【摘要】：背景白細(xì)胞介素-33(IL-33)是一種多效細(xì)胞因子,與多種腫瘤的發(fā)生發(fā)展密切相關(guān),在乳腺癌、肺癌、胃癌、卵巢癌等腫瘤疾病中呈現(xiàn)異常高表達(dá)。作為2型免疫反應(yīng)關(guān)鍵的促發(fā)介質(zhì),IL-33顯著刺激Th2及2型巨噬細(xì)胞的分化與增殖,并促進(jìn)MDSCs與Treg向腫瘤組織浸潤(rùn),從而在腫瘤微環(huán)境中可能發(fā)揮了重要的促腫瘤免疫抑制及免疫逃逸作用。因此,IL-33是潛在的腫瘤免疫治療靶點(diǎn),針對(duì)IL-33的免疫干預(yù)研究,可能為腫瘤的預(yù)防和治療提供新的思路。目的本研究擬以抗細(xì)胞因子主動(dòng)免疫方式下調(diào)腫瘤小鼠體內(nèi)IL-33水平,考察由此對(duì)腫瘤生長(zhǎng)及抗腫瘤免疫應(yīng)答的影響,揭示IL-33在乳腺癌形成與發(fā)展中的作用及靶向其主動(dòng)免疫干預(yù)策略在乳腺癌等腫瘤臨床治療中的應(yīng)用潛能。方法本研究利用乙肝核心抗原(HBcAg)病毒樣顆粒(VLPs)呈遞IL-33全分子,在大腸桿菌中誘導(dǎo)重組蛋白表達(dá),經(jīng)硫酸銨鹽析法和碘克沙醇密度梯度離心純化,并經(jīng)透射電鏡進(jìn)行分析鑒定。VLPs形式的疫苗經(jīng)預(yù)防性或治療性免疫策略分別對(duì)荷瘤小鼠進(jìn)行免疫干預(yù)。研究檢測(cè)包括:以ELISA檢測(cè)了免疫小鼠血清中IL-33特異的IgG抗體水平;每2日測(cè)量小鼠皮下腫瘤大小;記錄肺腫瘤結(jié)節(jié)數(shù)目以考察肺轉(zhuǎn)移情況;以ELISA分析血清IgG1、IgG2a抗體亞型水平;脾細(xì)胞分離后經(jīng)PMA刺激,以ELISA檢測(cè)IFN-γ表達(dá),以ELISPOT檢測(cè)分泌IFN-Y的脾細(xì)胞,以流式細(xì)胞分析儀檢測(cè)CD8+IFN-γ+CTL細(xì)胞。此外,腫瘤組織浸潤(rùn)的淋巴細(xì)胞分離后用于Treg、MDSCs的流式細(xì)胞分析。結(jié)果重組的HBcAg與IL-33嵌合蛋白HBcAg-33在大腸桿菌DH5α中獲得了高效表達(dá),純化的蛋白主要以VLPs形式存在。經(jīng)預(yù)防性策略免疫小鼠后,顯著抑制了乳腺腫瘤的生長(zhǎng)。治療性干預(yù)策略中,在腫瘤生長(zhǎng)早期HBcAg-33 VLPs免疫明顯抑制腫瘤增長(zhǎng),但隨著腫瘤生長(zhǎng)變大,抑制作用減弱,但肺部的腫瘤轉(zhuǎn)移在免疫小鼠中獲得了顯著抑制。免疫小鼠血清中可檢測(cè)到高滴度IL-33特異抗體應(yīng)答。進(jìn)一步的免疫應(yīng)答分析顯示,HBcAg-33 VLPs疫苗刺激機(jī)體產(chǎn)生顯著增強(qiáng)的脾細(xì)胞INF-γ表達(dá)及增多的表達(dá)INF-γ的脾細(xì)胞數(shù)目;流式檢測(cè)顯示脾細(xì)胞中 CD8+INF-γ+CTL 細(xì)胞數(shù)、CD4+INF-γ+Th1顯著增加,而 CD4+IL-4+Th2 細(xì)胞水平有一定程度下降。此外,腫瘤組織中的MDSCs、Treg細(xì)胞數(shù)目在疫苗接種小鼠中顯著下調(diào)。對(duì)應(yīng)于細(xì)胞因子表達(dá)及流式檢測(cè)免疫細(xì)胞情況所反映的Th1/CTL免疫應(yīng)答偏向性,血清中IgG1/IgG2a比例顯著降低。結(jié)論呈遞IL-33全分子的HBcAgVLPs疫苗能夠激發(fā)持續(xù)的特異抗體應(yīng)答,在乳腺癌模型中能夠顯著抑制腫瘤生長(zhǎng)及轉(zhuǎn)移,可能的機(jī)制是調(diào)控T細(xì)胞應(yīng)答向Th1/CTLs型偏向。研究結(jié)果有助于揭示IL-33在乳腺癌形成、生長(zhǎng)及轉(zhuǎn)移中可能的促進(jìn)作用,以及抗IL-33疫苗主動(dòng)免疫策略在乳腺癌臨床治療中潛在應(yīng)用前景。
[Abstract]:Background Interleukin-33 (IL-33) is a multipotent cytokine, which is closely related to the occurrence and development of many kinds of tumors. It is highly expressed in breast cancer, lung cancer, gastric cancer, ovarian cancer and other tumor diseases. As a key stimulator of type 2 immune response, IL-33 significantly stimulated the differentiation and proliferation of Th2 and type 2 macrophages, and promoted the infiltration of MDSCs and Treg into tumor tissues. It may play an important role in tumor immune suppression and immune escape in tumor microenvironment. Therefore, IL-33 is a potential target of tumor immunotherapy. The immunological intervention of IL-33 may provide new ideas for the prevention and treatment of tumor. Objective to study the effect of anti-cytokine active immunization on tumor growth and anti-tumor immune response in mice by down-regulating the level of IL-33 in vivo. To reveal the role of IL-33 in the formation and development of breast cancer and the potential application of