建立强直性脊柱炎患者ERAP变异基因功能体外研究细胞系

发布时间：2018-05-03 04:26

本文选题：强直性脊柱炎 + 内质网内氨基肽酶　；参考：《第二军医大学》2016年硕士论文

【摘要】：强直性脊柱炎(ankylosing spondylitis,AS)是一种常见的系统性自身免疫性关节炎,与银屑病关节炎、炎性肠病性关节炎、反应性关节炎等同属于血清阴性脊柱关节病(seronegative spondyloarthritis,SpA),有共同的疾病特点。AS好发于青壮年男性,起病隐匿,虽然诊断方法和手段的改进提高了早期诊断率,但还是有很多患者由于延误诊断和治疗而导致关节功能丧失、残疾,严重影响患者的生活质量,造成其家庭和社会的沉重的经济负担。AS是一种慢性炎症性关节炎,以脊柱和骶髂关节为著,表现为关节疼痛、晨僵,并导致关节融合和脊柱强直。因此,炎症是疾病发病机制中的重要因素,但对其发生的原因和过程的认知有限。目前尚无方法治愈AS,现有的治疗措施只能缓解疼痛、抑制炎症发展的进程,且只对部分病人有效。目的:强直性脊柱炎发病机制不明。40余年前发现其与HLA-B27强相关,但其在AS发病机制中的作用尚不清楚。全基因组关联研究(genome-wide association studies,GWAS)的发展发掘出越来越多与AS相关的基因位点。研究发现内质网内氨基肽酶(endoplasmic reticulum resident aminopeptidases,ERAP)也与疾病显著相关,且与HLA-B27存在基因间的相互作用。两者均位于内源性抗原递呈途径中,功能上也存在交互作用。本研究拟建立该交互作用的体外研究体系。方法:HLA-B27中与AS相关且常见的亚型为HLA-B27:04(汉族人群)和B27:05(高加索人种)。选择HLA-B27:04/B27:05+的AS患者和健康对照,用EBV将其B细胞转化为B淋巴母细胞(B lymphoblastoid cell line,B-LCL)。为将自然存在的ERAP基因变异与其抗原肽段的剪切功能相联系,将mRNA逆转录成cDNA后进行Sanger测序。通过CRISPR/Cas9技术分别或同时敲除这ERAP1和ERAP2基因,以下一步将野生型和测序获得的变异基因序列转染至上述细胞,比较其对HLA-B27限制性的自然抗原肽剪切功能的不同。为尽量体现其剪切功能的差异,需建立多种不同特异性的T细胞,其中包括HLA-B27:04/B27:05限制性异体特异性T细胞,故克隆HLA-B27:04/B27:05分子,建立C1R-B27:04/B27:05细胞系,予以刺激上述T细胞。结果:我们成功地将健康对照和AS患者来源的B细胞转化为B-LCL,用CRISPR/Cas9技术敲除了B-LCL的ERAP2基因。测序患者和对照外周血样本mRNA获得自然存在的ERAP1和ERAP2基因序列,ERAP1的5个AS相关单核苷酸多态性(single nucleotide polymorphism,SNP)的组成在患者和对照样本中未见明显差异,但ERAP2在AS患者中的变异较明显,且可能存在新的可变剪切。我们从患者B-LCL中克隆了HLA-B27:04基因,以建立C1R-B27:04细胞系,用于刺激HLA-B27:04限制性异体抗原特异性的T细胞。结论:本研究建立ERAP变异基因功能的体外研究体系的方法可行,待下一步建立HLA-B27:04/B27:05限制性的肽段特异性的T细胞,即可比较ERAP1基因变异所导致的肽段剪切功能变化,并可进一步按原计划敲除ERAP1基因。
[Abstract]:Ankylosing spondylitis (AS) is a common systemic autoimmune arthritis. It is associated with psoriatic arthritis, inflammatory enteritis, and reactive arthritis (reactive arthritis) as seronegative spinal arthropathy (seronegative spondyloarthritis, SpA). There is a common disease characteristic of.AS in young and young men, and the onset of disease is hidden. Although the improvement of diagnostic methods and methods improves the early diagnosis rate, there are still many patients with loss of joint function due to delayed diagnosis and treatment, which seriously affect the quality of life of the patients, and cause the heavy economic burden of their family and society.AS is a chronic inflammatory arthritis, with the spinal and sacroiliac joint as a kind of arthritis. As a result of joint pain, morning stiffness, joint fusion and spinal rigidity, inflammation is an important factor in the pathogenesis of the disease, but the causes and processes of the disease are limited. At present, there is no cure for AS. The existing treatment measures can only relieve pain, inhibit the progress of inflammation, and only be effective for some patients. The pathogenesis unknown.40 of ankylosing spondylitis has been found to be strongly associated with HLA-B27, but its role in the pathogenesis of AS is not clear. The development of genome-wide association studies (GWAS) has discovered more and more gene sites associated with AS. The study found that the endoplasmic reticulum aminopeptidase (endoplasmic re) is found. Ticulum resident aminopeptidases, ERAP) is also significantly associated with the disease, and the interaction between genes and HLA-B27. Both are located in the endogenous antigen presentation pathway, and there are interaction functions. This study intends to establish an in vitro research system for this interaction. Method: the common subtype of AS in HLA-B27 is HLA-B27:04 (HLA-B27:04). The Han population and the B27:05 (Caucasus). Select the AS patients and healthy controls of HLA-B27:04/B27:05+ and convert their B cells into B lymphoblastic cells (B lymphoblastoid cell line, B-LCL) with EBV. After CRISPR/Cas9, the ERAP1 and ERAP2 genes are knocked out, respectively. The following step is to transfect the mutant gene sequences of wild type and sequencing to the above cells to compare the different shear function of the natural antigen peptide to the HLA-B27 restriction. In order to reflect the difference of its shear function, a variety of different specific T cells need to be established. HLA-B27:04/B27:05 restrictive allogeneic T cells, therefore, clone HLA-B27:04/B27:05 molecules and establish C1R-B27:04/B27:05 cell lines to stimulate the T cells. Results: we successfully transformed healthy controls and B cells from AS patients to B-LCL, and the ERAP2 gene of B-LCL was knocked out with CRISPR/Cas9 technology. The peripheral blood sample mRNA obtained the natural ERAP1 and ERAP2 gene sequences, and the composition of the 5 AS related single nucleotide polymorphisms (single nucleotide polymorphism, SNP) of ERAP1 was not significantly different in the patient and the control sample, but the ERAP2 in the AS patient was more obvious, and there might be a new variable shear. We were from the patient B-LCL. The HLA-B27:04 gene was augmentation to establish the C1R-B27:04 cell line to stimulate the HLA-B27:04 restrictive alloantigen specific T cells. Conclusion: This study is feasible to establish an in vitro study system for the function of ERAP variant gene. The next step of establishing a HLA-B27:04/B27:05 restrictive peptide specific T cell can be used to compare the variation of ERAP1 gene. The changes of peptide shear function can be induced and the ERAP1 gene can be knocked out according to the original plan.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R593.23

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