本文關鍵詞： 麻風 CARD9基因 差異表達　出處：《山東大學》2017年碩士論文　論文類型：學位論文

【摘要】：研究背景:麻風病又稱漢森氏病,是由麻風分枝桿菌感染易感個體導致的慢性肉芽腫性傳染病,主要侵犯皮膚和外周神經(jīng),晚期可產(chǎn)生不可逆性畸殘發(fā)生。在機體識別和抵御麻風分枝桿菌的過程中,固有免疫和適應性免疫都發(fā)揮了重要的作用。由于機體對麻風分枝桿菌的免疫反應不同,使麻風臨床表現(xiàn)分為五型,且呈連續(xù)漸變的系譜狀分布。麻風分枝桿菌至今未被成功地體外培養(yǎng),我們對于麻風分枝桿菌的認識還很有限,這也限制了我們對麻風發(fā)病機制的深入研究�；駽ARD9位于9號染色體上,編碼胱天蛋白酶募集域蛋白9(caspase recruitment domain protein 9,CARD9)。CARD9蛋白是CARD蛋白家族成員之一,是一種重要的銜接蛋白,能通過蛋白-蛋白相互作用調節(jié)細胞內(nèi)信號傳導。CARD9是胞漿蛋白,在多種組織中都有表達,如:胸腺、脾、肺、骨髓等,尤其在骨髓來源巨噬細胞和樹突狀細胞中大量表達。它能調節(jié)炎癥反應,高效整合多種固有免疫受體傳導的信號,參與機體抵御真菌、細菌、病毒等多種病原微生物的免疫應答。研究目的:分析麻風患者與健康對照在皮膚組織和外周血中基因CARD9及其所在通路相關基因的表達差異,并利用小鼠巨噬細胞系進行感染實驗驗證,明確基因CARD9與麻風病之間的相關性,并對其作用機制進行初步的探討。研究方法::1.采用轉錄組測序(RNA-Seq)技術對27例(多菌型19例,少菌型8例)麻風患者的皮損組織及18例健康志愿者的正常皮膚組織進行轉錄組測序,分析CARD9的表達差異;采用qPCR技術檢測并比較11例患者皮損和5例健康對照正常皮膚組織中CARD9的mRNA表達水平;采用qPCR技術檢測并比較10例患者和11例健康對照全血中CARD9的mRNA的表達水平。2.從27例麻風患者及18例健康志愿者皮膚組織轉錄組測序數(shù)據(jù)中選取與CARD9蛋白參與通路相關基因進行表達差異分析。3.采用qPCR技術測定用不同感染濃度的海魚分枝桿菌刺激小鼠RAW264.7細胞后Card9的mRNA的表達水平。結果:1.CARD9基因在麻風病例皮損中的表達明顯高于健康對照正常皮膚中,且此差異有統(tǒng)計學意義(RNA-Seq:p7.92E-5,qPCR:p0.007),多菌型和少菌型患者之間的表達差異也存在統(tǒng)計學意義(p0.022),在外周全血中CARD9基因表達未見明顯差異(p0.124)。2.13個與CARD9通路相關的基因在麻風病例皮損與健康對照正常皮膚中表達存在顯著差異(p0.05),但是在多菌性和少菌型患者之間大部分基因無明顯差異。3.海魚分枝桿菌刺激小鼠巨噬細胞12小時后,Card9的mRNA表達水平明顯升高。結論:在麻風病例皮損中CARD9的mRNA表達水平顯著高于健康對照正常皮膚中,這一結果在小鼠巨噬細胞感染實驗中也得到了驗證。CARD9與明確參與麻風的發(fā)病機制。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R755

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