targeted active immune intervention strategies in clinical treatment of breast cancer and other tumors. Methods the recombinant protein was expressed in Escherichia coli and purified by ammonium sulfate salting out method and density gradient centrifugation with iodoxacin. The VLPs vaccine was immunized by prophylactic or therapeutic immunization strategy in mice bearing tumor. ELISA was used to detect the level of IL-33 specific IgG antibody in the serum of immunized mice; the size of subcutaneous tumor was measured every 2 days; the number of pulmonary tumor nodules was recorded to investigate lung metastasis. The expression of IFN- 緯 in spleen cells was detected by ELISA, IFN-Y secreted by ELISPOT and CD8 IFN- 緯 CTL cells by flow cytometry. In addition, lymphocytes infiltrated by tumor tissue were separated and used for flow cytometry analysis of Treg,MDSCs. Results the recombinant HBcAg and IL-33 chimeric protein HBcAg-33 were highly expressed in Escherichia coli DH5 偽, and the purified protein mainly existed in the form of VLPs. After immunizing mice with prophylactic strategy, the growth of breast tumor was significantly inhibited. In the therapeutic intervention strategy, HBcAg-33 VLPs immunity significantly inhibited tumor growth in the early stage of tumor growth, but the inhibitory effect was weakened with the tumor growing larger, but lung metastasis was significantly inhibited in immunized mice. High titer IL-33 specific antibody response could be detected in the serum of immunized mice. Further analysis of immune response showed that HBcAg-33 VLPs vaccine stimulated spleen cells to produce significantly enhanced INF- 緯 expression and increased number of spleen cells expressing INF- 緯. Flow cytometry showed that the number of CD8 INF- 緯 CTL cells and CD4 INF- 緯 Th1 in spleen cells increased significantly, while the level of CD4 IL-4 Th2 cells decreased to a certain extent. In addition, the number of MDSCs,Treg cells in tumor tissues was significantly down-regulated in vaccinated mice. According to the bias of Th1/CTL immune response reflected by cytokine expression and flow cytometry, the proportion of IgG1/IgG2a in serum decreased significantly. Conclusion the HBcAgVLPs vaccine presented with the whole IL-33 molecule can stimulate a sustained specific antibody response and inhibit tumor growth and metastasis significantly in breast cancer model. The possible mechanism is to regulate the T cell response to the Th1/CTLs type bias. The results are helpful to reveal the possible role of IL-33 in the formation, growth and metastasis of breast cancer, and the potential application prospect of active immunization strategy against IL-33 vaccine in the clinical treatment of breast cancer.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9;R-332

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