金黄色葡萄球菌肠毒素B引起THP-1细胞凋亡的作用与机制研究
发布时间：2018-07-13 16:35
【摘要】：细胞凋亡(Apoptosis)是多细胞生物中重要的生物学过程,具有影响组织分化、新陈代谢及免疫调节等多方面功能。在细菌引起的感染性疾病中,细菌的特定组分可以通过多种途径调控宿主细胞的凋亡,而这也被证实对疾病的进程有着至关重要的影响。金黄色葡萄球菌(Staphylococcus aureus)作为重要的人类病原菌,在其感染过程中也可引起宿主细胞凋亡。在许多与金黄色葡萄球菌密切相关的疾病,如特应性皮炎(Atopic Dermatitis,AD)和脓毒症(sepsis)中,异常的宿主细胞凋亡现象可显著影响疾病的进程、严重程度和预后。金黄色葡萄球菌的一个重要特点是其可分泌多种毒素,包括溶血素、杀白细胞素、肠毒素及分泌酶等。其中肠毒素是一种重要的金黄色葡萄球菌超抗原,在很多与金黄色葡萄球菌密切相关的疾病中发挥着重要的作用。肠毒素的天然受体包括T细胞受体(TCR)和主要组织相容性复合体II类分子(major histocompatibility complex II,MHCII),金葡菌肠毒素结合TCR可持续激活T细胞引起超敏反应;而结合MHCII则具有促细胞凋亡作用。在肠毒素家族中,金黄色葡萄球菌肠毒素B(staphylococcal enterotoxin B,SEB)分布较广、研究也较为深入。SEB已被证实能够引起T细胞和外周血单核细胞(PBMC)凋亡,且这种细胞凋亡与金黄色葡萄球菌引起的疾病,如AD等密切相关。但SEB是否能引起单核-巨噬细胞凋亡,以及其分子机制目前仍不清楚。本研究从我室分离并完成全基因组测序的金黄色葡萄球菌XQ株基因组中克隆、表达并纯化了具有生物学活性的重组SEB蛋白,开展了SEB引起THP-1单核-巨噬细胞凋亡的作用及其机制研究,发现了一个依赖于TNFα的正反馈回路在此过程中起到非常重要的作用;同时发现金属硫蛋白2A(metallothionein 2A,MT2A)可通过促进SEB引起的TNFα表达上调,从而促进TNFα正反馈回路的形成,在SEB引起THP-1细胞凋亡的过程中具有重要地位。所得研究结果为将来深入阐明金黄色葡萄球菌肠毒素的作用机制以及宿主细胞应对毒素攻击的机制提供了重要参考。本论文的主要研究方法和结果如下:1.重组SEB蛋白的表达载体构建、表达与纯化以金黄色葡萄球菌XQ株全基因组为模板,利用PCR技术获得SEB编码序列,再通过BamHI+Xho I双酶切以及采用T4连接酶与表达载体连接,成功构建了表达质粒pET30a-SEB。将表达质粒p ET30a-SEB转化入表达菌株大肠埃希菌C43后,成功诱导表达出重组SEB蛋白。表达的重组SEB蛋白经过镍柱亲和层析纯化后,利用去内毒素凝胶柱去除内毒素、超滤柱浓缩并置换缓冲液为PBS缓冲液,最终以SDS-PAGE测定重组蛋白纯度、Bradford法测定蛋白质浓度及鲎试剂反应实验检测残留内毒素,结果重组SEB蛋白的纯度在95%以上,浓度为539.2μg/ml,毒素含量低于2EU/ml,满足后续细胞实验要求。2.重组SEB蛋白具有引起THP-1细胞凋亡的作用委托北京阅微基因技术有限公司对我们获赠的THP-1细胞系进行STR检测,细胞中没有发现人类细胞交叉污染,与美国模式培养物保存中心(American type culture collection,ATCC)中THP-1细胞的STR数据匹配率为93.33%,证实我们的细胞系为THP-1的衍生细胞系。利用Annexin V+PI双染色及流式细胞分析,确定了重组SEB蛋白具有引起THP-1细胞凋亡作用,细胞凋亡比例约为3%,并且具有时间和剂量依赖效应。利用广谱caspase抑制剂Z-VAD-FMK,我们证实重组SEB蛋白引起的THP-1细胞凋亡依赖caspase的级联激活。在IFN-γ激活及PMA诱导分化后的THP-1细胞中,利用Annexin V+PI双染色及流式细胞分析,检测到重组SEB蛋白引起的细胞凋亡比例更高,分别约为7.5%和15%。内毒素激活THP-1细胞后并不增强重组SEB蛋白引起THP-1细胞凋亡的作用。然而IFN-γ激活和PMA诱导分化后,阴性对照组中THP-1细胞凋亡也有所上升。为避免额外因素的干扰,我们在接下来的研究中均直接使用重组SEB蛋白作用于THP-1细胞而不再进行额外的细胞激活或诱导分化。3.重组SEB蛋白引起THP-1细胞凋亡依赖于一个TNFα的正反馈回路经典的细胞凋亡包括外源性(caspase 8激活)和内源性(caspase 9激活)两条途径,我们通过caspase-3,-8,-9活性检测试剂盒对重组SEB蛋白作用后的TPH-1细胞之相应酶活性检测发现,重组SEB蛋白引起的THP-1细胞凋亡依赖caspase-3和-8的激活而不依赖caspase-9的激活,说明外源性通路在SEB引起THP-1细胞凋亡过程中发挥重要作用。利用RT-q PCR检测重组SEB蛋白处理后THP-1细胞中凋亡相关分子的转录水平,结果发现重组SEB蛋白处理后,THP-1细胞中TNFα和HLA-DRa的转录水平显著升高。进一步利用ELISA和Western blot实验,我们证实了重组SEB蛋白在THP-1细胞中引起的TNFα和HLA-DRa表达升高。利用anti-TNFα单克隆抗体中和TNFα作用后,检测到重组SEB蛋白引起的THP-1细胞凋亡比例下降至约1%,与重组SEB蛋白直接作用后的3.5%存在显著差异,而同型对照抗体则无此作用,证实TNFα在SEB引起的THP-1细胞凋亡过程中发挥关键作用。利用si RNA干扰HLA-DRa和TNFR1的表达,我们发现在干扰后的THP-1细胞中,重组SEB蛋白引起的凋亡细胞比例(约2.5%)显著低于用无目标si RNA干扰的对照组细胞(约9%)。同时,HLA-DRa和TNFR1 siRNA干扰的THP-1细胞在重组SEB蛋白作用后,TNFα分泌水平也显著低于无目标siRNA干扰的对照组细胞。而TNFα本身又会上调HLA-DRa的表达,从而增加了重组SEB蛋白与HLA-DRa的结合,使得TNFα的分泌进一步增加,于是形成了一个依赖于TNFα的正反馈回路。重组SEB蛋白引起的THP-1细胞凋亡依赖于这一正反馈回路的形成和完整。4.MT2A通过促进TNFα的正反馈回路从而促进重组SEB蛋白引起的THP-1细胞凋亡作为SEB的受体,THP-1细胞的HLA-DRa仅有的胞质区仅含15个氨基酸残基,且不含任何已知的结构域,HLA-DRa与SEB结合后,后续的信号转导很可能依靠与HLA-DRa直接结合的其他膜蛋白来实现。且SEB结合HLA-DRa以后如何上调TNFα的表达机制也不清楚。我们利用免疫共沉淀技术,通过anti-HLA-DRa单克隆抗体去捕捉与HLA-DRa相互作用的蛋白质,发现了一个金属硫蛋白(metallothioneins,MTs)MT2A可能参与了SEB引起的THP-1细胞凋亡作用过程。我们利用RT-q PCR分析了THP-1细胞中MTs的表达谱及其在重组SEB蛋白作用后转录水平的改变,结果发现THP-1细胞中表达的MTs包括MT1E、F、G、X和MT2A,在重组SEB蛋白作用后,仅MT2A表达上升。Western blot实验进一步证实了重组SEB蛋白作用后THP-1细胞中MT2A的表达上升约50%。利用Annexin V+PI双染色及流式细胞分析,我们发现在siRNA干扰MT2A表达的THP-1细胞中,重组SEB蛋白作用后的细胞凋亡比例(约4%)明显少于无目标si RNA干扰的对照组细胞(约8%)。利用ELISA和Western blot实验,我们进一步检测到在siRNA干扰MT2A表达的THP-1细胞中,重组SEB蛋白作用后TNFα和HLA-DRa表达水平均降低,说明MT2A通过促进TNFα正反馈回路形成从而在重组SEB蛋白引起的THP-1细胞凋亡发挥重要作用。这是初次发现细菌SEB对MTs的表达存在调控作用,为将来深入研究细菌毒素调控MTs的作用和机制奠定了理论基础。
