基于纳米系统的免疫、化疗联合治疗晚期黑色素瘤

发布时间：2018-05-19 02:42

本文选题：姜黄素胶束 + 肿瘤纳米疫苗　；参考：《华中科技大学》2016年博士论文

【摘要】：研究目的:肿瘤免疫治疗是应用免疫学方法,激发体内免疫系统,产生杀伤肿瘤细胞的特异性因子杀伤肿瘤细胞、从而抑制肿瘤生长的治疗方式,近年来备受关注。但是单独使用免疫治疗时反应率不是很高,尤其对于晚期恶性黑色素瘤的治疗效果明显降低。因为随着肿瘤生长,到晚期时,肿瘤微环境呈免疫抑制状态,阻止细胞毒性T淋巴细胞(CTL)向肿瘤组织浸润。比较可行的方法是将这些能够刺激免疫反应的分子或抗体或核酸等制成纳米形式,增强抗原递呈等免疫作用的同时与化学预防药物或者化学治疗药物联合使用,降低肿瘤微环境中的免疫抑制因子水平、加强免疫治疗的效果、有效遏制恶性黑色素瘤生长。而对于肿瘤抗原明确的模型以及临床上没有找到合适的肿瘤抗原时的模型采取不同的联合给药方式或许更有临床实际应用价值。方法:对于肿瘤抗原明确的模型,我们采用了磷酸钙核-壳型纳米粒包裹肿瘤多肽分子与佐剂,制成纳米疫苗(Trp-Vac);与化学预防药物姜黄素纳米胶束(Cur PM)联合给予小鼠。我们首先合成了Cur PM,对其表征,测试了其在荷瘤小鼠中的组织分布。再联合给药,进行肿瘤生长抑制实验,体内细胞毒性T淋巴细胞(in vivo CTL)杀伤效应实验和酶联免疫斑点检测(ELISPOT)等实验,评估细胞毒性T细胞增殖效果。接着用流式细胞术测定肿瘤浸润免疫细胞数目变化,实时荧光定量PCR测定肿瘤组织中细胞因子水平等,最后经血液化学分析评估其毒性作用。对于临床上找不到合适肿瘤抗原的模型,我们可利用化疗药物如顺铂等药物原位诱导凋亡细胞产生免疫原性的能力,将顺铂制成纳米粒(LPC),靶向到肿瘤组织内诱发肿瘤细胞凋亡,待其产生有免疫原性的自身肿瘤抗原后,再静脉注射免疫佐剂的脂质体(Cp G-Lipo)加强抗原递呈。测量肿瘤体积,观测处理后小鼠平均生存时间,HE染色切片评估其毒性作用。并进行ELISPOT等实验判定T细胞增殖效果,并用流式细胞术测定肿瘤浸润免疫细胞数目、ELISA测定肿瘤组织中细胞因子水平等。结果与结论:第一种给药方式结果表明,在晚期黑色素瘤荷瘤小鼠中,联合给予了纳米胶束和纳米疫苗的小鼠抗肿瘤作用明显高于单独给予药物的小组以及对照组。在免疫器官中,联合给药方式显著增强体内CTL效应以及γ干扰素(IFN-γ)这种效应细胞因子的水平。在肿瘤组织内,联合给药方式一方面显著降低免疫与肿瘤共同通路-信号转导及转录激活因子3-STAT3体内表达水平,另一方面联合给药方式导致了许多免疫抑制细胞或因子如骨髓源性抑制细胞(MDSC)、调节性T细胞(Treg)、白细胞介素6(IL-6)和趋化因子配体2(CCL2)等的显著性降低;而许多促炎因子如肿瘤坏死因子(TNF-α)和IFN-γ升高,因而减除了微环境中的免疫抑制状态,T细胞浸润增多,利于化学预防性药物及疫苗发挥作用。第二种给药方式结果表明,此联合给药方式产生了很强的诱导肿瘤组织细胞凋亡以及抑制晚期恶性黑色素瘤生长的作用。这种很强的协同抗肿瘤生长作用很大程度上是因为细胞毒性T细胞增殖加强,以及免疫器官、肿瘤微环境中的免疫抑制因子减少。因为这些作用,联合给药大大增强了小鼠生存时间。这种基于纳米系统的LPC联合Cp G-Lipo减小药物毒性的同时延长了荷瘤鼠生存时间、减少了肿瘤肺转移、显示了原位诱导产生肿瘤抗原也是一种有效可行的肿瘤治疗方式。创新点:(1)本研究中使用的药物、肿瘤疫苗以及免疫佐剂等都采用了纳米递释系统,并且根据药物性质的不同设计了不同的纳米形式,从而靶向到不同器官。(2)这些经由纳米系统递释的药物对晚期难以治疗的恶性黑色素瘤肿瘤微环境有很好的靶向聚集与调节作用,可以有效解除肿瘤微环境中免疫抑制状态,使之有免疫应答能力。当与纳米化的疫苗或者免疫佐剂联合使用时,可以促进细胞毒性T淋巴细胞向肿瘤组织浸润,有效遏制晚期恶性黑色素瘤生长。(3)本研究探索了基于纳米递释系统的两种联合给药模式。一种模式是对于肿瘤抗原明确的模型,采用了靶向树突状细胞(DC)的纳米肿瘤抗原疫苗(TrpVac)与姜黄素两亲性纳米胶束(Cur PM)联合给药的模式;另一种是当临床上没有找到适合的肿瘤抗原时适用的联合给药模式,利用核-壳纳米递释系统将化疗药物顺铂靶向到肿瘤组织中,原位诱导肿瘤自身抗原的产生,再辅以纳米免疫佐剂,增强肿瘤抗原递呈,启动小鼠适应性免疫应答对抗肿瘤。
[Abstract]:Objective: tumor immunotherapy is the application of immunology, to stimulate the immune system in the body, to kill tumor cell specific factors to kill tumor cells, and to inhibit the growth of tumor, and has attracted much attention in recent years. However, the response rate of immunotherapy alone is not very high, especially for advanced malignant melanoma. The effect of the treatment is obviously reduced, because with the growth of the tumor, the tumor microenvironment is immunosuppressive and the cytotoxic T lymphocyte (CTL) is prevented from infiltrating to the tumor tissue at the late stage. The more feasible method is to make the molecules or antibodies or nucleic acid, which can stimulate the immune response, to make nanoscale form and enhance the immunization of the antigen presenting. At the same time, combined with chemical prophylaxis or chemical treatment drugs, reducing the level of immunosuppressive factors in the tumor microenvironment, strengthening the effect of immunotherapy, effectively inhibiting the growth of malignant melanoma, and taking different combinations of the tumor antigen model and the clinical model that did not find the appropriate tumor antigen. The combination of drug delivery methods may be more practical and practical. Method: for the clear model of tumor antigen, we used the calcium phosphate core shell nanoparticles to wrap the tumor polypeptide molecules and adjuvant to make the nano vaccine (Trp-Vac), and the chemical prophylactic drug curcumin nano micelle (Cur PM) was combined to give mice. We first synthesized Cur PM, and The tissue distribution in the tumor bearing mice was tested, and then combined with the drug, the tumor growth inhibition test, the cytotoxic T lymphocyte (in vivo CTL) killing effect and the enzyme linked immunosorbent assay (ELISPOT) were used to evaluate the proliferation of cytotoxic T cells. Then the tumor infiltration immunity was measured by flow cytometry. Changes in the number of cells and the determination of cytokine levels in tumor tissues by real time fluorescence quantitative PCR, and finally by hematological analysis to evaluate their toxic effects. For the clinical model, we can use chemotherapeutic drugs such as cisplatin and other drugs in situ to induce apoptotic cells to