B-1 B细胞表面吞噬相关分子分析

发布时间：2018-03-02 07:43

本文关键词： B-1 B细胞吞噬细胞膜表面分子金黄色葡萄球菌脂多糖　出处：《第四军医大学》2009年硕士论文　论文类型：学位论文

【摘要】： 关于B1细胞的特性和功能等的研究一直是近10年来免疫学领域的重要课题。目前已明确,B1细胞是独立的B细胞亚群。与传统的B2细胞不同,B1细胞主要位于腹腔,表面标记特点为B220lowIgMhiIgDlowCD23-CD43+Mac-1+。B1细胞在天然免疫中具有重要的作用。B1细胞不需要经过体细胞突变,在接触抗原后数小时内即可扩增、活化而大量分泌抗体,在适应性免疫应答建立之前发挥抗感染作用。同时,B1细胞通过分泌IgA而参与粘膜免疫。另外B1细胞在自身免疫中也具有非常重要的作用,并参与缺血再灌注、动脉粥样硬化、自身免疫性贫血等的病理生理过程。以往关于B1细胞的天然免疫功能研究主要集中在其作为抗体分泌细胞和抗原呈递细胞的作用上。然而,B1细胞具有的一些独特特性如佛波酯刺激后活跃增生、表面表达吞噬相关分子CD11b、F4/80等提示B1细胞可能具有更加广泛的天然免疫功能。最近Sunyer研究组报告了硬骨鱼类(虹鳟鱼)的B细胞可以吞噬颗粒抗原,首次揭示了进化上较为原始的B细胞具有吞噬能力。而进化上高级的哺乳动物B细胞是否具有吞噬能力即成为关注的焦点。本研究组在前期分析转基因小鼠抗金葡菌感染机制的过程中发现腹腔接种绿色荧光染料——羟基荧光素二醋酸盐琥珀酰亚胺脂(Carboxy Fluoroscein Succinimidyl Ester, CFSE)染色的金葡菌后,CD19阳性的B细胞中存在着绿色荧光的细胞。这存在两种解释,一是B细胞表面黏附有金葡菌,二是B细胞吞噬有金葡菌,也可能二者兼而有之。为了澄清这一现象,我们采用激光共聚焦显微镜和透射电镜继续进行了分析。共聚焦显微镜的结果发现在转基因小鼠有较多CD19阳性的细胞内有绿色荧光染色的细菌颗粒;透射电镜的结果显示,在淋巴样细胞的胞浆内有较多的球状细菌。这些结果高度提示3B4转基因小鼠的腹腔B细胞吞噬了金葡菌。在形态学方面发现小鼠腹腔B细胞具有吞噬功能之后,接下来的问题是,B细胞吞噬的可能机制是什么?什么受体介导了吞噬?本研究从细胞膜表面吞噬相关分子的角度探索了可能参与B细胞吞噬过程的分子,对B细胞吞噬的机制进行初步的探索。一.小鼠腹腔B1细胞表面吞噬相关分子表达的研究取C57BL/6小鼠的腹腔灌洗液细胞及脾脏细胞,以Biotin-rat anti mouse IgM及亲和素标记的磁珠进行免疫磁珠分选,分选后细胞分别提取总RNA并进行RT-PCR,凝胶电泳比较分析各膜表面分子的表达情况。并通过流式细胞术对CD14、CD36在小鼠B-1 B细胞及B-2 B细胞的表达进行了分析。发现CD206、CD14、TLR-2、TLR-4、CD36、MARCO、CD32、CD11b、CD18、ABCA1均表达于正常小鼠腹腔B1细胞,但其表达量较腹腔吞噬细胞低,其中CD14、CD36、MARCO、CD11b在B1细胞的表达水平高于在B2细胞的表达水平。流式细胞术结果发现CD14及CD36在B-1 B细胞及B-2 B细胞低水平表达,且与RT-PCR结果基本一致。在此实验基础上我们以金葡菌和LPS为模式抗原观察上述膜表面分子在抗原刺激后的表达变化。二.金葡菌刺激后B-1 B细胞表面吞噬相关分子的表达变化小鼠腹腔内注射金葡菌,于注射后1h、3h、6h处死小鼠,收集腹腔及脾脏细胞,免疫磁珠分选后提取总RNA并进行RT-PCR。结果发现CD14、CD206在金葡菌刺激后在B-1B细胞的表达量较未刺激时升高,且在B-1 B细胞的表达水平高于在B2 B细胞的表达水平。三.LPS刺激后B-1 B细胞表面吞噬相关分子的表达变化小鼠腹腔内注射LPS,于注射后1h、3h、6h处死小鼠,收集腹腔及脾脏细胞,免疫磁珠分选后提取总RNA并进行RT-PCR。CD11b、CD14在LPS刺激后在B-1B细胞的表达量较未刺激时升高,且在B-1 B细胞的表达水平高于在B2 B细胞的表达水平。四.腹腔B细胞表面CD14、CD11b阻断实验在前期实验的基础上,我们选取CD14、CD11b进行阻断,观察阻断后腹腔B细胞对金葡菌吞噬能力的变化。收获小鼠腹腔细胞后静置贴壁2h以获取B细胞,随后将收获的小鼠腹腔B细胞分别与CD14和(或)CD11b的阻断抗体共同孵育后加入Co60灭活的金葡菌,2h后流式细胞仪检测有吞噬能力的B细胞的比率。阻断后各组之间吞噬率无统计学差异。综上所述,我们的研究结果首次发现CD206、CD14、CD36、MARCO、CD32、CD18、ABCA1均表达于正常B-1 B细胞。上述多个吞噬相关分子在B-1 B细胞均有表达提示B-1 B细胞可能具有吞噬能力。其中CD14、CD36、MARCO、CD11b在B1细胞的表达水平高于在B2细胞的表达水平,推测其可能参与B-1 B细胞区别于传统B-2 B细胞的独特生物学功能的发挥。进一步的实验结果说明CD206和MARCO可能参与了B-1 B细胞对金葡菌的识别和应答而CD11b、MARCO、CD14可能在B-1 B细胞对LPS的识别中起到较为重要的作用。
