基因CARD9在麻风病例及分枝杆菌感染的巨噬细胞中的表达研究

发布时间：2018-02-23 20:03

本文关键词： 麻风 CARD9基因差异表达　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:麻风病又称汉森氏病,是由麻风分枝杆菌感染易感个体导致的慢性肉芽肿性传染病,主要侵犯皮肤和外周神经,晚期可产生不可逆性畸残发生。在机体识别和抵御麻风分枝杆菌的过程中,固有免疫和适应性免疫都发挥了重要的作用。由于机体对麻风分枝杆菌的免疫反应不同,使麻风临床表现分为五型,且呈连续渐变的系谱状分布。麻风分枝杆菌至今未被成功地体外培养,我们对于麻风分枝杆菌的认识还很有限,这也限制了我们对麻风发病机制的深入研究。基因CARD9位于9号染色体上,编码胱天蛋白酶募集域蛋白9(caspase recruitment domain protein 9,CARD9)。CARD9蛋白是CARD蛋白家族成员之一,是一种重要的衔接蛋白,能通过蛋白-蛋白相互作用调节细胞内信号传导。CARD9是胞浆蛋白,在多种组织中都有表达,如:胸腺、脾、肺、骨髓等,尤其在骨髓来源巨噬细胞和树突状细胞中大量表达。它能调节炎症反应,高效整合多种固有免疫受体传导的信号,参与机体抵御真菌、细菌、病毒等多种病原微生物的免疫应答。研究目的:分析麻风患者与健康对照在皮肤组织和外周血中基因CARD9及其所在通路相关基因的表达差异,并利用小鼠巨噬细胞系进行感染实验验证,明确基因CARD9与麻风病之间的相关性,并对其作用机制进行初步的探讨。研究方法::1.采用转录组测序(RNA-Seq)技术对27例(多菌型19例,少菌型8例)麻风患者的皮损组织及18例健康志愿者的正常皮肤组织进行转录组测序,分析CARD9的表达差异;采用qPCR技术检测并比较11例患者皮损和5例健康对照正常皮肤组织中CARD9的mRNA表达水平;采用qPCR技术检测并比较10例患者和11例健康对照全血中CARD9的mRNA的表达水平。2.从27例麻风患者及18例健康志愿者皮肤组织转录组测序数据中选取与CARD9蛋白参与通路相关基因进行表达差异分析。3.采用qPCR技术测定用不同感染浓度的海鱼分枝杆菌刺激小鼠RAW264.7细胞后Card9的mRNA的表达水平。结果:1.CARD9基因在麻风病例皮损中的表达明显高于健康对照正常皮肤中,且此差异有统计学意义(RNA-Seq:p7.92E-5,qPCR:p0.007),多菌型和少菌型患者之间的表达差异也存在统计学意义(p0.022),在外周全血中CARD9基因表达未见明显差异(p0.124)。2.13个与CARD9通路相关的基因在麻风病例皮损与健康对照正常皮肤中表达存在显著差异(p0.05),但是在多菌性和少菌型患者之间大部分基因无明显差异。3.海鱼分枝杆菌刺激小鼠巨噬细胞12小时后,Card9的mRNA表达水平明显升高。结论:在麻风病例皮损中CARD9的mRNA表达水平显著高于健康对照正常皮肤中,这一结果在小鼠巨噬细胞感染实验中也得到了验证。CARD9与明确参与麻风的发病机制。
[Abstract]:Background: leprosy, also known as Hansen's disease, is a chronic granulomatous infectious disease caused by the susceptible individuals of Mycobacterium leprosy, mainly invading the skin and peripheral nerves. Both innate and adaptive immunity play an important role in the process of identification and resistance to Mycobacterium leprosy. Due to the different immune responses of the body to Mycobacterium leprosy, The clinical manifestations of leprosy are divided into five types, with a continuous and gradual pedigree distribution. Mycobacterium leprosy has not been successfully cultured in vitro, and our understanding of Mycobacterium leprosy is very limited. This also limits our in-depth study of leprosy pathogenesis. The gene CARD9 is located on chromosome 9 and encodes Caspase recruitment domain protein 9CARD9 protein, which is a member of CARD protein family and is an important link protein. Regulation of intracellular signal transduction by protein-protein interaction. CARD9 is a cytoplasmic protein that is expressed in a variety of tissues, such as thymus, spleen, lung, bone marrow, and so on. Especially in the bone marrow-derived macrophages and dendritic cells, it can regulate inflammatory response, effectively integrate the signals of various inherent immune receptors, participate in the body to resist fungi, bacteria, Objective: to investigate the expression of gene CARD9 and its related genes in skin and peripheral blood of leprosy patients and healthy controls. The relationship between CARD9 gene and leprosy was confirmed by using mouse macrophage cell line, and the mechanism of its action was preliminarily discussed. Methods: 1. 27 cases (19 cases of multibacterial type) were studied by RNA-Seq technique of transcriptome sequencing. The tissue of leprosy patients and normal skin tissues of 18 healthy volunteers were sequenced to analyze the difference of CARD9 expression. QPCR technique was used to detect and compare the mRNA expression of CARD9 in skin lesions of 11 patients and normal skin tissues of 5 healthy controls. QPCR technique was used to detect and compare the expression of CARD9 mRNA in whole blood of 10 patients and 11 healthy controls. From 27 leprosy patients and 18 healthy volunteers, CARD9 protein was selected to participate in the transcriptional sequence data of skin tissue. QPCR technique was used to detect the expression of Card9 mRNA in mouse RAW264.7 cells stimulated with different concentrations of Mycobacterium martensii. Results 1. The expression of CARD9 gene was significantly higher in leprosy skin lesions. In healthy control normal skin, The difference was statistically significant (RNA-Seq: p7.92E-5N qPCR: p0.007). There was also a statistically significant difference in the expression of CARD9 gene between the multicellular and oligozygote types. There was no significant difference in the expression of CARD9 gene in peripheral blood. There was no significant difference in the expression of CARD9 genes associated with CARD9 pathway in leprosy cases. There was a significant difference in the expression of P0.05G between the damaged and healthy normal skin, but there was no significant difference in the majority of genes between the multicellular and oligococcal patients. 3.The expression level of Card9 mRNA in murine macrophages stimulated by Mycobacterium martensii for 12 hours. Conclusion: the expression of CARD9 mRNA in leprosy is significantly higher than that in normal skin. This result was also verified in mouse macrophage infection experiment. CARD9 was involved in the pathogenesis of leprosy.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R755

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