偶发分枝杆菌对THP-1细胞凋亡影响的初步研究
发布时间：2018-08-09 19:44
【摘要】：非结核分枝杆菌(Nontuberculous mycobacteria,NTM)一直被认为是仅限于与临床相关的环境性细菌,且因为人们对结核分枝杆菌(Mycobacterium tuberculosis,MTB)的高度关注而更加忽视了它的存在,直到艾滋病的大肆流行,NTM才被医疗卫生界所重视。NTM是机会致病菌,且具有多种可变的致病潜力,大部分NTM是腐物寄生菌,其在自然环境中分布广泛。迄今为止已发现170多种,其中约1/3与人类疾病有关。巨噬细胞的吞噬作用可以有效抑制MTB在体内的增殖,当然MTB亦可干扰巨噬细胞的凋亡,以逃逸免疫系统对MTB的清除作用。近年研究表明NTM的临床表现和致病机制与MTB十分相似,鉴于国内外关于NTM研究的报道相对较少,所以本文主要参考了一些MTB的相关研究。偶发分枝杆菌(M.fortuitum)为一种快速生长型的NTM,多分离自土壤及水体中,且常造成创伤部位和软组织感染,当宿主免疫力较低时即可导致发病。为探讨M.fortuitum对巨噬细胞凋亡的具体影响,进行了以下试验研究:首先,将分离自牛肠系膜淋巴结中的偶发分枝杆菌重新进行培养鉴定,构建偶发分枝杆菌感染THP-1细胞的感染模型,利用Hoechst 33258染色和透射电镜对M.fortuitum感染后THP-1细胞的凋亡情况进行观察,发现M.fortuitum感染可以造成THP-1细胞的凋亡,主要是在感染后6h至24h这个时间段细胞的凋亡变化较为明显。利用荧光定量PCR方法、Western-blot检测M.fortuitum感染后凋亡相关基因caspase-3、caspase-8及bcl-2在mRNA转录水平以及蛋白水平的变化,结果显示:偶发分枝杆菌感染后caspase-3、caspase-8的表达量在mRNA转录水平以及蛋白水平会随着作用时间延长而不断升高,具有一定的时间依赖性,不同的是caspase-3的mRNA转录水平于感染后12h时最高,而其蛋白水平则于24h时达到最高,这可能是因为蛋白水平的表达与mRNA转录水平相比,具有一定的时间延后性;Bcl-2的表达量则与caspase-3、caspase-8不同,从mRNA转录水平来看,在感染的起始阶段,其Bcl-2的表达量较高,从4h起逐渐下降,12h时最低,尤以分离株的下降最为明显,并且与对照组差异显著,蛋白水平上,偶发分枝杆菌感染THP-1细胞后,其Bcl-2的表达低于对照组。上述结果说明M.fortuitum感染后能引起THP-1细胞的凋亡,使得caspase-3、caspase-8的表达量升高,而减少了抗凋亡基因Bcl-2的表达。采用ELISA方法检测M.fortuitum感染后,与THP-1凋亡有关的细胞因子TNF-α、IL-6和IL-10分泌量的变化,结果显示TNF-α的分泌量随着作用时间加长而不断升高,12h时达到最高,呈现一定的时间依赖性;IL-6在0h-4h表达量较高,随后开始下降;在感染的初始阶段,IL-10的表达量较高,从感染后的6h开始则出现明显的下降趋势。这一结果说明M.fortuitum感染THP-1后能使促凋亡细胞因子TNF-α大量分泌,有助于细胞内凋亡过程的继续进行,而IL-6和IL-10表达量的降低可能是偶发分枝杆菌并没能抵抗巨噬细胞的清除作用。本试验初步研究了M.fortuitum感染对THP-1凋亡的影响。结果发现,M.fortuitum感染THP-1后经上调凋亡相关基因caspase-3、caspase-8的表达,减少抗凋亡基因Bcl-2的表达,调控相关细胞因子TNF-α、IL-6及IL-10的分泌,最终引起巨噬细胞的凋亡。这些试验结果将为揭示NTM对巨噬细胞凋亡影响的机制奠定基础,并为进一步研究NTM病免疫机制提供一定理论依据。
[Abstract]:Nontuberculous mycobacteria (NTM) has been considered to be limited to clinical related environmental bacteria, and because of the high attention to Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), the existence of it is ignored. Until the epidemic of AIDS, NTM is valued by the health community as a.NTM. It is an opportunistic pathogen and has a variety of variable pathogenicity potential. Most NTM is a parasitic fungus, which is widely distributed in the natural environment. Up to now, more than 170 are found, about which about 1/3 are related to human disease. Macrophage phagocytosis can effectively inhibit the proliferation of MTB in the body, and of course MTB can also interfere with the apoptosis of macrophages. The scavenging effect of escaping immune system on MTB. Recent studies have shown that the clinical and pathogenic mechanisms of NTM are very similar to that of MTB. In view of the relatively few reports about the NTM study at home and abroad, this paper mainly refers to some related studies of MTB. Mycobacterium sporogenes (M.fortuitum) is a fast growing NTM, which is separated from the soil and is more isolated from the soil. In water, it often causes the site of trauma and the infection of soft tissue. When the host immunity is low, the specific effects of M.fortuitum on the apoptosis of macrophages are discussed. The following experimental studies have been carried out. First, the isolated mycobacteria isolated from the mesenteric lymph nodes were recultured and identified to construct the Mycobacterium even. The infection model of THP-1 cells was observed by Hoechst 33258 staining and transmission electron microscopy. The apoptosis of THP-1 cells after M.fortuitum infection was observed. It was found that M.fortuitum infection could cause the apoptosis of THP-1 cells, mainly in the period of 6h to 24h after infection, the apoptosis of cells was more obvious. The fluorescence quantitative PCR method, We, was used. Stern-blot detected the changes of Caspase-3, Caspase-8 and Bcl-2 at mRNA transcriptional level