【摘要】： 天然抗體(natural antibodies, NAbs)是指在沒有任何抗原免疫的情況下,正常機(jī)體產(chǎn)生的針對一種或多種自身或外源抗原的抗體[1],主要為IgM同種型,由未突變的胚系基因編碼,具有維持自身穩(wěn)定、抗感染、參與移植排斥、腫瘤監(jiān)視等作用[2-4]。 抗感染是天然抗體的重要功能,天然抗體與補體、單核-巨噬細(xì)胞等一起構(gòu)成了機(jī)體抵御感染的第一道防線。通過多反應(yīng)性,天然抗體可以和多種病原體結(jié)合。天然抗體主要結(jié)合微生物表面保守的結(jié)構(gòu),通過與Toll樣受體等PRR相似的方式發(fā)揮對病原體的識別,并在細(xì)菌、病毒、真菌、寄生蟲等的感染中發(fā)揮重要的保護(hù)作用。 B1細(xì)胞是產(chǎn)生天然抗體的主要細(xì)胞。目前研究認(rèn)為成熟B細(xì)胞分為B1細(xì)胞、濾泡B細(xì)胞和邊緣區(qū)B細(xì)胞三個亞群,其中B1細(xì)胞是近10余年來發(fā)現(xiàn)并命名的B細(xì)胞亞群,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]。以往研究發(fā)現(xiàn)B1細(xì)胞缺陷的小鼠對細(xì)菌等病原微生物高度易感,然而,關(guān)于天然抗體和B1細(xì)胞在病原體感染時的效應(yīng)機(jī)制尚不明確[7]。 以往主要應(yīng)用混合的血清天然抗體、分泌型IgM缺陷的小鼠和CD19轉(zhuǎn)基因小鼠模型[8]以及B1細(xì)胞移植實驗進(jìn)行天然抗體及B1細(xì)胞在抗感染中作用的研究,取得了很多重要的結(jié)論。然而,由于沒有純化的特異性天然抗體及高天然抗體滴度轉(zhuǎn)基因小鼠模型,使得關(guān)于天然抗體和B1細(xì)胞的作用機(jī)制的研究一直受到制約。 近年來,隨著耐藥金黃色葡萄球菌的不斷出現(xiàn)以及免疫受損人群增多,耐藥金黃色葡萄球菌引起的感染越來越受到人們重視,成為醫(yī)院感染的重要死亡原因之一。天然免疫特別是天然抗體在抗金黃色葡萄球菌感染中的作用引起了越來越多學(xué)者的興趣。 本課題組在前期工作中發(fā)現(xiàn),來自未免疫小鼠B細(xì)胞的天然抗角蛋白自身抗體3B4可以識別金黃色葡萄球菌,并可以促進(jìn)巨噬細(xì)胞對其的吞噬,從而提示天然抗體3B4在抵御金黃色葡萄球菌感染中可能具有的重要作用。結(jié)合天然抗體3B4轉(zhuǎn)基因小鼠,我們對天然抗體的抗細(xì)菌感染作用進(jìn)行了系統(tǒng)研究,并利用這一小鼠模型對B1細(xì)胞的應(yīng)答進(jìn)行了深入探索。一.天然抗體3B4的體外抗金黃色葡萄球菌作用 采用ELISA、流式細(xì)胞術(shù)檢測3B4對金黃色葡萄球菌的結(jié)合,結(jié)果發(fā)現(xiàn)天然抗體3B4可以結(jié)合于金黃色葡萄球菌表面的抗原。 天然抗體3B4及對照抗體(MOPC)與金黃色葡萄球菌作用后,分別與小鼠腹腔細(xì)胞共孵育,結(jié)果菌落形成實驗顯示3B4作用組菌落形成單位少于對照組,流式細(xì)胞術(shù)檢測到3B4作用組的吞噬指數(shù)顯著高于同型對照,表明天然抗體可以促進(jìn)巨噬細(xì)胞對金黃色葡萄球菌的吞噬。 二.利用轉(zhuǎn)基因小鼠進(jìn)行抗金黃色葡萄球菌感染的在體研究 金黃色葡萄球菌腹腔接種后觀察小鼠菌負(fù)荷、中性粒細(xì)胞浸潤、炎癥因子濃度等。結(jié)果發(fā)現(xiàn),轉(zhuǎn)基因小鼠的腹腔、腎臟細(xì)菌負(fù)荷顯著低于對照小鼠;轉(zhuǎn)基因小鼠腹腔中性粒細(xì)胞數(shù)顯著低于對照小鼠,炎癥因子IL-12、IFN-γ濃度與對照鼠相比無明顯差異,而IL-6、IL-10、MCP-1、TNF-α濃度顯著低于陰性對照小鼠。表明轉(zhuǎn)基因小鼠對于金黃色葡萄球菌腹腔感染具有良好的抵抗。 三.天然抗體3B4及B1細(xì)胞的作用機(jī)制研究 流式細(xì)胞儀檢測發(fā)現(xiàn)轉(zhuǎn)基因小鼠腹腔細(xì)胞吞噬指數(shù)顯著高于陰性對照小鼠。ELISA法檢測轉(zhuǎn)基因小鼠,陰性對照小鼠腹腔沖洗液總IgM、抗角蛋白IgM、抗金黃色葡萄球菌IgM水平。結(jié)果顯示,轉(zhuǎn)基因小鼠與陰性小鼠的總IgM水平無顯著差異,但轉(zhuǎn)基因小鼠的腹腔抗角蛋白IgM水平顯著高于對照小鼠,并且轉(zhuǎn)基因小鼠的腹腔沖洗液中抗金黃色葡萄球菌的IgM濃度顯著高于對照小鼠。 與腹腔抗角蛋白/金黃色葡萄球菌滴度相一致,感染后轉(zhuǎn)基因小鼠的腹腔漿細(xì)胞數(shù)顯著增多,提示細(xì)菌刺激后導(dǎo)致B細(xì)胞活化并轉(zhuǎn)化成漿細(xì)胞。進(jìn)一步檢測了B1細(xì)胞的增殖,發(fā)現(xiàn)轉(zhuǎn)基因小鼠的B1a細(xì)胞增殖顯著強于對照小鼠,提示基于對病原體的識別,轉(zhuǎn)基因小鼠的腹腔B1細(xì)胞發(fā)生了顯著的活化和增殖,分泌大量的抗體,在抗細(xì)菌感染中發(fā)揮了重要的作用。 同時,我們發(fā)現(xiàn)轉(zhuǎn)基因小鼠腹腔內(nèi)的B1細(xì)胞對細(xì)菌的結(jié)合/吞噬明顯增加,為了區(qū)別是結(jié)合還是吞噬,進(jìn)一步應(yīng)用激光共聚焦顯微鏡進(jìn)行了觀察,發(fā)現(xiàn)B細(xì)胞胞漿內(nèi)的綠色熒光顆粒,提示B1細(xì)胞可能吞噬了顆粒性細(xì)菌抗原,然而這一結(jié)果仍需大量的吞噬實驗予以證實和完善。 本研究從體外和在體水平揭示了天然抗體3B4對金黃色葡萄球菌感染的保護(hù)作用,并觀察到B1細(xì)胞對金黃色葡萄球菌感染的應(yīng)答,初步闡明了天然抗體及B1細(xì)胞抗感染作用的機(jī)制,豐富了對天然抗體的功能的認(rèn)識,具有重要的理論創(chuàng)新意義。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392.1

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