDAI评分之间的相关性。方法收集SLE女性患者119例和女性正常对照129例的外周静脉血样本及临床病历信息,从PBMC中提取出RNA并经逆转录反应获得模板c DNA(complementary DNA),加入荧光染料试剂和基因CREBL2,CDKN1B,GPR19及内参基因GAPDH对应的引物,通过ABI 7500HT测定PBMC中CREBL2,CDKN1B,GPR19的相对表达量;应用ABI 3730XL测序仪对所有研究对象的SNP点rs12822507,rs10845606,rs34330进行基因分型;收集整理基因表达、基因分型结果以及临床资料信息,用SPSS17.0软件做统计学分析。结果SLE病例组中CREBL2和CDKN1B的相对表达量明显低于对照组,且差异具有统计学意义(P0.001)。未发现SNP点rs12822507,rs34330,rs10845606与基因CREBL2,CDKN1B,GPR19表达量之间存在e QTL作用,差异无统计学意义(P0.05)。CREBL2在CRP升高,或抗SSA/Ro抗体阳性,或光敏感阴性的SLE患者中表达水平降低,CDKN1B在C3正常或升高,或抗SSA/Ro抗体阳性,或SLEDAI10的SLE患者中表达水平降低(P0.05)。未发现基因表达量与SLEDAI之间存在线性相关(P0.05)。结论CREBL2,CDKN1B与SLE患病存在相关性,且CREBL2,CDKN1B与SLE的临床表型存在相关性,暂未发现SNP rs12822507,rs10845606,rs34330与三个基因表达之间存在e QTL效应。
[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.
【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】：R593.241

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