天然抗体3B4的抗金黄色葡萄球菌作用及其机制研究
发布时间：2018-07-24 10:20
【摘要】： 天然抗体(natural antibodies, NAbs)是指在没有任何抗原免疫的情况下,正常机体产生的针对一种或多种自身或外源抗原的抗体[1],主要为IgM同种型,由未突变的胚系基因编码,具有维持自身稳定、抗感染、参与移植排斥、肿瘤监视等作用[2-4]。 抗感染是天然抗体的重要功能,天然抗体与补体、单核-巨噬细胞等一起构成了机体抵御感染的第一道防线。通过多反应性,天然抗体可以和多种病原体结合。天然抗体主要结合微生物表面保守的结构,通过与Toll样受体等PRR相似的方式发挥对病原体的识别,并在细菌、病毒、真菌、寄生虫等的感染中发挥重要的保护作用。 B1细胞是产生天然抗体的主要细胞。目前研究认为成熟B细胞分为B1细胞、滤泡B细胞和边缘区B细胞三个亚群,其中B1细胞是近10余年来发现并命名的B细胞亚群,主要位于腹腔和胸膜腔,在天然免疫、自身免疫和粘膜免疫中具有重要的作用[5-6]。以往研究发现B1细胞缺陷的小鼠对细菌等病原微生物高度易感,然而,关于天然抗体和B1细胞在病原体感染时的效应机制尚不明确[7]。 以往主要应用混合的血清天然抗体、分泌型IgM缺陷的小鼠和CD19转基因小鼠模型[8]以及B1细胞移植实验进行天然抗体及B1细胞在抗感染中作用的研究,取得了很多重要的结论。然而,由于没有纯化的特异性天然抗体及高天然抗体滴度转基因小鼠模型,使得关于天然抗体和B1细胞的作用机制的研究一直受到制约。 近年来,随着耐药金黄色葡萄球菌的不断出现以及免疫受损人群增多,耐药金黄色葡萄球菌引起的感染越来越受到人们重视,成为医院感染的重要死亡原因之一。天然免疫特别是天然抗体在抗金黄色葡萄球菌感染中的作用引起了越来越多学者的兴趣。 本课题组在前期工作中发现,来自未免疫小鼠B细胞的天然抗角蛋白自身抗体3B4可以识别金黄色葡萄球菌,并可以促进巨噬细胞对其的吞噬,从而提示天然抗体3B4在抵御金黄色葡萄球菌感染中可能具有的重要作用。结合天然抗体3B4转基因小鼠,我们对天然抗体的抗细菌感染作用进行了系统研究,并利用这一小鼠模型对B1细胞的应答进行了深入探索。一.天然抗体3B4的体外抗金黄色葡萄球菌作用 采用ELISA、流式细胞术检测3B4对金黄色葡萄球菌的结合,结果发现天然抗体3B4可以结合于金黄色葡萄球菌表面的抗原。 天然抗体3B4及对照抗体(MOPC)与金黄色葡萄球菌作用后,分别与小鼠腹腔细胞共孵育,结果菌落形成实验显示3B4作用组菌落形成单位少于对照组,流式细胞术检测到3B4作用组的吞噬指数显著高于同型对照,表明天然抗体可以促进巨噬细胞对金黄色葡萄球菌的吞噬。 二.利用转基因小鼠进行抗金黄色葡萄球菌感染的在体研究 金黄色葡萄球菌腹腔接种后观察小鼠菌负荷、中性粒细胞浸润、炎症因子浓度等。结果发现,转基因小鼠的腹腔、肾脏细菌负荷显著低于对照小鼠;转基因小鼠腹腔中性粒细胞数显著低于对照小鼠,炎症因子IL-12、IFN-γ浓度与对照鼠相比无明显差异,而IL-6、IL-10、MCP-1、TNF-α浓度显著低于阴性对照小鼠。表明转基因小鼠对于金黄色葡萄球菌腹腔感染具有良好的抵抗。 三.天然抗体3B4及B1细胞的作用机制研究 流式细胞仪检测发现转基因小鼠腹腔细胞吞噬指数显著高于阴性对照小鼠。ELISA法检测转基因小鼠,阴性对照小鼠腹腔冲洗液总IgM、抗角蛋白IgM、抗金黄色葡萄球菌IgM水平。结果显示,转基因小鼠与阴性小鼠的总IgM水平无显著差异,但转基因小鼠的腹腔抗角蛋白IgM水平显著高于对照小鼠,并且转基因小鼠的腹腔冲洗液中抗金黄色葡萄球菌的IgM浓度显著高于对照小鼠。 与腹腔抗角蛋白/金黄色葡萄球菌滴度相一致,感染后转基因小鼠的腹腔浆细胞数显著增多,提示细菌刺激后导致B细胞活化并转化成浆细胞。进一步检测了B1细胞的增殖,发现转基因小鼠的B1a细胞增殖显著强于对照小鼠,提示基于对病原体的识别,转基因小鼠的腹腔B1细胞发生了显著的活化和增殖,分泌大量的抗体,在抗细菌感染中发挥了重要的作用。 同时,我们发现转基因小鼠腹腔内的B1细胞对细菌的结合/吞噬明显增加,为了区别是结合还是吞噬,进一步应用激光共聚焦显微镜进行了观察,发现B细胞胞浆内的绿色荧光颗粒,提示B1细胞可能吞噬了颗粒性细菌抗原,然而这一结果仍需大量的吞噬实验予以证实和完善。 本研究从体外和在体水平揭示了天然抗体3B4对金黄色葡萄球菌感染的保护作用,并观察到B1细胞对金黄色葡萄球菌感染的应答,初步阐明了天然抗体及B1细胞抗感染作用的机制,丰富了对天然抗体的功能的认识,具有重要的理论创新意义。
[Abstract]:Natural antibodies (NAbs) is an antibody [1] produced by the normal body against one or more of its own or exogenous antigens in the absence of any antigen immunization, mainly the IgM homohomoisoform, encoded by the non mutant embryo gene, and has the role of maintaining self stability, anti infection, involvement in transplant rejection, and tumor surveillance, and so on.