and protein level after M.fortuitum infection. The results showed that the expression of Caspase-3, Caspase-8, at mRNA transcriptional level and protein level, increased with the time of action. The difference is that the mRNA transcriptional level of Caspase-3 is highest at 12h after infection, and the protein level reaches the highest at 24h, which may be because the expression of the protein level has a certain time delay compared to the mRNA transcriptional level, and the expression of Bcl-2 is different from that of Caspase-3, Caspase-8, from the mRNA transcriptional level, in the sense of sense. The expression of Bcl-2 was higher in the initial stage of dyeing, decreased gradually from 4h, and the lowest in 12h, especially with the drop of isolated strains, and the difference was significant with the control group. On the protein level, the expression of Bcl-2 was lower than that of the control group after infection of THP-1 cells. The results indicated that M.fortuitum infection could cause THP-1 cells after infection. Apoptosis, which increased the expression of Caspase-3 and caspase-8, reduced the expression of the anti apoptotic gene Bcl-2. The changes in the cytokine TNF- a, IL-6 and IL-10 secretion related to THP-1 apoptosis were detected by ELISA method. The results showed that the secretion of TNF- a increased with the longer time of action, and reached the highest in 12h. IL-6 expressed a certain time dependence; the expression of 0h-4h was higher and then began to decline; in the initial stage of infection, the expression of IL-10 was higher, and a significant decline from the beginning of 6h after infection showed that the M.fortuitum infection of THP-1 could make the apoptotic cell factor TNF- alpha secreted in large quantities and contribute to the apoptosis in the cells. The decrease in the expression of IL-6 and IL-10 may be the decrease in the expression of Mycobacterium tumefaciens and did not resist the scavenging effect of macrophages. The effect of M.fortuitum infection on the apoptosis of THP-1 was preliminarily studied. The results showed that the expression of Caspase-3, Caspase-8 was reduced by M.fortuitum infection after THP-1 infection, and the anti apoptotic basis was reduced. The expression of Bcl-2 regulates the secretion of related cytokines, TNF- a, IL-6 and IL-10, and eventually induces apoptosis of macrophages. These results will provide a basis for revealing the mechanism of NTM on the apoptosis of macrophages and provide a theoretical basis for further study of the immune mechanism of NTM disease.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61
本文编号：2175118
[Abstract]:Nontuberculous mycobacteria (NTM) has been considered to be limited to clinical related environmental bacteria, and because of the high attention to Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), the existence of it is ignored. Until the epidemic of AIDS, NTM is valued by the health community as a.NTM. It is an opportunistic pathogen and has a variety of variable pathogenicity potential. Most NTM is a parasitic fungus, which is widely distributed in the natural environment. Up to now, more than 170 are found, about which about 1/3 are related to human disease. Macrophage phagocytosis can effectively inhibit the proliferation of MTB in the body, and of course MTB can also interfere with the apoptosis of macrophages. The scavenging effect of escaping immune system on MTB. Recent studies have shown that the clinical and pathogenic mechanisms of NTM are very similar to that of MTB. In view of the relatively few reports about the NTM study at home and abroad, this paper mainly refers to some related studies of MTB. Mycobacterium