胰腺癌肿瘤微环境的纳米免疫体系调控及其协同增强放化疗机制的研究

发布时间：2018-02-13 00:35

本文关键词： 胰腺癌肿瘤微环境纳米药物抗PD-L1抗体放化疗　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：胰腺癌细胞由于缺少特异性的肿瘤标志物,前期症状又隐匿,因此治疗窗口小;另外胰腺癌解剖位置特殊,造成手术难度大,而且术后易复发,并不延长生存期。因此,对于绝大多数的中晚期胰腺癌,放化疗以及综合疗法成为优选方案。然而,临床胰腺癌的特殊的肿瘤微环境明显降低了这些治疗的效果。胰腺癌的肿瘤微环境包括富含透明质酸的稠密基质屏障、血管生长分布异质性等严重阻碍了药物的瘤内富集,降低了免疫细胞(如T和NK细胞)的浸润导致肿瘤细胞免疫耐受;而基质内的胰腺星状细胞及还原环境又促进胰腺癌细胞耐辐射损伤;同时,肿瘤组织内PD-L1的表达及其他负向调控因子也抑制了效应T细胞的杀伤作用。因此,通过打破基质屏障促进药物渗透,抑制胰腺星状细胞,调节免疫环境来调控胰腺癌肿瘤微环境来提高放化疗治疗效果显得尤为重要。该设想的关键是如何针对上述不同靶点设计体系,协同治疗。纳米药物由于独特的增强渗透阻滞(EPR)效应以及环境响应性等特点为上述设想提供了完美的解决方案。本研究中,我们针对胰腺癌微环境中的基质成分透明质酸、谷胱甘肽、胰腺星状细胞、效应T细胞等因素,巧妙利用二硫键将支化-聚乙烯亚胺(b-PEI)偶联,并负载星状细胞抑制剂LDE225,同时在载体表面修饰抗PD-L1抗体(Avelumab)及透明质酸酶,设计靶向这些因素的可瘤内崩解的纳米免疫体系。动静态激光光散射仪(Dynamic/Static laser light scattering,DLLS/SLLS),HPLC(高效液相色谱),透射电子显微镜(TEM)及Zeta电位等检测结果都提示了载药纳米免疫体系的成功合成,并且得到了载体类似凝胶的拓扑结构,载体体系的分子量,抗体和酶的负载总数量为130个左右;同时,ELISA及酶功能试验都表明载体上修饰的抗体及透明质酸酶都保持了良好的功能,而借助免疫荧光我们发现纳米免疫体系在模拟细胞外基质环境中有更好的渗透能力。因此课题所制备的纳米免疫体系具有良好的结构和生物功能。我们验证了胰腺癌肿瘤微环境的基质屏障影响了小分子药物的渗透,同时胰腺星状细胞对胰腺癌细胞的放射损伤有一定保护作用。胰腺癌PANC-1细胞及SW1990细胞PD-L1表达不明显,胰腺癌BxPC-3细胞和耐辐射的PANC-1细胞的PD-L1却高表达PD-L1。细胞水平的评价发现,纳米免疫体系的生物相容性良好,流式及激光共聚焦的结果验证了载药纳米免疫体系与细胞表面PD-L1良好结合。载药纳米免疫体系能对胰腺星状细胞或者胰腺癌细胞产生杀伤作用,同时联合放疗后能明显提高杀伤效果。3D共培养克隆形成实验表明在同一照射剂量下,载药纳米免疫体系能突破表面的基质屏障渗透进入共培养环境,释放药物抑制细胞,从而增强了放疗敏感性。我们用人外周血的单个核细胞评价了纳米免疫体系诱导的ADCC(Antibody-dependent cellular cytotoxicity,ADCC)作用,在利用IFN-γ使胰腺癌细胞PD-L1上调后,纳米免疫体系产生了更强的ADCC作用,这可能由于胰腺癌细胞表面结合的纳米免疫体系偶联着大量抗体,引起多个NK细胞的识别而释放更多的穿孔素和颗粒酶,产生了更强的免疫杀伤效果。而当用渗透过基质屏障的纳米免疫体系和抗体来进行ADCC实验时,我们发现纳米免疫体系的ADCC作用要更强,这也从侧面反映了纳米免疫体系的良好渗透能力。我们进一步通过皮下荷瘤小鼠(Bal B/c裸鼠)系统研究了纳米免疫体系联合放疗的体内抑瘤效果,结果表明载药纳米免疫体系具有较为突出的抑瘤效果,而与放疗联用后,抑瘤效果也增强了,说明了载药纳米免疫体系联合放疗有一定协同增效的效果。本课题的通过精细构建的纳米免疫体系消融肿瘤组织中丰富的透明质酸,抑制活化的胰腺星状细胞,调节肿瘤内的免疫环境,有效实现协同增强放化疗效果。本课题的顺利开展为设计新型纳米免疫抗体,提升临床胰腺癌的综合治疗效果提供了新的思路和方案,对临床治疗胰腺癌及机制研究具有重要的指导意义。
[Abstract]:Pancreatic cancer cells due to the lack of specific tumor markers, early symptoms and hidden, so the treatment of small window; in addition to pancreatic cancer special anatomical position, resulting in difficult surgery, and postoperative recurrence, does not prolong the survival period. Therefore, for the vast majority of advanced pancreatic cancer, chemotherapy and combined therapy as the preferred scheme. However, clinical pancreatic cancer specific tumor microenvironment significantly reduces the effectiveness of treatment of pancreatic cancer. The tumor microenvironment including hyaluronic acid rich dense matrix barrier, blood vessel growth heterogeneity has seriously hindered the intratumoral drug accumulation, decreased immune cells (such as T and NK cells). The infiltration of tumor cells induced immune tolerance; and pancreatic stellate cells within the stroma and reducing environment and promote pancreatic cancer cells resistant to radiation damage; at the same time, the expression of PD-L1 in tumor tissues and other negative regulatory factors Also inhibits the cytotoxic function of T cells. Therefore, by breaking the matrix barrier to promote drug permeation, inhibition of pancreatic stellate cells, regulating the immune environment to regulate pancreatic tumor microenvironment is particularly important to improve the therapeutic effect of chemotherapy is put. The key idea is how to according to the different target system design, collaborative nano treatment. The drug due to the enhanced permeability block unique (EPR) effect and environmental response characteristics provides a perfect solution for the above assumption. In this study, we aimed at the matrix components of pancreatic cancer microenvironment in hyaluronic acid, glutathione, pancreatic stellate cells, T cells and other effect factors, use of two disulfide bonds will branched polyethylenimine (b-PEI) coupling, and load stellate cell inhibitor LDE225, and modification of anti PD-L1 antibody on the surface of the carrier (Avelumab) and