Anti infection is an important function of natural antibodies. Natural antibodies, complement, mononuclear macrophages, and so on constitute the first line of defense against infection. By multi reactivity, natural antibodies can be combined with a variety of pathogens. Natural antibodies are mainly combined with microbiological surface conserved structures, and are produced in a similar manner to the Toll like receptor, such as PRR. It plays an important role in the identification of pathogens and in the infection of bacteria, viruses, fungi, parasites and so on.
B1 cells are the main cells that produce natural antibodies. It is believed that mature B cells are divided into three subgroups of B1 cells, follicular B cells and marginal zone B cells, of which B1 cells are the subgroups of B cells discovered and named in the last 10 years, mainly in the abdominal cavity and the pleural cavity, which play an important role in natural immunity, autoimmune and mucosal immunity. -6]. previously found that mice with B1 cell defects were highly susceptible to bacterial and other pathogenic microorganisms. However, the mechanism of the effects of natural and B1 cells on pathogens was not yet clear [7].
In the past, many important conclusions have been made in the study of natural antibodies mixed with mixed natural antibodies, IgM deficient mice and CD19 transgenic mice model [8] and B1 cell transplantation in the anti infection effect of natural antibodies and B1 cells. The gene mouse model has restricted the research on the mechanism of natural antibodies and B1 cells.
In recent years, with the continuous emergence of drug-resistant staphylococcus aureus and the increase of immune impaired population, the infection caused by drug-resistant staphylococcus aureus has been paid more and more attention and one of the important causes of death in hospital infection. Natural immunity, especially natural antibody, has caused the increasing effect in the anti Staphylococcus aureus infection. The more scholars are interested.
In our previous work, we found that natural anti keratin autoantibody 3B4 from unimmunized mouse B cells can identify Staphylococcus aureus and promote macrophage phagocytosis, suggesting that the natural antibody 3B4 can play an important role in resisting Staphylococcus aureus infection. Combined with natural antibody 3B4 translocation In mice, we systematically studied the anti bacterial infection of natural antibodies and explored the response of B1 cells with this mouse model. 1. The effect of natural antibody 3B4 on Staphylococcus aureus in vitro
ELISA was used to detect the binding of 3B4 to Staphylococcus aureus by flow cytometry. It was found that the natural antibody 3B4 could bind to the surface antigen of Staphylococcus aureus.
The natural antibody 3B4 and the control antibody (MOPC) were incubated with the mouse celiac cells after the action of the Staphylococcus aureus. The result of the colony formation experiment showed that the colony forming unit of the 3B4 group was less than the control group. The phagocytosis index of the 3B4 group was significantly higher than that of the same type control, indicating that the natural antibody could promote the mega bite. Phagocytosis of the cell to Staphylococcus aureus.
Two. In vivo study of transgenic mice against Staphylococcus aureus infection.
The bacterial load, neutrophils infiltration and the concentration of inflammatory factors were observed after the peritoneal inoculation of Staphylococcus aureus. The results showed that the bacterial load in the peritoneal cavity and kidney of the transgenic mice was significantly lower than that of the control mice, and the number of neutrophils in the peritoneal cavity of transgenic mice was significantly lower than that of the control mice, and the concentration of inflammatory factors IL-12 and IFN- gamma was no more than that of the control mice. The concentration of IL-6, IL-10, MCP-1, TNF- alpha was significantly lower than that of the negative control mice, indicating that the transgenic mice had good resistance to Staphylococcus aureus intraperitoneal infection.