[Abstract]:Apoptosis (Apoptosis) is an important biological process in multicellular organisms, which has many functions that affect tissue differentiation, metabolism and immunomodulation. In the infectious diseases caused by bacteria, the specific components of bacteria can regulate the apoptosis of host cells through a variety of ways, and this has also been proved to be crucial to the process of disease. Important effects. Staphylococcus aureus, as an important human pathogen, can also cause cell apoptosis in the process of infection. In many diseases closely related to Staphylococcus aureus, such as atopic dermatitis (Atopic Dermatitis, AD) and sepsis (sepsis), abnormal host cell apoptosis can be found. The important characteristic of Staphylococcus aureus is that it can secrete a variety of toxins, including hemolysin, leukin, enterotoxin and secretase. Enterotoxin is an important Staphylococcus aureus superantigen, in many diseases closely related to Staphylococcus aureus. The natural receptors of enterotoxin include the T cell receptor (TCR) and the major histocompatibility complex II (major histocompatibility complex II, MHCII). Staphylococcus aureus enterotoxin combined with TCR to activate the hypersensitivity of T cells, while MHCII has the role of promoting apoptosis. In the enterotoxin family, gold The distribution of staphylococcal enterotoxin B (staphylococcal enterotoxin B, SEB) is widely distributed, and the research is also in depth that.SEB has been proved to cause apoptosis in T cells and peripheral blood mononuclear cells (PBMC), and this cell apoptosis is closely related to the disease caused by Staphylococcus aureus, such as AD, but whether SEB can cause mononuclear macrophage apoptosis, And its molecular mechanism is still unclear. This study cloned the genome of Staphylococcus aureus XQ strains isolated and completed genome sequencing from our lab, expressed and purified the recombinant SEB protein with biological activity, and carried out the study on the use and mechanism of SEB to induce THP-1 mononuclear macrophage apoptosis, and found a dependence on TN. The positive feedback loop of F alpha plays a very important role in this process. At the same time, it is found that metallothionein 2A (metallothionein 2A, MT2A) can increase the expression of TNF alpha induced by SEB, thus promoting the formation of TNF alpha positive feedback loop, which is important in the process of SEB induced apoptosis of THP-1 cells. The results of this study are in depth in the future. The mechanism of the action of Staphylococcus aureus enterotoxin and the host cells provide important reference for the mechanism of the toxin attack. The main research methods and results in this paper are as follows: 1. the expression vector of recombinant SEB protein was constructed, the whole genome of Staphylococcus aureus XQ strain was expressed and purified as a template, and the SEB coding was obtained by PCR technology. The expression plasmid pET30a-SEB. was successfully constructed and the expression plasmid P ET30a-SEB was transformed into the expression strain of Escherichia coli C43 by BamHI+Xho I double enzyme digestion and the expression plasmid pET30a-SEB. was successfully constructed, and the recombinant SEB protein was successfully induced. The recombinant SEB protein expressed in the expression was purified by nickel column affinity chromatography and used to remove the endotoxin. The endotoxin was removed by the plain gel column. The ultrafiltration column was concentrated and replaced by PBS buffer solution. The purity of the recombinant protein was determined by SDS-PAGE. The concentration of protein and the reaction test of the reagents were detected by Bradford. The purity of the recombinant SEB protein was above 95%, the concentration was 539.2 Mu g/ml and the content of the toxin was lower than 2EU/ml. Cell experiments require that.2. recombinant SEB protein have the role of inducing apoptosis of THP-1 cells, and the Beijing microgene Technology Co., Ltd. is entrusted with the STR detection of the THP-1 cell lines we have received. No human cell cross contamination is found in the cells, and the THP-1 cells in the American model culture center (American type culture collection, ATCC) are not found. The matching rate of STR data was 93.33%, which confirmed that our cell line was a derived cell line of THP-1. Using Annexin V+PI double staining and