本文關(guān)鍵詞： B-1 B細(xì)胞 吞噬 細(xì)胞膜表面分子 金黃色葡萄球菌 脂多糖　出處：《第四軍醫(yī)大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 關(guān)于B1細(xì)胞的特性和功能等的研究一直是近10年來免疫學(xué)領(lǐng)域的重要課題。目前已明確,B1細(xì)胞是獨立的B細(xì)胞亞群。與傳統(tǒng)的B2細(xì)胞不同,B1細(xì)胞主要位于腹腔,表面標(biāo)記特點為B220lowIgMhiIgDlowCD23-CD43+Mac-1+。B1細(xì)胞在天然免疫中具有重要的作用。B1細(xì)胞不需要經(jīng)過體細(xì)胞突變,在接觸抗原后數(shù)小時內(nèi)即可擴增、活化而大量分泌抗體,在適應(yīng)性免疫應(yīng)答建立之前發(fā)揮抗感染作用。同時,B1細(xì)胞通過分泌IgA而參與粘膜免疫。另外B1細(xì)胞在自身免疫中也具有非常重要的作用,并參與缺血再灌注、動脈粥樣硬化、自身免疫性貧血等的病理生理過程。 以往關(guān)于B1細(xì)胞的天然免疫功能研究主要集中在其作為抗體分泌細(xì)胞和抗原呈遞細(xì)胞的作用上。然而,B1細(xì)胞具有的一些獨特特性如佛波酯刺激后活躍增生、表面表達(dá)吞噬相關(guān)分子CD11b、F4/80等提示B1細(xì)胞可能具有更加廣泛的天然免疫功能。最近Sunyer研究組報告了硬骨魚類(虹鱒魚)的B細(xì)胞可以吞噬顆�？乖�,首次揭示了進(jìn)化上較為原始的B細(xì)胞具有吞噬能力。而進(jìn)化上高級的哺乳動物B細(xì)胞是否具有吞噬能力即成為關(guān)注的焦點。 本研究組在前期分析轉(zhuǎn)基因小鼠抗金葡菌感染機制的過程中發(fā)現(xiàn)腹腔接種綠色熒光染料——羥基熒光素二醋酸鹽琥珀酰亞胺脂(Carboxy Fluoroscein Succinimidyl Ester, CFSE)染色的金葡菌后,CD19陽性的B細(xì)胞中存在著綠色熒光的細(xì)胞。這存在兩種解釋,一是B細(xì)胞表面黏附有金葡菌,二是B細(xì)胞吞噬有金葡菌,也可能二者兼而有之。為了澄清這一現(xiàn)象,我們采用激光共聚焦顯微鏡和透射電鏡繼續(xù)進(jìn)行了分析。共聚焦顯微鏡的結(jié)果發(fā)現(xiàn)在轉(zhuǎn)基因小鼠有較多CD19陽性的細(xì)胞內(nèi)有綠色熒光染色的細(xì)菌顆粒;透射電鏡的結(jié)果顯示,在淋巴樣細(xì)胞的胞漿內(nèi)有較多的球狀細(xì)菌。這些結(jié)果高度提示3B4轉(zhuǎn)基因小鼠的腹腔B細(xì)胞吞噬了金葡菌。 在形態(tài)學(xué)方面發(fā)現(xiàn)小鼠腹腔B細(xì)胞具有吞噬功能之后,接下來的問題是,B細(xì)胞吞噬的可能機制是什么?什么受體介導(dǎo)了吞噬?本研究從細(xì)胞膜表面吞噬相關(guān)分子的角度探索了可能參與B細(xì)胞吞噬過程的分子,對B細(xì)胞吞噬的機制進(jìn)行初步的探索。 一.小鼠腹腔B1細(xì)胞表面吞噬相關(guān)分子表達(dá)的研究 取C57BL/6小鼠的腹腔灌洗液細(xì)胞及脾臟細(xì)胞,以Biotin-rat anti mouse IgM及親和素標(biāo)記的磁珠進(jìn)行免疫磁珠分選,分選后細(xì)胞分別提取總RNA并進(jìn)行RT-PCR,凝膠電泳比較分析各膜表面分子的表達(dá)情況。并通過流式細(xì)胞術(shù)對CD14、CD36在小鼠B-1 B細(xì)胞及B-2 B細(xì)胞的表達(dá)進(jìn)行了分析。