[Abstract]:Study on the characteristics and function of B1 cells is an important subject in the field of immunology in recent 10 years. It is now clear that B1 cells are independent B cell subsets. Unlike traditional B2 cells, B1 cells were mainly located in the abdominal cavity, the surface marker characteristics of B220lowIgMhiIgDlowCD23-CD43+ Mac-1+.B1 cells plays an important role in natural.B1 cells immunity without somatic mutations that can be amplified in a few hours after exposure to antigen, activation and secretion of antibodies play a role of anti infection before the adaptive immune response is established. At the same time, B1 cells by secreting IgA in mucosal immune B1 cells. In addition also has a very important role in autoimmunity, and in ischemia reperfusion, atherosclerosis, pathophysiology of autoimmune anemia.
The previous research on the immune function of B1 cells mainly concentrated in as antibody secreting cells and antigen presenting cells. However, B1 cells have some unique characteristics such as phorbol ester stimulated active proliferation, phagocytosis related molecules expressed on the surface of CD11b F4/80, suggesting that B1 cells may have innate immune function more widely recently. Sunyer research group reported teleost fish (rainbow trout) B cells phagocytize particulate antigen, revealed for the first time on the evolution of primitive B cells with phagocytosis. The evolution of advanced mammalian B cell phagocytosis is whether it has become the focus of attention.
Found that intraperitoneal inoculation of green fluorescent dye carboxyfluorescein succinimidyl ester: two this research group analysis of transgenic mice in the early stage of anti Staphylococcus aureus infection mechanism in (Carboxy Fluoroscein Succinimidyl Ester, CFSE) were Staphylococcus aureus, there are green fluorescent cells CD19 positive B cells. There are two possible explanations, one is adhered on the surface of B cells with Staphylococcus aureus, two B phagocytosis of Staphylococcus aureus, may also be the combination of the two. In order to clarify this phenomenon, we used laser confocal microscopy and transmission electron microscopy to were analyzed. Results of confocal microscopy showed that in transgenic mice have more CD19 positive cells bacteria particle green fluorescence staining; transmission electron microscopy showed that there are more spherical bacteria in the cytoplasm of lymphoid cells. These results suggested that 3B4 transgenic mice The B cells in the abdominal cavity phagocytic Staphylococcus aureus.
