【摘要】：細(xì)胞凋亡(Apoptosis)是多細(xì)胞生物中重要的生物學(xué)過程,具有影響組織分化、新陳代謝及免疫調(diào)節(jié)等多方面功能。在細(xì)菌引起的感染性疾病中,細(xì)菌的特定組分可以通過多種途徑調(diào)控宿主細(xì)胞的凋亡,而這也被證實(shí)對疾病的進(jìn)程有著至關(guān)重要的影響。金黃色葡萄球菌(Staphylococcus aureus)作為重要的人類病原菌,在其感染過程中也可引起宿主細(xì)胞凋亡。在許多與金黃色葡萄球菌密切相關(guān)的疾病,如特應(yīng)性皮炎(Atopic Dermatitis,AD)和膿毒癥(sepsis)中,異常的宿主細(xì)胞凋亡現(xiàn)象可顯著影響疾病的進(jìn)程、嚴(yán)重程度和預(yù)后。金黃色葡萄球菌的一個(gè)重要特點(diǎn)是其可分泌多種毒素,包括溶血素、殺白細(xì)胞素、腸毒素及分泌酶等。其中腸毒素是一種重要的金黃色葡萄球菌超抗原,在很多與金黃色葡萄球菌密切相關(guān)的疾病中發(fā)揮著重要的作用。腸毒素的天然受體包括T細(xì)胞受體(TCR)和主要組織相容性復(fù)合體II類分子(major histocompatibility complex II,MHCII),金葡菌腸毒素結(jié)合TCR可持續(xù)激活T細(xì)胞引起超敏反應(yīng);而結(jié)合MHCII則具有促細(xì)胞凋亡作用。在腸毒素家族中,金黃色葡萄球菌腸毒素B(staphylococcal enterotoxin B,SEB)分布較廣、研究也較為深入。SEB已被證實(shí)能夠引起T細(xì)胞和外周血單核細(xì)胞(PBMC)凋亡,且這種細(xì)胞凋亡與金黃色葡萄球菌引起的疾病,如AD等密切相關(guān)。但SEB是否能引起單核-巨噬細(xì)胞凋亡,以及其分子機(jī)制目前仍不清楚。本研究從我室分離并完成全基因組測序的金黃色葡萄球菌XQ株基因組中克隆、表達(dá)并純化了具有生物學(xué)活性的重組SEB蛋白,開展了SEB引起THP-1單核-巨噬細(xì)胞凋亡的作用及其機(jī)制研究,發(fā)現(xiàn)了一個(gè)依賴于TNFα的正反饋回路在此過程中起到非常重要的作用;同時(shí)發(fā)現(xiàn)金屬硫蛋白2A(metallothionein 2A,MT2A)可通過促進(jìn)SEB引起的TNFα表達(dá)上調(diào),從而促進(jìn)TNFα正反饋回路的形成,在SEB引起THP-1細(xì)胞凋亡的過程中具有重要地位。所得研究結(jié)果為將來深入闡明金黃色葡萄球菌腸毒素的作用機(jī)制以及宿主細(xì)胞應(yīng)對毒素攻擊的機(jī)制提供了重要參考。本論文的主要研究方法和結(jié)果如下:1.重組SEB蛋白的表達(dá)載體構(gòu)建、表達(dá)與純化以金黃色葡萄球菌XQ株全基因組為模板,利用PCR技術(shù)獲得SEB編碼序列,再通過BamHI+Xho I雙酶切以及采用T4連接酶與表達(dá)載體連接,成功構(gòu)建了表達(dá)質(zhì)粒pET30a-SEB。將表達(dá)質(zhì)粒p ET30a-SEB轉(zhuǎn)化入表達(dá)菌株大腸埃希菌C43后,成功誘導(dǎo)表達(dá)出重組SEB蛋白。表達(dá)的重組SEB蛋白經(jīng)過鎳柱親和層析純化后,利用去內(nèi)毒素凝膠柱去除內(nèi)毒素、超濾柱濃縮并置換緩沖液為PBS緩沖液,最終以SDS-PAGE測定重組蛋白純度、Bradford法測定蛋白質(zhì)濃度及鱟試劑反應(yīng)實(shí)驗(yàn)檢測殘留內(nèi)毒素,結(jié)果重組SEB蛋白的純度在95%以上,濃度為539.2μg/ml,毒素含量低于2EU/ml,滿足后續(xù)細(xì)胞實(shí)驗(yàn)要求。2.重組SEB蛋白具有引起THP-1細(xì)胞凋亡的作用委托北京閱微基因技術(shù)有限公司對我們獲贈(zèng)的THP-1細(xì)胞系進(jìn)行STR檢測,細(xì)胞中沒有發(fā)現(xiàn)人類細(xì)胞交叉污染,與美國模式培養(yǎng)物保存中心(American type culture collection,ATCC)中THP-1細(xì)胞的STR數(shù)據(jù)匹配率為93.33%,證實(shí)我們的細(xì)胞系為THP-1的衍生細(xì)胞系。利用Annexin V+PI雙染色及流式細(xì)胞分析,確定了重組SEB蛋白具有引起THP-1細(xì)胞凋亡作用,細(xì)胞凋亡比例約為3%,并且具有時(shí)間和劑量依賴效應(yīng)。利用廣譜caspase抑制劑Z-VAD-FMK,我們證實(shí)重組SEB蛋白引起的THP-1細(xì)胞凋亡依賴caspase的級聯(lián)激活。在IFN-γ激活及PMA誘導(dǎo)分化后的THP-1細(xì)胞中,利用Annexin V+PI雙染色及流式細(xì)胞分析,檢測到重組SEB蛋白引起的細(xì)胞凋亡比例更高,分別約為7.5%和15%。內(nèi)毒素激活THP-1細(xì)胞后并不增強(qiáng)重組SEB蛋白引起THP-1細(xì)胞凋亡的作用。