Three. The mechanism of natural antibodies 3B4 and B1 cells
Flow cytometry showed that the phagocytosis index of the celiac cells in transgenic mice was significantly higher than that of negative control mice by.ELISA method, and the total IgM, anti keratin IgM and Staphylococcus aureus IgM level in the negative control mice. The results showed that there was no significant difference in the total IgM level between the transgenic mice and the negative mice, but the change of the total IgM level was not significant. The anti keratin IgM level in the abdominal cavity of the gene mice was significantly higher than that of the control mice, and the IgM concentration of the anti Staphylococcus aureus in the peritoneal lavage fluid of the transgenic mice was significantly higher than that of the control mice.
The number of celiac plasma cells in the transgenic mice increased significantly after the infection of the peritoneal anti keratin / Staphylococcus aureus, suggesting that the B cells were activated and transformed into plasma cells after the bacterial stimulation. The proliferation of B1 cells was further detected, and the proliferation of B1a cells in the transgenic mice was significantly stronger than that of the control mice. In the transgenic mice, the B1 cells of the transgenic mice showed significant activation and proliferation, and secreted a large number of antibodies, which played an important role in the anti bacterial infection.
At the same time, we found that the B1 cells in the peritoneal cavity of the transgenic mice increased significantly to the binding / phagocytosis of the bacteria. In order to distinguish whether the cells were binding or phagocytic, the laser confocal microscopy was further observed and the green fluorescent particles in the cytoplasm of the B cells were found, suggesting that the B1 cells may have phagocytic bacterial antigens. However, this result is still a result. A large number of phagocytic experiments are required to be confirmed and perfected.
This study revealed the protective effect of natural antibody 3B4 on Staphylococcus aureus infection in vitro and at the body level, and observed the response of B1 cells to Staphylococcus aureus infection, preliminarily clarified the anti infection mechanism of natural antibodies and B1 cells, enriched the understanding of the function of natural anti body, and had important theoretical innovation. Righteousness.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392.1
本文编号：2141067
[Abstract]:Natural antibodies (NAbs) is an antibody [1] produced by the normal body against one or more of its own or exogenous antigens in the absence of any antigen immunization, mainly the IgM homohomoisoform, encoded by the non mutant embryo gene, and has the role of maintaining self stability, anti infection, involvement in transplant rejection, and tumor surveillance, and so on.
Anti infection is an important function of natural antibodies. Natural antibodies, complement, mononuclear macrophages, and so on constitute the first line of defense against infection. By multi reactivity, natural antibodies can be combined with a variety of pathogens. Natural antibodies are mainly combined with microbiological surface conserved structures, and are produced in a similar manner to the Toll like receptor, such as PRR. It plays an important role in the identification of pathogens and in the infection of bacteria, viruses, fungi, parasites and so on.
B1 cells are the main cells that produce natural antibodies. It is believed that mature B cells are divided into three subgroups of B1 cells, follicular B cells and marginal zone B cells, of which B1 cells are the subgroups of B cells discovered and named in the last 10 years, mainly in the abdominal cavity and the pleural cavity, which play an important role in natural immunity, autoimmune and mucosal immunity. -6]. previously found that mice with B1 cell defects were highly susceptible to bacterial and other pathogenic microorganisms. However, the mechanism of the effects of natural and B1 cells on pathogens was not yet clear [7].
In the past, many important conclusions have been made in the study of natural antibodies mixed with mixed natural antibodies, IgM deficient mice and CD19 transgenic mice model [8] and B1 cell transplantation in the anti infection effect of natural antibodies and B1 cells. The gene mouse model has restricted the research on the mechanism of natural antibodies and B1 cells.
In recent years, with the continuous emergence of drug-resistant staphylococcus aureus and the increase of immune impaired population, the infection caused by drug-resistant staphylococcus aureus has been paid more and more attention and one of the important causes of death in hospital infection. Natural immunity, especially natural antibody, has caused the increasing effect in the anti Staphylococcus aureus infection. The more scholars are interested.
In our previous work, we found that natural anti keratin autoantibody 3B4 from unimmunized mouse B cells can identify Staphylococcus aureus and promote macrophage phagocytosis, suggesting that the natural antibody 3B4 can play an important role in resisting Staphylococcus aureus infection. Combined with natural antibody 3B4 translocation In mice, we systematically studied the anti bacterial infection of natural antibodies and explored the response of B1 cells with this mouse model. 1. The effect of natural antibody 3B4 on Staphylococcus aureus in vitro
ELISA was used to detect the binding of 3B4 to Staphylococcus aureus by flow cytometry. It was found that the natural antibody 3B4 could bind to the surface antigen of Staphylococcus aureus.