Found in the morphology of mouse peritoneal B cells with phagocytosis, the next question is, what is the possible mechanism of B cell phagocytosis? What receptor mediated phagocytosis? Molecules related to this study from the cell membrane surface phagocytosis explored may be involved in the process of phagocytosis of B molecules, makes a preliminary exploration on the mechanism of B phagocytosis.
Study on the expression of phagocytic molecules on the surface of B1 cells in mouse abdominal cavity
From C57BL/6 mouse peritoneal cells and spleen cells by immunomagnetic separation with Biotin-rat anti mouse IgM and affinity labeled beads, total RNA was extracted from the sorted cells and the expression of RT-PCR, comparative analysis of surface molecules of each membrane. Through gel electrophoresis and flow cytometry of CD14, CD36 were analysis on B-1 expression of B cells and B-2 cells of mice with B. CD206, CD14, TLR-2, TLR-4, CD36, MARCO, CD32, CD11b, CD18, ABCA1 were expressed in normal mouse peritoneal B1 cells, but its expression is relatively low phagocyte, including CD14, CD36, MARCO, CD11b expression level in B1 higher cell expression level in B2 cells. Flow cytometry results showed that the expression of CD14 and CD36 in the low level of B-1 B cells and B-2 B cells, which is consistent with the RT-PCR results. On the basis of the experiment we to Staphylococcus aureus and LPS as a model antigen observed above The changes in the expression of the membrane surface molecules after the stimulation of the antigen.
Two. Changes in the expression of phagocytic molecules on the surface of B-1 B cells after Staphylococcus aureus
Intraperitoneal injection of Staphylococcus aureus, after injection of 1H, 3h, 6h mice were killed, were collected and spleen cells, immunomagnetic separation after extraction of total RNA and RT-PCR. results showed that CD14 increased CD206 in Staphylococcus aureus stimulated expression in B-1B cells was not stimulated, and the expression level of B-1 B the expression level of B2 cells than in B cells.
Changes in the expression of phagocytic molecules on the surface of B-1 B cells after three.LPS stimulation
Intraperitoneal injection of LPS after injection of 1H, 3h, 6h mice were killed, were collected and spleen cells, immunomagnetic separation after extraction of total RNA and RT-PCR.CD11b increased at CD14 after the stimulation of LPS expression in B-1B cells was not stimulated, and the expression level of B-1 in B cells than in the expression level of B2 B cells.
Four. CD14, CD11b blocking experiment on the surface of B cells in abdominal cavity
Based on the previous experiment, we selected CD14, CD11b block, to observe the changes of B cells after intraperitoneal on phagocytosis of Staphylococcus aureus. The blocking of harvest mouse peritoneal cells after incubation of adherent 2H to B cells, then harvest mouse peritoneal B cells respectively with CD14 and (or) CD11b blocking antibody after CO incubation adding Co60 inactivated Staphylococcus aureus, 2h flow cytometry ratio of phagocytosis of B cells. Between the blocking group and negative control group was not statistically significant.
In summary, our results for the first time that CD206, CD14, CD36, MARCO, CD32, CD18, ABCA1 were expressed in normal B-1 B cells. The phagocytosis related molecules expression of B-1 B cells may have phagocytosis in both B-1 cells. B CD14, CD36, MARCO, CD11b expression level in B1 cells higher expression level in B2 cells, that unique biological function might be involved in the difference between B-1 B cells from the traditional B-2 B cells play. Further experimental results show that CD206 and MARCO may be involved in the recognition of B-1 B cells of Staphylococcus aureus and a CD11b, MARCO, CD14 may play a more important role in the B-1 B cell recognition of the LPS.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【共引文献】