抗白细胞介素-33主动免疫在小鼠乳腺癌肿瘤模型中的干预研究

发布时间：2018-11-04 17:58

【摘要】：背景白细胞介素-33(IL-33)是一种多效细胞因子,与多种肿瘤的发生发展密切相关,在乳腺癌、肺癌、胃癌、卵巢癌等肿瘤疾病中呈现异常高表达。作为2型免疫反应关键的促发介质,IL-33显著刺激Th2及2型巨噬细胞的分化与增殖,并促进MDSCs与Treg向肿瘤组织浸润,从而在肿瘤微环境中可能发挥了重要的促肿瘤免疫抑制及免疫逃逸作用。因此,IL-33是潜在的肿瘤免疫治疗靶点,针对IL-33的免疫干预研究,可能为肿瘤的预防和治疗提供新的思路。目的本研究拟以抗细胞因子主动免疫方式下调肿瘤小鼠体内IL-33水平,考察由此对肿瘤生长及抗肿瘤免疫应答的影响,揭示IL-33在乳腺癌形成与发展中的作用及靶向其主动免疫干预策略在乳腺癌等肿瘤临床治疗中的应用潜能。方法本研究利用乙肝核心抗原(HBcAg)病毒样颗粒(VLPs)呈递IL-33全分子,在大肠杆菌中诱导重组蛋白表达,经硫酸铵盐析法和碘克沙醇密度梯度离心纯化,并经透射电镜进行分析鉴定。VLPs形式的疫苗经预防性或治疗性免疫策略分别对荷瘤小鼠进行免疫干预。研究检测包括:以ELISA检测了免疫小鼠血清中IL-33特异的IgG抗体水平;每2日测量小鼠皮下肿瘤大小;记录肺肿瘤结节数目以考察肺转移情况;以ELISA分析血清IgG1、IgG2a抗体亚型水平;脾细胞分离后经PMA刺激,以ELISA检测IFN-γ表达,以ELISPOT检测分泌IFN-Y的脾细胞,以流式细胞分析仪检测CD8+IFN-γ+CTL细胞。此外,肿瘤组织浸润的淋巴细胞分离后用于Treg、MDSCs的流式细胞分析。结果重组的HBcAg与IL-33嵌合蛋白HBcAg-33在大肠杆菌DH5α中获得了高效表达,纯化的蛋白主要以VLPs形式存在。经预防性策略免疫小鼠后,显著抑制了乳腺肿瘤的生长。治疗性干预策略中,在肿瘤生长早期HBcAg-33 VLPs免疫明显抑制肿瘤增长,但随着肿瘤生长变大,抑制作用减弱,但肺部的肿瘤转移在免疫小鼠中获得了显著抑制。免疫小鼠血清中可检测到高滴度IL-33特异抗体应答。进一步的免疫应答分析显示,HBcAg-33 VLPs疫苗刺激机体产生显著增强的脾细胞INF-γ表达及增多的表达INF-γ的脾细胞数目;流式检测显示脾细胞中 CD8+INF-γ+CTL 细胞数、CD4+INF-γ+Th1显著增加,而 CD4+IL-4+Th2 细胞水平有一定程度下降。此外,肿瘤组织中的MDSCs、Treg细胞数目在疫苗接种小鼠中显著下调。对应于细胞因子表达及流式检测免疫细胞情况所反映的Th1/CTL免疫应答偏向性,血清中IgG1/IgG2a比例显著降低。结论呈递IL-33全分子的HBcAgVLPs疫苗能够激发持续的特异抗体应答,在乳腺癌模型中能够显著抑制肿瘤生长及转移,可能的机制是调控T细胞应答向Th1/CTLs型偏向。研究结果有助于揭示IL-33在乳腺癌形成、生长及转移中可能的促进作用,以及抗IL-33疫苗主动免疫策略在乳腺癌临床治疗中潜在应用前景。
[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- 纬 in spleen cells was detected by ELISA, IFN-Y secreted by ELISPOT and CD8 IFN- 纬 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 伪, 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- 纬 expression and increased number of spleen cells expressing INF- 纬. Flow cytometry showed that the number of CD8 INF- 纬 CTL cells and CD4 INF- 纬 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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9;R-332

【相似文献】