The natural antibody 3B4 and the control antibody (MOPC) were incubated with the mouse celiac cells after the action of the Staphylococcus aureus. The result of the colony formation experiment showed that the colony forming unit of the 3B4 group was less than the control group. The phagocytosis index of the 3B4 group was significantly higher than that of the same type control, indicating that the natural antibody could promote the mega bite. Phagocytosis of the cell to Staphylococcus aureus.
Two. In vivo study of transgenic mice against Staphylococcus aureus infection.
The bacterial load, neutrophils infiltration and the concentration of inflammatory factors were observed after the peritoneal inoculation of Staphylococcus aureus. The results showed that the bacterial load in the peritoneal cavity and kidney of the transgenic mice was significantly lower than that of the control mice, and the number of neutrophils in the peritoneal cavity of transgenic mice was significantly lower than that of the control mice, and the concentration of inflammatory factors IL-12 and IFN- gamma was no more than that of the control mice. The concentration of IL-6, IL-10, MCP-1, TNF- alpha was significantly lower than that of the negative control mice, indicating that the transgenic mice had good resistance to Staphylococcus aureus intraperitoneal infection.
Three. The mechanism of natural antibodies 3B4 and B1 cells
Flow cytometry showed that the phagocytosis index of the celiac cells in transgenic mice was significantly higher than that of negative control mice by.ELISA method, and the total IgM, anti keratin IgM and Staphylococcus aureus IgM level in the negative control mice. The results showed that there was no significant difference in the total IgM level between the transgenic mice and the negative mice, but the change of the total IgM level was not significant. The anti keratin IgM level in the abdominal cavity of the gene mice was significantly higher than that of the control mice, and the IgM concentration of the anti Staphylococcus aureus in the peritoneal lavage fluid of the transgenic mice was significantly higher than that of the control mice.
The number of celiac plasma cells in the transgenic mice increased significantly after the infection of the peritoneal anti keratin / Staphylococcus aureus, suggesting that the B cells were activated and transformed into plasma cells after the bacterial stimulation. The proliferation of B1 cells was further detected, and the proliferation of B1a cells in the transgenic mice was significantly stronger than that of the control mice. In the transgenic mice, the B1 cells of the transgenic mice showed significant activation and proliferation, and secreted a large number of antibodies, which played an important role in the anti bacterial infection.
At the same time, we found that the B1 cells in the peritoneal cavity of the transgenic mice increased significantly to the binding / phagocytosis of the bacteria. In order to distinguish whether the cells were binding or phagocytic, the laser confocal microscopy was further observed and the green fluorescent particles in the cytoplasm of the B cells were found, suggesting that the B1 cells may have phagocytic bacterial antigens. However, this result is still a result. A large number of phagocytic experiments are required to be confirmed and perfected.
This study revealed the protective effect of natural antibody 3B4 on Staphylococcus aureus infection in vitro and at the body level, and observed the response of B1 cells to Staphylococcus aureus infection, preliminarily clarified the anti infection mechanism of natural antibodies and B1 cells, enriched the understanding of the function of natural anti body, and had important theoretical innovation. Righteousness.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392.1
【参考文献】
相关期刊论文 前7条
1 虞红,刘玉峰,赵小东,张亮;抗角蛋白自身抗体对培养人舌鳞癌细胞表达表皮生长因子受体的影响[J];第四军医大学学报;1998年06期
2 武彩霞,李巍,刘玉峰,李承新,卢宁,万业宏;抗角蛋白抗体进入活细胞的共聚焦显微镜观察[J];细胞与分子免疫学杂志;2002年05期
3 王文清,刘玉峰,赵小东,马翠玲;天然抗角蛋白自身抗体抑制角质形成细胞及鳞状细胞癌细胞增殖的作用[J];中华皮肤科杂志;2001年03期
4 付萌,李巍,王刚,张衍国,李承新,樊平申,刘玉峰;天然属性的抗角蛋白单克隆IgM抗体的制备[J];中华皮肤科杂志;2005年04期
5 管海宏,付萌,夏汝山,王刚,刘玉峰;天然抗角蛋白单克隆抗体3B4的多反应性鉴定及其分子机制的初步探讨[J];中华皮肤科杂志;2005年06期
6 安金刚;李巍;付萌;王刚;李承新;刘玉峰;;天然抗角蛋白IgM调理巨噬细胞吞噬金黄色葡萄球菌的作用[J];中华皮肤科杂志;2005年10期
7 李巍;韩骅;邢影;匡颖;麻孙恺;付萌;王刚;李承新;刘玉峰;;高滴度天然抗角蛋白自身抗体转基因小鼠模型的建立[J];中华皮肤科杂志;2005年12期
，本文编号：2141067
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2141067.html
最近更新
教材专著