flow cytometry, the recombinant SEB protein could induce apoptosis of THP-1 cells, the proportion of apoptosis was about 3%, and it had time and dose dependent effects. The broad-spectrum caspase inhibitor Z-VAD-FMK, I was used. We confirmed that the apoptosis of THP-1 cells induced by recombinant SEB protein is dependent on the cascade activation of caspase. In THP-1 cells activated by IFN- gamma and PMA induced THP-1 cells, the proportion of apoptosis induced by recombinant SEB protein is higher by Annexin V+PI double staining and flow cytometry, which is about 7.5% and 15%. endotoxin activates THP-1 cells, respectively. The apoptosis of THP-1 cells was enhanced by the enhanced recombinant SEB protein. However, after IFN- gamma activation and PMA induced differentiation, the apoptosis of THP-1 cells in the negative control group was also increased. In order to avoid the interference of additional factors, we have used the recombinant SEB protein directly to act on THP-1 cells in the next study without any additional cell activation or induction. Differentiation of.3. recombinant SEB protein induced THP-1 cell apoptosis dependent on a TNF alpha positive feedback loop classic apoptosis including exogenous (caspase 8 activation) and endogenous (caspase 9 activation) two pathways. We detected the enzyme activity of the enzyme activity of TPH-1 cells after the action of recombinant SEB protein by Caspase-3, -8, and -9 activity detection kits The apoptosis of THP-1 cells induced by recombinant SEB protein is dependent on the activation of Caspase-3 and -8 without relying on the activation of caspase-9, indicating that the exogenous pathway plays an important role in the apoptosis of THP-1 cells induced by SEB. The transcriptional level of the apoptotic phase molecules in the THP-1 cells after the recombinant SEB protein is detected by the RT-q PCR, and the recombinant SEB eggs are found. After white treatment, the transcriptional level of TNF alpha and HLA-DRa in THP-1 cells increased significantly. Further using ELISA and Western blot experiments, we confirmed that the expression of TNF alpha and HLA-DRa induced by recombinant SEB protein increased in THP-1 cells. The apoptosis induced by recombinant protein was detected by the action of anti-TNF alpha monoclonal antibody and TNF alpha. The ratio decreased to about 1%, which was significantly different from the 3.5% of the recombinant SEB protein, while the same type control antibody had no effect. It proved that TNF alpha played a key role in the apoptosis of THP-1 cells induced by SEB. Using Si RNA to interfere with the expression of HLA-DRa and TNFR1, we found that the recombinant SEB protein was caused in the interfered THP-1 cells. The percentage of apoptotic cells (about 2.5%) was significantly lower than that of the control group (about 9%) with non target Si RNA interference. At the same time, the level of TNF a secreted by HLA-DRa and TNFR1 siRNA interfered with the recombinant SEB protein was also significantly lower than that of the control group without the target siRNA interference. And TNF a itself would increase the HLA-DRa expression, thereby increasing the weight. The combination of group SEB protein and HLA-DRa makes the secretion of TNF a further increase, thus forming a positive feedback loop dependent on TNF alpha. The apoptosis of THP-1 cells caused by recombinant SEB protein depends on the formation of this positive feedback loop and the complete.4.MT2A by promoting the positive feedback pathway of TNF alpha to promote THP-1 fineness caused by the recombinant SEB protein. Apoptosis is the receptor of SEB, the only cytoplasmic region of THP-1 cell HLA-DRa contains only 15 amino acid residues and does not contain any known domain. After the combination of HLA-DRa and SEB, the subsequent signal transduction may depend on other membrane proteins which are directly combined with