sporogenes (M.fortuitum) is a fast growing NTM, which is separated from the soil and is more isolated from the soil. In water, it often causes the site of trauma and the infection of soft tissue. When the host immunity is low, the specific effects of M.fortuitum on the apoptosis of macrophages are discussed. The following experimental studies have been carried out. First, the isolated mycobacteria isolated from the mesenteric lymph nodes were recultured and identified to construct the Mycobacterium even. The infection model of THP-1 cells was observed by Hoechst 33258 staining and transmission electron microscopy. The apoptosis of THP-1 cells after M.fortuitum infection was observed. It was found that M.fortuitum infection could cause the apoptosis of THP-1 cells, mainly in the period of 6h to 24h after infection, the apoptosis of cells was more obvious. The fluorescence quantitative PCR method, We, was used. Stern-blot detected the changes of Caspase-3, Caspase-8 and Bcl-2 at mRNA transcriptional level and protein level after M.fortuitum infection. The results showed that the expression of Caspase-3, Caspase-8, at mRNA transcriptional level and protein level, increased with the time of action. The difference is that the mRNA transcriptional level of Caspase-3 is highest at 12h after infection, and the protein level reaches the highest at 24h, which may be because the expression of the protein level has a certain time delay compared to the mRNA transcriptional level, and the expression of Bcl-2 is different from that of Caspase-3, Caspase-8, from the mRNA transcriptional level, in the sense of sense. The expression of Bcl-2 was higher in the initial stage of dyeing, decreased gradually from 4h, and the lowest in 12h, especially with the drop of isolated strains, and the difference was significant with the control group. On the protein level, the expression of Bcl-2 was lower than that of the control group after infection of THP-1 cells. The results indicated that M.fortuitum infection could cause THP-1 cells after infection. Apoptosis, which increased the expression of Caspase-3 and caspase-8, reduced the expression of the anti apoptotic gene Bcl-2. The changes in the cytokine TNF- a, IL-6 and IL-10 secretion related to THP-1 apoptosis were detected by ELISA method. The results showed that the secretion of TNF- a increased with the longer time of action, and reached the highest in 12h. IL-6 expressed a certain time dependence; the expression of 0h-4h was higher and then began to decline; in the initial stage of infection, the expression of IL-10 was higher, and a significant decline from the beginning of 6h after infection showed that the M.fortuitum infection of THP-1 could make the apoptotic cell factor TNF- alpha secreted in large quantities and contribute to the apoptosis in the cells. The decrease in the expression of IL-6 and IL-10 may be the decrease in the expression of Mycobacterium tumefaciens and did not resist the scavenging effect of macrophages. The effect of M.fortuitum infection on the apoptosis of THP-1 was preliminarily studied. The results showed that the expression of Caspase-3, Caspase-8 was reduced by M.fortuitum infection after THP-1 infection, and the anti apoptotic basis was reduced. The expression of Bcl-2 regulates the secretion of related cytokines, TNF- a, IL-6 and IL-10, and eventually induces apoptosis of macrophages. These results will provide a basis for revealing the mechanism of NTM on the apoptosis of macrophages and provide a theoretical basis for further study of the immune mechanism of NTM disease.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61
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