SIRPα及LincRNA-Cox2在结核菌感染中的作用研究

发布时间：2018-02-05 23:18

本文关键词： 结核分枝杆菌(Mtb) 信号调节蛋白α(SIRPα) Linc RNA-Cox2 自噬 TLR信号转导通路 Cox-2 TNF-α　出处：《河北北方学院》2015年硕士论文　论文类型：学位论文

【摘要】：结核病是严重威胁人类健康的全球性问题。近年来随着临床结核病耐药的不断增加以及艾滋病的广泛流行,结核病的发病率和死亡率又有上升的趋势。结核分枝杆菌(Mycobacterium tuberculosis,Mtb)是结核病的病原菌,其侵入人体后主要被单核巨噬细胞吞噬。Mtb很难被彻底清除,极易在细胞内持续潜伏,一旦被激活,即形成复发感染。结核病的许多症状并非Mtb的直接毒力所致,而是宿主免疫反应的表现。因此,WHO提倡采用化学药物疗法与免疫疗法相结合的治疗方案,促使我们进一步研究结核病的发病机制和保护性免疫机制。基因芯片技术是近几年发展起来的新兴技术,应用该技术进行高通量筛选及检测分析,在寻找和筛查新基因、进行基因突变体和基因多态性检测以及靶基因的预测等方面有着广泛的应用前景。本研究应用全基因组测序技术,对结核菌感染模型的转录RNA(mRNA)、微小RNA(microRNA)和长链非编码RNA(lincRNA)进行了筛选及鉴定,发现了结核菌感染中的关键蛋白-信号调节蛋白α(SIRPα)和关键基因lincRNA-Cox2。信号调节蛋白α(signal regulatory proteinα,SIRPα)是一种主要发挥抑制性作用的免疫球蛋白受体。已有研究发现,脂多糖(LPS)引起的天然免疫应答中SIRPα是一个重要的免疫反应抑制子。LPS诱导的SIRPα表达下调能激活Toll样受体(TLR)信号通路中天然免疫应答,对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感染的小鼠巨噬细胞及结核病患者肺组织中验证了Mtb感染下调SIRPα的现象。应用稳定低表达SIRPα的小鼠巨噬细胞(SIRPα-KD),研究了SIRPα对巨噬细胞中结核菌生存率的影响,初步探讨了下调SIRPα与自噬激活之间的内在联系,并利用信号转导通路中关键酶的磷酸化水平来研究下调SIRPα激活的tlr信号通路,初步研究了sirpα在mtb感染巨噬细胞后发挥负性调控作用的分子机制。lincrna-cox2是一条位于cox-2上游51kd的基因,在细胞初级免疫应答中起重要的作用。对感染与非感染mtb的巨噬细胞模型进行全基因组测序,经pcr验证发现,lincrna-cox2的表达差异较为显著,感染组较非感染组上调幅度为10倍以上。利用信号转导通路抑制剂来研究lincrna-cox2杀菌的信号通路,发现tlr中p38mapk、jnk通路是mtb感染后上调lincrna-cox2的关键通路。本研究应用sirna转染技术,建立瞬转低表达lincrna-cox2的巨噬细胞系,研究了lincrna-cox2对mtb感染后炎症因子tnf-α的影响,初步揭示了lincrna-cox2在mtb感染巨噬细胞后发挥杀菌作用的分子机制。本论文主要研究结果如下:1、建立了bcg感染小鼠巨噬细胞模型,从炎症因子、炎症反应关键酶两个方面,验证感染模型建立成功与否,为后续研究提供基础。2、经基因芯片筛选出差异表达基因,并从中选取sirpα和lincrna-cox2做进一步深入研究。经过对细胞感染模型和结核病患者肺组织检测,进一步验证了基因芯片结果:感染mtb会下调sirpα的表达。3、sirpα在mtb感染中发挥负性调控作用。下调sirpα后会抑制mtb在巨噬细胞内的生存,这种抑菌作用有可能通过激活自噬而实现。4、下调sirpα后会激活p38mapk通路,并且引起下游tnf-α和炎症反应关键酶cox-2的表达升高,又进一步促进了对mtb的杀灭。5、lincrna-cox2的杀菌作用可能是通过激活p38mapk、jnk通路,进而促进转录因子tnf-α的释放,起到控制mtb感染的作用。综上所述,sirpα在mtb感染巨噬细胞的过程中发挥负性调节作用。根据已有结果推测,下调sirpα可能通过激活p38mapk信号转导通路诱导自噬的发生,参与结核菌感染中的炎症反应的调节过程,为进一步阐明结核菌感染的免疫调控机制以及研发抗结核药物提供了新的靶标。mtb感染可能通过激活tlr中p38mapk、jnk通路上调Linc RNA-Cox2的表达,进而促进前炎症因子TNF-α的释放,起到控制Mtb感染的作用,为结核病在基因水平上的诊断和治疗提供参考。
[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.

【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R440

【共引文献】