HLA-DRa. And how to increase the expression mechanism of TNF alpha after SEB binding HLA-DRa It is not clear that we use immunoprecipitation technique to capture proteins interacting with HLA-DRa by anti-HLA-DRa monoclonal antibody, and we found that a metallothioneins (MTs) MT2A may be involved in the apoptosis process of THP-1 cells induced by SEB. We use RT-q PCR to analyze MTs expression profiles in THP-1 cells and The transcriptional level changes after the recombination of SEB protein, the results showed that the expression of MTs in THP-1 cells included MT1E, F, G, X and MT2A. After the recombination of SEB protein, only MT2A expression rose and.Western blot experiment further confirmed the expression of the recombinant protein. In cell analysis, we found that the apoptosis ratio of recombinant SEB protein (about 4%) in THP-1 cells interfered with MT2A expression (about 4%) was significantly less than that of the control group without target Si RNA interference (about 8%). Using ELISA and Western blot experiments, we further detected the effect of recombinant protein on siRNA interfering MT2A expression of THP-1 cells. The expression level of NF alpha and HLA-DRa decreased, indicating that MT2A can play an important role in the apoptosis of THP-1 cells induced by recombinant SEB protein by promoting the formation of TNF alpha positive feedback loop. This is the first time that the expression of SEB has a regulatory role in the expression of MTs, which provides a theoretical basis for further study of the role and mechanism of bacterial toxin regulating MTs in the future.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R378.11
本文编号：2120059
[Abstract]:Apoptosis (Apoptosis) is an important biological process in multicellular organisms, which has many functions that affect tissue differentiation, metabolism and immunomodulation. In the infectious diseases caused by bacteria, the specific components of bacteria can regulate the apoptosis of host cells through a variety of ways, and this has also been proved to be crucial to the process of disease. Important effects. Staphylococcus aureus, as an important human pathogen, can also cause cell apoptosis in the process of infection. In many diseases closely related to Staphylococcus aureus, such as atopic dermatitis (Atopic Dermatitis, AD) and sepsis (sepsis), abnormal host cell apoptosis can be found. The important characteristic of Staphylococcus aureus is that it can secrete a variety of toxins, including hemolysin, leukin, enterotoxin and secretase. Enterotoxin is an important Staphylococcus aureus superantigen, in many diseases closely related to Staphylococcus aureus. The natural receptors of enterotoxin include the T cell receptor (TCR) and the major histocompatibility complex II (major histocompatibility complex II, MHCII). Staphylococcus aureus enterotoxin combined with TCR to activate the hypersensitivity of T cells, while MHCII has the role of promoting apoptosis. In the enterotoxin family, gold The distribution of staphylococcal enterotoxin B (staphylococcal enterotoxin B, SEB) is widely distributed, and the research is also in depth that.SEB has been proved to cause apoptosis in T cells and peripheral blood mononuclear cells (PBMC), and this cell apoptosis is closely related to the disease caused by Staphylococcus aureus, such as AD, but whether SEB can cause mononuclear macrophage apoptosis, And its molecular mechanism is still unclear. This study cloned the genome of Staphylococcus aureus XQ strains isolated and completed genome sequencing from our lab, expressed and purified the recombinant SEB protein with biological activity, and carried out the study on the use and mechanism of SEB to induce THP-1 mononuclear macrophage apoptosis, and found a dependence on TN. The positive feedback loop of F alpha plays a very important role in this process. At the same time, it is found that