發(fā)布時間：2018-02-05 23:18

  本文關鍵詞： 結核分枝桿菌(Mtb) 信號調節(jié)蛋白α(SIRPα) Linc RNA-Cox2 自噬 TLR信號轉導通路 Cox-2 TNF-α　出處：《河北北方學院》2015年碩士論文　論文類型：學位論文

【摘要】：結核病是嚴重威脅人類健康的全球性問題。近年來隨著臨床結核病耐藥的不斷增加以及艾滋病的廣泛流行,結核病的發(fā)病率和死亡率又有上升的趨勢。結核分枝桿菌(Mycobacterium tuberculosis,Mtb)是結核病的病原菌,其侵入人體后主要被單核巨噬細胞吞噬。Mtb很難被徹底清除,極易在細胞內持續(xù)潛伏,一旦被激活,即形成復發(fā)感染。結核病的許多癥狀并非Mtb的直接毒力所致,而是宿主免疫反應的表現(xiàn)。因此,WHO提倡采用化學藥物療法與免疫療法相結合的治療方案,促使我們進一步研究結核病的發(fā)病機制和保護性免疫機制�；蛐酒夹g是近幾年發(fā)展起來的新興技術,應用該技術進行高通量篩選及檢測分析,在尋找和篩查新基因、進行基因突變體和基因多態(tài)性檢測以及靶基因的預測等方面有著廣泛的應用前景。本研究應用全基因組測序技術,對結核菌感染模型的轉錄RNA(mRNA)、微小RNA(microRNA)和長鏈非編碼RNA(lincRNA)進行了篩選及鑒定,發(fā)現(xiàn)了結核菌感染中的關鍵蛋白-信號調節(jié)蛋白α(SIRPα)和關鍵基因lincRNA-Cox2。信號調節(jié)蛋白α(signal regulatory proteinα,SIRPα)是一種主要發(fā)揮抑制性作用的免疫球蛋白受體。已有研究發(fā)現(xiàn),脂多糖(LPS)引起的天然免疫應答中SIRPα是一個重要的免疫反應抑制子。LPS誘導的SIRPα表達下調能激活Toll樣受體(TLR)信號通路中天然免疫應答,對抵抗病原生物的入侵起到重要作用。但目前,SIRPα在Mtb感染中的作用尚不清楚。因此本研究在Mtb感染的小鼠巨噬細胞及結核病患者肺組織中驗證了Mtb感染下調SIRPα的現(xiàn)象。應用穩(wěn)定低表達SIRPα的小鼠巨噬細胞(SIRPα-KD),研究了SIRPα對巨噬細胞中結核菌生存率的影響,初步探討了下調SIRPα與自噬激活之間的內在聯(lián)系,并利用信號轉導通路中關鍵酶的磷酸化水平來研究下調SIRPα激活的tlr信號通路,初步研究了sirpα在mtb感染巨噬細胞后發(fā)揮負性調控作用的分子機制。lincrna-cox2是一條位于cox-2上游51kd的基因,在細胞初級免疫應答中起重要的作用。對感染與非感染mtb的巨噬細胞模型進行全基因組測序,經(jīng)pcr驗證發(fā)現(xiàn),lincrna-cox2的表達差異較為顯著,感染組較非感染組上調幅度為10倍以上。利用信號轉導通路抑制劑來研究lincrna-cox2殺菌的信號通路,發(fā)現(xiàn)tlr中p38mapk、jnk通路是mtb感染后上調lincrna-cox2的關鍵通路。本研究應用sirna轉染技術,建立瞬轉低表達lincrna-cox2的巨噬細胞系,研究了lincrna-cox2對mtb感染后炎癥因子tnf-α的影響,初步揭示了lincrna-cox2在mtb感染巨噬細胞后發(fā)揮殺菌作用的分子機制。本論文主要研究結果如下:1、建立了bcg感染小鼠巨噬細胞模型,從炎癥因子、炎癥反應關鍵酶兩個方面,驗證感染模型建立成功與否,為后續(xù)研究提供基礎。2、經(jīng)基因芯片篩選出差異表達基因,并從中選取sirpα和lincrna-cox2做進一步深入研究。經(jīng)過對細胞感染模型和結核病患者肺組織檢測,進一步驗證了基因芯片結果:感染mtb會下調sirpα的表達。3、sirpα在mtb感染中發(fā)揮負性調控作用。下調sirpα后會抑制mtb在巨噬細胞內的生存,這種抑菌作用有可能通過激活自噬而實現(xiàn)。4、下調sirpα后會激活p38mapk通路,并且引起下游tnf-α和炎癥反應關鍵酶cox-2的表達升高,又進一步促進了對mtb的殺滅。5、lincrna-cox2的殺菌作用可能是通過激活p38mapk、jnk通路,進而促進轉錄因子tnf-α的釋放,起到控制mtb感染的作用。綜上所述,sirpα在mtb感染巨噬細胞的過程中發(fā)揮負性調節(jié)作用。根據(jù)已有結果推測,下調sirpα可能通過激活p38mapk信號轉導通路誘導自噬的發(fā)生,參與結核菌感染中的炎癥反應的調節(jié)過程,為進一步闡明結核菌感染的免疫調控機制以及研發(fā)抗結核藥物提供了新的靶標。mtb感染可能通過激活tlr中p38mapk、jnk通路上調Linc RNA-Cox2的表達,進而促進前炎癥因子TNF-α的釋放,起到控制Mtb感染的作用,為結核病在基因水平上的診斷和治療提供參考。
[Abstract]:Tuberculosis is a global problem that threaten human health. In recent years, with the increase of clinical drug resistance tuberculosis and widespread AIDS, TB incidence and mortality have increased. Mycobacterium tuberculosis (Mycobacterium tuberculosis Mtb) is the pathogen of tuberculosis, it invades the human body mainly mononuclear macrophage phagocytosis of.Mtb is very difficult is completely clear, easily in the cells continued to lurk, once activated, the formation of recurrent infection caused by tuberculosis virulence directly. Many of the symptoms is not Mtb, but the host immune response. Therefore, WHO advocated treatment was chemotherapy and immunotherapy combined, prompts us to further investigate the tuberculosis mechanism and protective immune mechanism. Gene chip technology is a new technology developed in recent years, the application of the technology of high throughput screening Analysis and testing, in the search and screening new genes, has a broad application prospect for predicting gene mutants and gene polymorphism detection and target gene and so on. The research and application of whole genome sequencing technology, the transcription of RNA of Mycobacterium tuberculosis infection model (mRNA), micro RNA (microRNA) and long chain non encoding RNA (lincRNA) were screened and identified, found the key protein - TB infection in signal regulated protein alpha (SIRP alpha) and key gene lincRNA-Cox2. signal regulatory protein (signal regulatory protein SIRP alpha, alpha) is a main to exert the inhibitory effect of the immunoglobulin receptor. It has been found that lipopolysaccharide (LPS) SIRP alpha induced innate immune response is an important immune response suppressor.LPS induced down-regulation of SIRP alpha can activate Toll like receptor (TLR) signaling pathway in the