hyaluronidase, designed to target these factors The immune system can be disintegrated in nano tumor. The static and dynamic laser light scattering (Dynamic/Static laser light scattering, DLLS/SLLS), HPLC (HPLC), transmission electron microscopy (TEM) and Zeta potential test results that the successful synthesis of the drug loaded nano immune system, and obtained the similar topology vector the gel carrier system, molecular weight, the total quantity of antibody and enzyme load was about 130; at the same time, ELISA and enzyme function test showed that the antibody carrier modification and hyaluronidase have maintained a good function, and by immunofluorescence. We found that the immune system has better ability to penetrate nano in simulated cell the matrix in the environment. Therefore the topic nano immune system prepared has good structure and biological function. We verified the matrix barrier of pancreatic cancer tumor microenvironment affects the permeability of small molecule drugs At the same time, the radiation damage of pancreatic stellate cells on pancreatic cancer cells had a protective effect on pancreatic carcinoma cell lines PANC-1 and SW1990. The expression of PD-L1 was not obvious, pancreatic carcinoma BxPC-3 cells and PANC-1 cells of the radiation resistant evaluation of PD-L1 is high expression level of PD-L1. cells, the immune system of nano good biocompatibility, flow cytometry and the results of confocal laser to verify the drug loaded immune system and cell surface PD-L1. Good combination of drug loaded immune system could produce cytotoxic effect on pancreatic stellate cells or pancreatic cancer cells, at the same time combined with radiotherapy can significantly improve the killing effect of.3D cloning experiment showed that in the same dose of co culture, drug loading nano immune system can break through the barrier on the surface of substrate penetration into the co culture environment, the release of drugs that inhibit cell, thereby enhancing the sensitivity of radiotherapy. We use mononuclear human peripheral blood To evaluate the immune system cells induced by nano ADCC (Antibody-dependent cellular cytotoxicity, ADCC), in the use of IFN- to gamma PD-L1 in pancreatic cancer cells increased after the immune system produce nano ADCC, this may be due to the nano immune system combined with the surface of pancreatic cancer cells is coupled to a large number of antibodies, by identifying a plurality of NK cells the release of perforin and granzyme more, have stronger immune effect. When using permeable matrix barrier nano immune system and antibody to ADCC experiments, we found that the effect of nano ADCC immune system is stronger, it also reflected from the good penetration ability of nano immune system we further by subcutaneous tumor bearing mice (Bal B/c mice) were studied by nano system combined with radiotherapy in vivo antitumor effect, the results showed that the drug loaded nano immune system The inhibitory effect is more prominent, and in combination with radiotherapy, anti-tumor effect was also enhanced, the drug loaded nano immune system combined with radiotherapy have synergistic effect. The immune system constructed by Nano fine ablation of hyaluronic acid rich in tumor tissue, inhibit the activation of pancreatic stellate cells in the regulation of tumor, immune environment, effectively realize the synergistic efficacy of radiotherapy and chemotherapy. The smooth development of this subject is the design of new nano antibody, provides new ideas and methods to enhance the clinical effect of comprehensive treatment of pancreatic cancer, has important guiding significance to study the mechanism and clinical treatment of pancreatic cancer.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9

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