| 题名 | 多佐剂复合型肿瘤全细胞疫苗的构建及应用探索 |
| 作者 | 刘世英 |
| 学位类别 | 硕士 |
| 答辩日期 | 2013-05 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 苏志国 ; 魏炜 |
| 关键词 | 肿瘤全细胞疫苗 佐剂 纳米球 细胞因子 |
| 其他题名 | Nanoparticles-based multi-adjuvant whole cell tumor vaccine for cancer immunotherapy |
| 学位专业 | 生物化工 |
| 中文摘要 | 肿瘤疫苗通过激发患者自身的免疫系统，从而诱导机体产生特异性的免疫应答，具有特异性强、副作用低的优点，是一种极具潜力的肿瘤生物治疗方法。由于目前多数肿瘤抗原未得到鉴定，因此，包含肿瘤全部抗原的肿瘤全细胞疫苗有望激发机体产生多价的免疫应答，具有最为广泛的应用前景。由于树突状细胞（DC）不能有效迁移、抗原提呈能力不足和T细胞增殖能力较弱，造成了传统的肿瘤全细胞疫苗免疫原性弱的缺点，所激发的免疫反应有限。本课题针对上述问题，借助肿瘤细胞对纳米球的摄取作用，将两种细胞因子佐剂，即粒细胞-巨噬细胞集落刺激因子（GM-CSF）及白细胞介素2（IL-2）成功引入肿瘤细胞内，经过灭活后构建了同时包括纳米球佐剂和两种细胞因子佐剂在内的多佐剂复合型肿瘤全细胞疫苗，从而程序性地提高了DC迁移能力、抗原提呈能力以及T淋巴细胞增殖能力，最大程度上激发有效的特异性免疫应答。论文主要包括以下四部分：1 制备粒径均一的PLGA纳米球，并在其表面完成了细胞穿膜肽CPP修饰。利用课题组开发的快速膜乳化技术，制备了粒径均一的PLGA纳米球（pristine nanoparticles, p-NP）。为了进一步增强细胞对纳米球的摄取，在纳米球表面进行CPP修饰。通过采用定点修饰策略，使用间臂NAEM（N-（2-氨基乙基）马来亚胺）进行两步反应，成功制备了CPP修饰的纳米球（CNP）。细胞水平的研究表明，p-NP及CNP均不会对小鼠肺癌细胞Lewis lung carinoma（LLC）产生细胞毒性，CPP修饰显著地提高了LLC对纳米球的内吞速率以及内吞量。2 借助肿瘤细胞对纳米球的摄取作用，成功地将两种细胞因子佐剂协同导入肿瘤细胞，并通过反复冻融的灭活方法，制备了多佐剂复合型肿瘤全细胞疫苗。将修饰前后的纳米球与细胞因子复配，考察了细胞因子导入肿瘤细胞后的生物利用度、内吞情况及胞内定位情况。实验结果表明，CNP可以被LLC更高效地摄取，从而携带了更多的细胞因子进入，提高了细胞因子在胞内的浓度；此外，由于CPP的直接穿膜作用，细胞因子进入细胞后，可以回避溶酶体途径，从而避免了溶酶体的酸性环境及酶解作用对其活性的破坏。上述两方面原因使得细胞因子进入肿瘤细胞后仍然保持了较高的生物利用度。在此基础上，采用一种较为温和的灭活方法来制备疫苗，通过优化冻融次数，从而保证了疫苗的安全性以及细胞膜的完整性。3 细胞水平上考察了多佐剂复合型肿瘤全细胞疫苗对DC迁移及抗原提呈的影响。通过使用TRANSWELL体系进行体外迁移实验，研究结果发现，由于多佐剂复合型肿瘤全细胞疫苗对GM-CSF的持续释放作用，使得DC能够在较长的时间内迁移；通过检测不同制剂处理后DC的表面分子水平，发现CNP明显地上调了DC表面抗原提呈分子MHC II和MHC I水平，成功诱导交叉提呈，并提高了共刺激分子CD80和CD86的表达，为激发后续有效的免疫应答创造条件。4 动物水平上评价了多佐剂复合型肿瘤全细胞疫苗的治疗及预防效果。体内实验结果表明，所构建的多佐剂复合型肿瘤全细胞疫苗明显上调了淋巴结中总的T淋巴细胞所占比例，证明了疫苗中添加IL-2的必要性。本论文所设计的疫苗还产生了大量具有细胞杀伤作用的细胞毒性T淋巴细胞（CTL），抑制了肿瘤的生长，延长了小鼠的生存时间，并且具有防止肿瘤转移的效果。荷瘤小鼠的体重变化、小鼠血清中反映肝、肾和心肌毒性的指标以及组织切片，均证明了疫苗的安全性。最后，疫苗的预防效果研究表明，多佐剂复合型肿瘤全细胞疫苗通过激发有效的体液免疫应答，大大减缓了肿瘤的生长。 |
| 英文摘要 | As a potential treatment modality, tumor vaccine has gained significant attention. Because a majority of tumor antigens have not been identified, whole cell tumor vaccine (WCTV) including all the tumor antigens is promising to elicit a multi-valent immune response. However, traditional WCTV elicits limited immune response due to the poor immunogenicity. In order to address this issue, we introduced two cytokine adjuvants, GM-CSF and IL-2 into tumor cells by the cellular uptake of nanoparticles to construct a multi-adjuvant WCTV including a nanoparticle adjuvant and two cytokine adjuvants. After inactivation, as-designed multi-adjuvant WCTV exhibited programmed promotions on dendritic cells (DCs) recruitment, antigen presentation, and T cell activation, thus eliciting the maximum immune response in tumor-bearing mice.In detail, this thesis mainly included the following four issues:1 Uniform-sized PLGA nanoparticles were prepared and further modified with cell penetrating peptide (CPP). Using SPG premix membrane emulsification technique developed in our group, we successfully prepared pristine PLGA nanoparticles (p-NPs, ~ 248 nm) with