innate immune response to pathogen resistance. The invasion has played an important role. However, the role of SIRP alpha in Mtb infection is not clear. So the study in Mtb infected macrophages and tuberculosis in lung tissues of patients with confirmed Mtb infection by SIRP alpha. Application of stable low expression of mouse macrophage SIRP alpha (SIRP alpha -KD), was studied SIRP effect on the survival rate of alpha of Mycobacterium tuberculosis in macrophages, discussed the relationship between the SIRP alpha and down-regulation of autophagy activation, and the key enzymes in the signal transduction pathway of phosphorylation of down-regulation of TLR signaling pathway SIRP alpha activation, preliminary research on.Lincrna-cox2 molecular mechanism of SIRP alpha played a negative role in MTB infection after macrophage is a COX-2 located upstream of 51kd gene, plays an important role in cell immune response in macrophages. The primary model of infection and non infection MTB by whole genome sequencing, the PCR results showed that the expression of lincrna-cox2 is significant. The infection group than in the non infected group increased by more than 10 times. To study the lincrna-cox2 signal pathway by bactericidal signal transduction pathway inhibitor, TLR p38MAPK, JNK pathway is a key upregulation of lincrna-cox2 after MTB infection. The research and application of siRNA technology to establish transient transfection. The low expression of lincrna-cox2 cells, studied the effects of lincrna-cox2 on inflammatory factor tnf- alpha MTB infection, reveal the molecular mechanism of lincrna-cox2 bacteria play a role in MTB infected macrophages after. The main results are as follows: 1, to establish a mouse model of BCG infection from macrophages, inflammatory cytokines, two key enzymes the inflammatory reaction, modeling the success of infection verification, provide the basis for further research of.2 by gene chip, we screened the differentially expressed genes, and choose from SIRP alpha and lincrna-cox2 for further study. After the cell infection model and lung tissue of patients with tuberculosis detection, further validation of the microarray results: the expression of.3 MTB infection will cut SIRP alpha, alpha SIRP played a negative role in MTB infection. The down-regulation of SIRP alpha after inhibition of MTB in macrophage survival this inhibitory effect is possible through the activation of autophagy and.4, downregulation of SIRP alpha activates the p38MAPK pathway, and increased expression of COX-2 alpha and tnf- key enzyme of downstream inflammatory reaction, and further promote the MTB to kill.5, bactericidal effect of lincrna-cox2 may be through the activation of p38MAPK JNK pathway, thereby promoting transcription factor tnf- alpha release, to control MTB infection. In conclusion, alpha SIRP play a negative regulatory role in the process of MTB infection of macrophages. Speculation on the basis of the results, SIRP may fall The occurrence of autophagy induced by activation of the p38MAPK signaling pathway, involved in the regulation of the inflammatory response to Mycobacterium tuberculosis infection, provide a new target of.Mtb infection may be through the activation of TLR in p38MAPK to further elucidate the immunoregulation mechanism of Mycobacterium tuberculosis infection and the development of anti tuberculosis drugs, the expression of JNK pathway upregulation of Linc RNA-Cox2, and then promote the proinflammatory factor TNF- alpha release to control Mtb infection, to provide reference for the diagnosis and treatment of tuberculosis at the gene level.

【學位授予單位】：河北北方學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R440

【共引文獻】

相關期刊論文 前2條

1 孫慧杰;董梅;王迪;;自噬參與結核桿菌感染免疫應答調控的研究進展[J];解放軍醫(yī)學院學報;2015年02期

2 金福姝;薛強;鄒明強;傅迎;孫福軍;李莉;;結核分枝桿菌臨床檢測技術的研究進展[J];中國冶金工業(yè)醫(yī)學雜志;2015年02期

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本文編號：1493018