然而IFN-γ激活和PMA誘導(dǎo)分化后,陰性對照組中THP-1細(xì)胞凋亡也有所上升。為避免額外因素的干擾,我們在接下來的研究中均直接使用重組SEB蛋白作用于THP-1細(xì)胞而不再進(jìn)行額外的細(xì)胞激活或誘導(dǎo)分化。3.重組SEB蛋白引起THP-1細(xì)胞凋亡依賴于一個(gè)TNFα的正反饋回路經(jīng)典的細(xì)胞凋亡包括外源性(caspase 8激活)和內(nèi)源性(caspase 9激活)兩條途徑,我們通過caspase-3,-8,-9活性檢測試劑盒對重組SEB蛋白作用后的TPH-1細(xì)胞之相應(yīng)酶活性檢測發(fā)現(xiàn),重組SEB蛋白引起的THP-1細(xì)胞凋亡依賴caspase-3和-8的激活而不依賴caspase-9的激活,說明外源性通路在SEB引起THP-1細(xì)胞凋亡過程中發(fā)揮重要作用。利用RT-q PCR檢測重組SEB蛋白處理后THP-1細(xì)胞中凋亡相關(guān)分子的轉(zhuǎn)錄水平,結(jié)果發(fā)現(xiàn)重組SEB蛋白處理后,THP-1細(xì)胞中TNFα和HLA-DRa的轉(zhuǎn)錄水平顯著升高。進(jìn)一步利用ELISA和Western blot實(shí)驗(yàn),我們證實(shí)了重組SEB蛋白在THP-1細(xì)胞中引起的TNFα和HLA-DRa表達(dá)升高。利用anti-TNFα單克隆抗體中和TNFα作用后,檢測到重組SEB蛋白引起的THP-1細(xì)胞凋亡比例下降至約1%,與重組SEB蛋白直接作用后的3.5%存在顯著差異,而同型對照抗體則無此作用,證實(shí)TNFα在SEB引起的THP-1細(xì)胞凋亡過程中發(fā)揮關(guān)鍵作用。利用si RNA干擾HLA-DRa和TNFR1的表達(dá),我們發(fā)現(xiàn)在干擾后的THP-1細(xì)胞中,重組SEB蛋白引起的凋亡細(xì)胞比例(約2.5%)顯著低于用無目標(biāo)si RNA干擾的對照組細(xì)胞(約9%)。同時(shí),HLA-DRa和TNFR1 siRNA干擾的THP-1細(xì)胞在重組SEB蛋白作用后,TNFα分泌水平也顯著低于無目標(biāo)siRNA干擾的對照組細(xì)胞。而TNFα本身又會上調(diào)HLA-DRa的表達(dá),從而增加了重組SEB蛋白與HLA-DRa的結(jié)合,使得TNFα的分泌進(jìn)一步增加,于是形成了一個(gè)依賴于TNFα的正反饋回路。重組SEB蛋白引起的THP-1細(xì)胞凋亡依賴于這一正反饋回路的形成和完整。4.MT2A通過促進(jìn)TNFα的正反饋回路從而促進(jìn)重組SEB蛋白引起的THP-1細(xì)胞凋亡作為SEB的受體,THP-1細(xì)胞的HLA-DRa僅有的胞質(zhì)區(qū)僅含15個(gè)氨基酸殘基,且不含任何已知的結(jié)構(gòu)域,HLA-DRa與SEB結(jié)合后,后續(xù)的信號轉(zhuǎn)導(dǎo)很可能依靠與HLA-DRa直接結(jié)合的其他膜蛋白來實(shí)現(xiàn)。且SEB結(jié)合HLA-DRa以后如何上調(diào)TNFα的表達(dá)機(jī)制也不清楚。我們利用免疫共沉淀技術(shù),通過anti-HLA-DRa單克隆抗體去捕捉與HLA-DRa相互作用的蛋白質(zhì),發(fā)現(xiàn)了一個(gè)金屬硫蛋白(metallothioneins,MTs)MT2A可能參與了SEB引起的THP-1細(xì)胞凋亡作用過程。我們利用RT-q PCR分析了THP-1細(xì)胞中MTs的表達(dá)譜及其在重組SEB蛋白作用后轉(zhuǎn)錄水平的改變,結(jié)果發(fā)現(xiàn)THP-1細(xì)胞中表達(dá)的MTs包括MT1E、F、G、X和MT2A,在重組SEB蛋白作用后,僅MT2A表達(dá)上升。Western blot實(shí)驗(yàn)進(jìn)一步證實(shí)了重組SEB蛋白作用后THP-1細(xì)胞中MT2A的表達(dá)上升約50%。利用Annexin V+PI雙染色及流式細(xì)胞分析,我們發(fā)現(xiàn)在siRNA干擾MT2A表達(dá)的THP-1細(xì)胞中,重組SEB蛋白作用后的細(xì)胞凋亡比例(約4%)明顯少于無目標(biāo)si RNA干擾的對照組細(xì)胞(約8%)。利用ELISA和Western blot實(shí)驗(yàn),我們進(jìn)一步檢測到在siRNA干擾MT2A表達(dá)的THP-1細(xì)胞中,重組SEB蛋白作用后TNFα和HLA-DRa表達(dá)水平均降低,說明MT2A通過促進(jìn)TNFα正反饋回路形成從而在重組SEB蛋白引起的THP-1細(xì)胞凋亡發(fā)揮重要作用。這是初次發(fā)現(xiàn)細(xì)菌SEB對MTs的表達(dá)存在調(diào)控作用,為將來深入研究細(xì)菌毒素調(diào)控MTs的作用和機(jī)制奠定了理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R378.11

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