metallothionein 2A (metallothionein 2A, MT2A) can increase the expression of TNF alpha induced by SEB, thus promoting the formation of TNF alpha positive feedback loop, which is important in the process of SEB induced apoptosis of THP-1 cells. The results of this study are in depth in the future. The mechanism of the action of Staphylococcus aureus enterotoxin and the host cells provide important reference for the mechanism of the toxin attack. The main research methods and results in this paper are as follows: 1. the expression vector of recombinant SEB protein was constructed, the whole genome of Staphylococcus aureus XQ strain was expressed and purified as a template, and the SEB coding was obtained by PCR technology. The expression plasmid pET30a-SEB. was successfully constructed and the expression plasmid P ET30a-SEB was transformed into the expression strain of Escherichia coli C43 by BamHI+Xho I double enzyme digestion and the expression plasmid pET30a-SEB. was successfully constructed, and the recombinant SEB protein was successfully induced. The recombinant SEB protein expressed in the expression was purified by nickel column affinity chromatography and used to remove the endotoxin. The endotoxin was removed by the plain gel column. The ultrafiltration column was concentrated and replaced by PBS buffer solution. The purity of the recombinant protein was determined by SDS-PAGE. The concentration of protein and the reaction test of the reagents were detected by Bradford. The purity of the recombinant SEB protein was above 95%, the concentration was 539.2 Mu g/ml and the content of the toxin was lower than 2EU/ml. Cell experiments require that.2. recombinant SEB protein have the role of inducing apoptosis of THP-1 cells, and the Beijing microgene Technology Co., Ltd. is entrusted with the STR detection of the THP-1 cell lines we have received. No human cell cross contamination is found in the cells, and the THP-1 cells in the American model culture center (American type culture collection, ATCC) are not found. The matching rate of STR data was 93.33%, which confirmed that our cell line was a derived cell line of THP-1. Using Annexin V+PI double staining and flow cytometry, the recombinant SEB protein could induce apoptosis of THP-1 cells, the proportion of apoptosis was about 3%, and it had time and dose dependent effects. The broad-spectrum caspase inhibitor Z-VAD-FMK, I was used. We confirmed that the apoptosis of THP-1 cells induced by recombinant SEB protein is dependent on the cascade activation of caspase. In THP-1 cells activated by IFN- gamma and PMA induced THP-1 cells, the proportion of apoptosis induced by recombinant SEB protein is higher by Annexin V+PI double staining and flow cytometry, which is about 7.5% and 15%. endotoxin activates THP-1 cells, respectively. The apoptosis of THP-1 cells was enhanced by the enhanced recombinant SEB protein. However, after IFN- gamma activation and PMA induced differentiation, the apoptosis of THP-1 cells in the negative control group was also increased. In order to avoid the interference of additional factors, we have used the recombinant SEB protein directly to act on THP-1 cells in the next study without any additional cell activation or induction. Differentiation of.3. recombinant SEB protein induced THP-1 cell apoptosis dependent on a TNF alpha positive feedback loop classic apoptosis including exogenous (caspase 8 activation) and endogenous (caspase 9 activation) two pathways. We detected the enzyme activity of the enzyme activity of TPH-1 cells after the action of recombinant SEB protein by Caspase-3, -8, and -9 activity detection kits The apoptosis of THP-1 cells induced by recombinant SEB protein is dependent on the activation of Caspase-3 and -8 without relying on the activation of caspase-9, indicating that the exogenous pathway plays an important role in the apoptosis of THP-1 