结核分枝杆菌感染后自噬基因的表达水平和二甲双胍对自噬的干预的研究

发布时间：2018-04-22 01:07

本文选题：结核分枝杆菌 + 结核病　；参考：《北京市结核病胸部肿瘤研究所》2017年硕士论文

【摘要】：结核分枝杆菌是一种胞内感染菌,故在体液免疫应答过程中产生的抗体对它的免疫作用较弱,而对细胞内的结核分枝杆菌无作用,因此天然免疫在结核分枝杆菌的感染过程中起到非常重要的作用。自噬作为天然免疫应答中非常重要的一部分,参与众多先天免疫信号的调控。自噬是低氧、营养缺乏、细胞器老化等各种原因导致的细胞的应激反应,自噬不仅能降解细胞内老化的细胞器和蛋白质,还能清除细胞内感染的细菌和病毒。自噬相关蛋白(autophagy related protein,ATG)之间互相协同,引起自噬体形成过程中的细胞膜变化,当抗原提呈细胞一旦吞噬了结核分枝杆菌由单层膜结构组成的吞噬囊泡在瞬间就会被LC3蛋白修饰,自噬蛋白相互激活修饰最终成熟为自噬小体,自噬小体与溶酶体互相融合形成自噬溶酶体,将吞噬的结核分枝杆菌降解。细胞的自噬除了对病原体具有直接的杀伤作用,而且自噬在抗原提成中发挥不可替代的作用,自噬作用可以促进位于自噬体表面的MHC-ΙΙ分子与病原微生物的抗原表位结合,并提呈给CD4+T细胞,自噬同时可以通过MHC-Ι将抗原提呈给CD8+T细胞。本研究收集了初治肺结核病患者和健康志愿者共24人,使用实时定量PCR的方法检测了初治结核病患者(病例组)和健康志愿者(对照组)之间的自噬基因的水平,本研究发现,对照组mTOR基因表达水平是病例组的(2.76±1.56)倍;对照组Rheb基因表达水平是病例组的(1.85±0.91)倍,可见初治结核病患者中这两种基因表达下调,可在一定程度上促进自噬水平,抵抗M.tb感染。除此之外,研究发现对照组BECN1基因表达水平是病例组的(2.23±1.52)倍,说明初治肺结核患者可以通过下调BECN1基因的表达从而抑制自噬的发生,通过这一基因水平的变化可以得出BECN1通路和mTOR通路对自噬的调节是不一致的,甚至有可能是相反的调节。本研究同时检测了使用H37Rv感染THP-1细胞后自噬相关基因ATG5、ATG7、ATG12和Rheb在感染后30min、90min、4h、10h和24h的表达水平,研究发现ATG5、ATG7和ATG12在THP-1细胞感染H37Rv后30min后即表达上调,ATG5和ATG7基因在感染10h左右达到了基因表达的最高峰,自噬的调节基因Rheb表达水平在实验早期并没有显著的升高或者下降,在感染的时间段内(30min到24h),自噬的基因Rheb表达水平与感染前相比变化在2倍以内,故可以认为在体外实验中在H37Rv感染早期,Rheb基因没有明显变化;使用二甲双胍干预后的THP-1细胞在感染了BCG菌株后,自噬相关基因Rheb的表达水平在实验组(使用二甲双胍干预)和对照组(不使用二甲双胍干预)并没有显著的变化,而自噬相关基因mTOR在感染30min时有显著的表达下调,故二甲双胍可能在THP-1细胞在感染了BCG菌株早期抑制了自噬基因mTOR表达;除此之外,使用活细胞工作站观察使用BCG菌株感染后含有二甲双胍和不含有二甲双胍药物的细胞中mRFP-GFP-LC3B的含量,发现二甲双胍还可以促进自噬小体和溶酶体的融合,促进了THP-1细胞对BCG菌株的自噬。
[Abstract]:Mycobacterium tuberculosis is a kind of intracellular infection bacteria, so the antibody produced in the humoral immune response to it is weak, but it does not affect the Mycobacterium tuberculosis in the cell. Therefore, the natural immunity plays a very important role in the infection process of Mycobacterium tuberculosis. Autophagy is very important in the natural immune response. Part one participates in the regulation of numerous innate immune signals. Autophagy is the stress response of cells, such as hypoxia, nutritional deficiency, organelle aging and so on. Autophagy can not only degrade intracellular organelles and proteins, but also remove intracellular infection bacteria and diseases. Autophagy related protein (autophagy related protein, ATG) The cell membrane changes in the process of the formation of autophagosomes. Once the antigen presenting cells have phagocytic phagocytic vesicles composed of single layer membrane structure, the phagocytic vesicles of Mycobacterium tuberculosis are instantly modified by LC3 protein. Autophagic proteins interact with each other to mature into autophagic bodies, and autophagosomes and lysosomes are fused to form autophagy. The lysosome degrades the phagocytic Mycobacterium tuberculosis. Autophagy has a direct killing effect on the pathogen, and autophagy plays an irreplaceable role in the antigen preparation. Autophagy can promote the combination of the MHC- molecules on the surface of the autophagosome and the epitopes of the pathogenic microorganism, and present it to CD4+T cells and autophagy. At the same time, the antigen can be presented to CD8+T cells by MHC-. This study collected 24 patients with pulmonary tuberculosis and healthy volunteers. The level of autophagy gene was detected by real-time quantitative PCR. This study found that the mTOR gene expressed water in the control group. The level of the case group was (2.76 + 1.56) times, and the expression level of Rheb gene in the control group was (1.85 + 0.91) times that of the case group. It was found that the expression of these two genes was downregulated in the patients with primary TB, which could promote autophagy to a certain extent and resist M.tb infection. In addition, the study found that the expression level of BECN1 gene in the control group was (2.23 + 1.52) times that of the case group. It is suggested that the primary treatment of tuberculosis patients can inhibit the occurrence of autophagy by downregulating the expression of the BECN1 gene. Through the change of this gene level, the regulation of autophagy between the BECN1 pathway and the mTOR pathway is inconsistent, and may even be the opposite regulation. This study also detected the autophagy associated with the use of H37Rv infected THP-1 cells. The expression level of 30min, 90min, 4h, 10h and 24h after infection of the gene ATG5, ATG7, ATG12 and Rheb was found to be up and up after the infection of ATG5, ATG7 and ATG12. In the period of infection (30min to 24h), the expression level of autophagy gene Rheb is less than 2 times compared with that before infection. Therefore, it can be considered that in the early stage of H37Rv infection, the Rheb gene did not change obviously. The THP-1 cells using metformin after the infection of the BCG strain, the autophagy related gene Rhe. There was no significant change in the expression level of B in the experimental group (using metformin intervention) and the control group (without the use of metformin intervention), but the autophagy related gene mTOR decreased significantly in 30min infection, so metformin may inhibit the expression of autophagy gene mTOR in the early stage of the infection of the BCG strain of the BCG strain; in addition, The content of mRFP-GFP-LC3B in cells containing metformin and no metformin containing metformin after infection with BCG strain was observed with a live cell workstation. It was found that metformin also promoted the fusion of autophagosomes and lysosomes, and promoted autophagy of THP-1 cells to BCG.

【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R52

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