【摘要】：在現(xiàn)代分子克隆中，人們普遍認(rèn)為大腸桿菌麥芽糖結(jié)合蛋白（maltose bindingprotein, MBP）無生物學(xué)活性或具有較低的生物學(xué)活性，故將其作為分子生物學(xué)常用的標(biāo)簽蛋白。各種實(shí)驗(yàn)性病原菌和病毒的亞單位疫苗即利用了它的這一特性來進(jìn)行疫苗研究。然而，最近有文獻(xiàn)報(bào)道MBP通過TLR4促進(jìn)DC細(xì)胞的成熟并分泌細(xì)胞因子。本課題組研究發(fā)現(xiàn)MBP能提高M(jìn)UC1的免疫原性，與BCG協(xié)同顯著增強(qiáng)細(xì)胞免疫應(yīng)答，明顯抑制小鼠皮下人乳腺癌移植瘤和小鼠Lewis肺癌移植瘤和轉(zhuǎn)移瘤的生長(zhǎng)。探討腫瘤生長(zhǎng)抑制機(jī)制的結(jié)果顯示，MBP與BCG聯(lián)合免疫組小鼠乳腺癌組織周圍有大量淋巴細(xì)胞浸潤(rùn)，且CD4+T淋巴細(xì)胞較MBP或BCG單獨(dú)免疫組顯著增多。研究也發(fā)現(xiàn)MBP不僅能增強(qiáng)巨噬細(xì)胞吞噬活性，誘導(dǎo)M1型巨噬細(xì)胞活化，也可促進(jìn)NK細(xì)胞活化。研究結(jié)果均提示MBP具有免疫增強(qiáng)作用，但其具體作用及其機(jī)制還不清楚。本研究通過探討MBP對(duì)小鼠脾臟淋巴細(xì)胞的作用，明確MBP對(duì)T細(xì)胞的作用及其誘導(dǎo)的免疫應(yīng)答類型。研究表明MBP非特異性刺激T淋巴細(xì)胞增殖，誘導(dǎo)Th1細(xì)胞活化，且MBP直接作用于淋巴細(xì)胞。本研究揭示MBP可直接刺激機(jī)體的免疫系統(tǒng)，誘導(dǎo)Th1方向的細(xì)胞免疫應(yīng)答，為MBP作為一種新型免疫增強(qiáng)劑應(yīng)用于腫瘤疫苗提供理論基礎(chǔ)。目前在腫瘤疫苗的研究中，力求能尋找到誘導(dǎo)Th1和CTL細(xì)胞應(yīng)答的佐劑，用于腫瘤疫苗中誘導(dǎo)細(xì)胞免疫應(yīng)答。MBP可誘導(dǎo)Th1細(xì)胞活化，其有望成為腫瘤疫苗的佐劑。本研究室已經(jīng)展開了這方面的工作，已對(duì)MBP作為腫瘤疫苗的佐劑刺激機(jī)體產(chǎn)生細(xì)胞免疫應(yīng)答而發(fā)揮抗腫瘤作用進(jìn)行了相關(guān)文獻(xiàn)報(bào)道。本研究擬從另一角度即MBP對(duì)腫瘤細(xì)胞的直接作用這一角度展開研究。文獻(xiàn)報(bào)道微生物佐劑可直接作用于腫瘤細(xì)胞發(fā)揮促凋亡或抗凋亡作用，尤其在血液腫瘤，故本研究將進(jìn)一步探討MBP是否可在體外直接作用于血液腫瘤細(xì)胞發(fā)揮生物學(xué)作用及其具體的機(jī)制，希望在探討MBP抗腫瘤作用的同時(shí)尋找MBP的作用靶點(diǎn)。本研究表明MBP直接作用于U937細(xì)胞，通過TLR2活化細(xì)胞的NF-κB和MAPK信號(hào)通道，并調(diào)節(jié)細(xì)胞表面TLRs和MyD88的表達(dá)，促進(jìn)U937細(xì)胞增殖和分化；MBP直接作用于Jurkat細(xì)胞，活化細(xì)胞的NF-κB和MAPK信號(hào)通道，并調(diào)節(jié)細(xì)胞表面TLRs和MyD88的表達(dá)，，促進(jìn)Jurkat細(xì)胞增殖，但這種作用未通過TLR2和TLR4發(fā)揮，同時(shí)研究發(fā)現(xiàn)MBP提高了U937和Jurkat細(xì)胞對(duì)化療藥物紫杉醇的敏感性。提示MBP直接作用于U937和Jurkat細(xì)胞，使細(xì)胞處于增殖狀態(tài)，與化療藥物聯(lián)合使用可提高化療藥物的作用效果。 本研究揭示MBP可直接增強(qiáng)機(jī)體的免疫應(yīng)答，為MBP作為一種新型免疫增強(qiáng)劑的廣泛推廣提供理論基礎(chǔ)，并為血液腫瘤的免疫治療提供新的佐劑和研究思路。
[Abstract]:In modern molecular cloning, it is generally believed that E. coli maltose binding protein (maltose bindingprotein, MBP) has no biological activity or low biological activity, so it is regarded as a commonly used tag protein in molecular biology. The subunit vaccines of various experimental pathogens and viruses use this property for vaccine research. Recently, however, it has been reported that MBP promotes the maturation and secretion of cytokines in DC cells through TLR4. Our study found that MBP could enhance the immunogenicity of MUC1, enhance the cellular immune response in combination with BCG, and inhibit the growth of subcutaneous human breast cancer xenografts in mice and Lewis lung cancer xenografts and metastases in mice. The results of the study on the mechanism of tumor growth inhibition showed that a large number of lymphocytes infiltrated around the breast cancer tissues of mice immunized with MBP and BCG, and CD4 T lymphocytes increased significantly compared with those of MBP or BCG alone. It was also found that