cells induced by SEB. The transcriptional level of the apoptotic phase molecules in the THP-1 cells after the recombinant SEB protein is detected by the RT-q PCR, and the recombinant SEB eggs are found. After white treatment, the transcriptional level of TNF alpha and HLA-DRa in THP-1 cells increased significantly. Further using ELISA and Western blot experiments, we confirmed that the expression of TNF alpha and HLA-DRa induced by recombinant SEB protein increased in THP-1 cells. The apoptosis induced by recombinant protein was detected by the action of anti-TNF alpha monoclonal antibody and TNF alpha. The ratio decreased to about 1%, which was significantly different from the 3.5% of the recombinant SEB protein, while the same type control antibody had no effect. It proved that TNF alpha played a key role in the apoptosis of THP-1 cells induced by SEB. Using Si RNA to interfere with the expression of HLA-DRa and TNFR1, we found that the recombinant SEB protein was caused in the interfered THP-1 cells. The percentage of apoptotic cells (about 2.5%) was significantly lower than that of the control group (about 9%) with non target Si RNA interference. At the same time, the level of TNF a secreted by HLA-DRa and TNFR1 siRNA interfered with the recombinant SEB protein was also significantly lower than that of the control group without the target siRNA interference. And TNF a itself would increase the HLA-DRa expression, thereby increasing the weight. The combination of group SEB protein and HLA-DRa makes the secretion of TNF a further increase, thus forming a positive feedback loop dependent on TNF alpha. The apoptosis of THP-1 cells caused by recombinant SEB protein depends on the formation of this positive feedback loop and the complete.4.MT2A by promoting the positive feedback pathway of TNF alpha to promote THP-1 fineness caused by the recombinant SEB protein. Apoptosis is the receptor of SEB, the only cytoplasmic region of THP-1 cell HLA-DRa contains only 15 amino acid residues and does not contain any known domain. After the combination of HLA-DRa and SEB, the subsequent signal transduction may depend on other membrane proteins which are directly combined with HLA-DRa. And how to increase the expression mechanism of TNF alpha after SEB binding HLA-DRa It is not clear that we use immunoprecipitation technique to capture proteins interacting with HLA-DRa by anti-HLA-DRa monoclonal antibody, and we found that a metallothioneins (MTs) MT2A may be involved in the apoptosis process of THP-1 cells induced by SEB. We use RT-q PCR to analyze MTs expression profiles in THP-1 cells and The transcriptional level changes after the recombination of SEB protein, the results showed that the expression of MTs in THP-1 cells included MT1E, F, G, X and MT2A. After the recombination of SEB protein, only MT2A expression rose and.Western blot experiment further confirmed the expression of the recombinant protein. In cell analysis, we found that the apoptosis ratio of recombinant SEB protein (about 4%) in THP-1 cells interfered with MT2A expression (about 4%) was significantly less than that of the control group without target Si RNA interference (about 8%). Using ELISA and Western blot experiments, we further detected the effect of recombinant protein on siRNA interfering MT2A expression of THP-1 cells. The expression level of NF alpha and HLA-DRa decreased, indicating that MT2A can play an important role in the apoptosis of THP-1 cells induced by recombinant SEB protein by promoting the formation of TNF alpha positive feedback loop. This is the first time that the expression of SEB has a regulatory role in the expression of MTs, which provides a theoretical basis for further study of the role and mechanism of bacterial toxin regulating MTs in the future.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R378.11
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