發(fā)現(xiàn)CD206、CD14、TLR-2、TLR-4、CD36、MARCO、CD32、CD11b、CD18、ABCA1均表達(dá)于正常小鼠腹腔B1細(xì)胞,但其表達(dá)量較腹腔吞噬細(xì)胞低,其中CD14、CD36、MARCO、CD11b在B1細(xì)胞的表達(dá)水平高于在B2細(xì)胞的表達(dá)水平。流式細(xì)胞術(shù)結(jié)果發(fā)現(xiàn)CD14及CD36在B-1 B細(xì)胞及B-2 B細(xì)胞低水平表達(dá),且與RT-PCR結(jié)果基本一致。在此實驗基礎(chǔ)上我們以金葡菌和LPS為模式抗原觀察上述膜表面分子在抗原刺激后的表達(dá)變化。 二.金葡菌刺激后B-1 B細(xì)胞表面吞噬相關(guān)分子的表達(dá)變化 小鼠腹腔內(nèi)注射金葡菌,于注射后1h、3h、6h處死小鼠,收集腹腔及脾臟細(xì)胞,免疫磁珠分選后提取總RNA并進(jìn)行RT-PCR。結(jié)果發(fā)現(xiàn)CD14、CD206在金葡菌刺激后在B-1B細(xì)胞的表達(dá)量較未刺激時升高,且在B-1 B細(xì)胞的表達(dá)水平高于在B2 B細(xì)胞的表達(dá)水平。 三.LPS刺激后B-1 B細(xì)胞表面吞噬相關(guān)分子的表達(dá)變化 小鼠腹腔內(nèi)注射LPS,于注射后1h、3h、6h處死小鼠,收集腹腔及脾臟細(xì)胞,免疫磁珠分選后提取總RNA并進(jìn)行RT-PCR。CD11b、CD14在LPS刺激后在B-1B細(xì)胞的表達(dá)量較未刺激時升高,且在B-1 B細(xì)胞的表達(dá)水平高于在B2 B細(xì)胞的表達(dá)水平。 四.腹腔B細(xì)胞表面CD14、CD11b阻斷實驗 在前期實驗的基礎(chǔ)上,我們選取CD14、CD11b進(jìn)行阻斷,觀察阻斷后腹腔B細(xì)胞對金葡菌吞噬能力的變化。收獲小鼠腹腔細(xì)胞后靜置貼壁2h以獲取B細(xì)胞,隨后將收獲的小鼠腹腔B細(xì)胞分別與CD14和(或)CD11b的阻斷抗體共同孵育后加入Co60滅活的金葡菌,2h后流式細(xì)胞儀檢測有吞噬能力的B細(xì)胞的比率。阻斷后各組之間吞噬率無統(tǒng)計學(xué)差異。 綜上所述,我們的研究結(jié)果首次發(fā)現(xiàn)CD206、CD14、CD36、MARCO、CD32、CD18、ABCA1均表達(dá)于正常B-1 B細(xì)胞。上述多個吞噬相關(guān)分子在B-1 B細(xì)胞均有表達(dá)提示B-1 B細(xì)胞可能具有吞噬能力。其中CD14、CD36、MARCO、CD11b在B1細(xì)胞的表達(dá)水平高于在B2細(xì)胞的表達(dá)水平,推測其可能參與B-1 B細(xì)胞區(qū)別于傳統(tǒng)B-2 B細(xì)胞的獨特生物學(xué)功能的發(fā)揮。進(jìn)一步的實驗結(jié)果說明CD206和MARCO可能參與了B-1 B細(xì)胞對金葡菌的識別和應(yīng)答而CD11b、MARCO、CD14可能在B-1 B細(xì)胞對LPS的識別中起到較為重要的作用。
[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.

【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】：2009
【分類號】：R392

【共引文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 鄭力強;天然抗角蛋白自身抗體在病理性自身免疫中的作用及機制的研究[D];第四軍醫(yī)大學(xué);2007年

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