MBP not only enhanced the phagocytic activity of macrophages, induced the activation of M1 macrophages, but also promoted the activation of NK cells. All the results suggest that MBP has immune enhancement effect, but its specific role and mechanism are not clear. The purpose of this study was to investigate the effect of MBP on T cells and the type of immune response induced by MBP. The results showed that MBP stimulated the proliferation of T lymphocytes and induced the activation of Th1 cells, and MBP acted directly on lymphocytes. This study revealed that MBP can directly stimulate the immune system and induce cellular immune response in the direction of Th1, which provides a theoretical basis for the application of MBP as a new type of immune enhancer in tumor vaccines. At present, in the research of tumor vaccine, we try to find the adjuvant to induce Th1 and CTL cell response, which can be used to induce cellular immune response in tumor vaccine. MBP can induce the activation of Th1 cells, which is expected to be the adjuvant of tumor vaccine. This work has been carried out in our laboratory, and it has been reported that MBP, as an adjuvant of tumor vaccine, can stimulate the cellular immune response and play an antitumor role in the body. The purpose of this study is to study the direct effect of MBP on tumor cells. It has been reported that microbial adjuvants can directly promote apoptosis or inhibit apoptosis in tumor cells, especially in blood tumors. Therefore, this study will further explore whether MBP can directly act on blood tumor cells in vitro and its specific mechanism. We hope to explore the anti-tumor effect of MBP and seek the target of MBP at the same time. This study showed that MBP acts directly on U937 cells, activates NF- 魏 B and MAPK signaling channels through TLR2, and regulates the expression of TLRs and MyD88 on the surface of U937 cells, thus promoting the proliferation and differentiation of U937 cells. MBP acts directly on Jurkat cells, activates NF- 魏 B and MAPK signal channels, regulates the expression of TLRs and MyD88 on the cell surface, and promotes the proliferation of Jurkat cells, but this effect is not played by TLR2 and TLR4. At the same time, MBP increased the sensitivity of U937 and Jurkat cells to paclitaxel. The results suggest that MBP acts directly on U937 and Jurkat cells, which makes the cells proliferate. The combination of MBP and chemotherapeutic drugs can improve the effect of chemotherapeutic drugs. This study revealed that MBP can directly enhance the immune response of the body, provide a theoretical basis for the wide application of MBP as a new immune enhancer, and provide a new adjuvant and research idea for immunotherapy of blood tumors.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.9

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