a narrow size distribution. In order to promote NP cellular uptake and efficiently import cytokines, these p-NPs were further modified by CPP, which was conjugated to p-NPs via a two-step coupling reaction by a NAEM (N-(2-aminoethyl) maleimide) spacer, so the CPP decorated NPs (CNP) were obtained. Then, cellular uptake of NPs in Lewis lung carcinoma（LLC）were investigated. Compared with p-NPs, CNPs revealed a faster uptake manner and a greater uptake amount.2 GM-CSF and IL-2 were imported into tumor cells by cellular uptake of nanoparticles. Then the multi-adjuvant WCTV was fabricated by inactivating tumor cells via freeze/thaw cycles. By mixing the cytokines with NPs, we investigated the internalization, intracellular trafficking, and bioavailabilities of GM-CSF and IL-2 in tumor cells. The results showed that CNP significantly improved bioavailabilities of imported GM-CSF and IL-2, which could be attributed to two factors. On one hand, CNP could transport more cytokines into LLC cells, which increased the intracellular concentrations of cytokines. On the other hand, CNP facilitated the transportation of cytokines by direct penetrating the cell membrane and helped them evade lysosome degradation to maintain their activities. Finally, this multi-adjuvant WCTV was prepared by inactivation through freeze/thaw cycles.3 Effects of multi-adjuvant WCTVs on DCs migration and antigen presentation in vitro. By mimicking the DCs migration through TRANSWELL system, sustained DC migration was observed in WCTV groups due to the released GM-CSF component. What’s more, after determination the surface markers of DCs when incubating with whole tumor cell lysate and NPs, we found CNP upregulated the expressions of MHC II, MHC I, CD80, and CD86 molecules and induced the cross-presentation, thus the efficacious immune response was awaited. 4 The therapeutic and protective effects of multi-adjuvant WCTV were evaluated in tumor-bearing mice in vivo. The results in vivo demonstrated as-designed multi-adjuvant WCTV increased the percentage of T cells in the lymph nodes, suggesting the necessity of IL-2 in the vaccine. We also found as-constructed WCTV produced enormous cytotoxic T lymphocytes (CTL) to eradicate tumor cells, which resulted in the satisfactory effects on tumor growth suppression and metastasis inhibition. The body weight change, serum chemistry values, and histological sections revealed that multi-adjuvant WCTV had little side effects. The preventive study showed the total IgG level in tumor-bearing mice immunized with as-designed WCTV was significantly increased, indicating the potential tumor-rejection capability to prevent tumor recurrence. |
| 语种 | 中文 |
| 公开日期 | 2015-07-08 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/15507]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 刘世英. 多佐剂复合型肿瘤全细胞疫苗的构建及应用探索[D]. 中国科学院研究生院. 2013. |
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