produce immunogenicity. Rice grain (LPC), target the tumor cells to induce apoptosis in the tumor tissue. After producing the immunogenicity of the tumor antigen, the antigen presentation is strengthened by the liposome (Cp G-Lipo) of the immuno adjuvant. The tumor volume is measured. The average survival time of the mice after the observation is observed and the HE staining section is used to evaluate the toxic effect of the tumor. And ELISPOT and so on are carried out. The effect of T cell proliferation was determined and the number of tumor infiltrating immune cells was measured by flow cytometry and the level of cytokine in tumor tissue was measured by ELISA. Results and conclusion: the first method of administration showed that in the advanced melanoma tumor bearing mice, the anti tumor effect of the combination of nano micelle and nano vaccine was significantly higher in mice with advanced melanoma. In the immune organs, the combined administration of drugs significantly enhanced the CTL effect and the level of interferon gamma (IFN- gamma) effect cytokine levels in the immune organs. In tumor tissue, combined administration of drugs significantly reduced the co pathway of immunization and tumor, signal transduction and transcription activator 3-STAT3 body. The level of internal expression, on the other hand, leads to a significant reduction in many immunosuppressive cells or factors such as myeloid suppressor cells (MDSC), regulatory T cells (Treg), interleukin 6 (IL-6) and chemokine ligand 2 (CCL2), while many proinflammatory factors such as tumor necrosis factor (TNF- alpha) and IFN- gamma are reduced. The immunosuppressive state in the microenvironment, the increase of T cell infiltration, is beneficial to the chemical prophylactic drugs and vaccines. The results of second drug delivery methods show that the combined administration of the drugs has a strong effect on inducing apoptosis of tumor tissue cells and inhibiting the growth of advanced malignant melanoma. This is a very strong synergistic antitumor growth. To a large extent, it is due to the proliferation of cytotoxic T cells, as well as immune organs, and the reduction of immunosuppressive factors in the microenvironment of the tumor. The combination of these effects greatly enhances the survival time of mice. This nano system based LPC combined with Cp G-Lipo reduces the drug toxicity and prolongs the survival time of the tumor bearing mice and reduces the survival time of the mice. Tumor lung metastasis, showing that in situ induction of tumor antigen is also an effective and feasible method of cancer treatment. Innovation: (1) the drugs used in this study, tumor vaccine and immune adjuvant have adopted the nano delivery system, and different nano forms are designed according to the different drug properties, so as to target different devices. (2) these drugs, which are delivered by the nano system, have good target aggregation and regulation on the malignant melanoma microenvironment of late treatment, which can effectively relieve the immunosuppressive state in the tumor microenvironment and make it immune response. When combined with the nanoscale vaccine or immune adjuvant, it can promote the refinement. Cytotoxic T lymphocytes infiltrate into tumor tissue and effectively inhibit the growth of advanced malignant melanoma. (3) this study explored the two modes of joint delivery based on the nano delivery system. A model is a clear model for tumor antigen, and the nano tumor antigen vaccine (TrpVac) and curcumin are two affinity with targeted dendritic cells (DC). The combination of nano micelle (Cur PM) combined with drug delivery; the other is a joint delivery mode when the suitable tumor antigen is not found in the clinic. The nuclear shell nano delivery system is used to target the chemotherapy drug cisplatin into the tumor tissue and in situ induce the production of the tumor autoantigen, and the nano immuno adjuvant is used to enhance the tumor antigen presentation. The adaptive immune response in mice was initiated against the tumor.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.5

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