磁性介孔氧化硅纳米粒子促进DC细胞对DRiPs抗原交叉递呈和凋亡的作用及机制研究

Biotherapy for cancer has been great concerned by the clinicians. Dendritic cells (DCs) vaccine loaded with the tumor antigen and then transfused into patients with cancer is one of the most potential strategies, which has the advantages of high efficiency, low toxicity, low side effects and so on. During DCs loaded, the types and the amount of tumor antigens are extremely crucial and directly affect the strength of immune responses. Short-lived proteins with defective ribosome products (DRiPs) could be regarded as tumor related antigens by inhibiting proteasome of tumor cells. We will synthesize magnetic mesoporous silica nanomaterials (MMSN) through the combination of the synthesis of magnetic nanoparticles with mesoporous silica nanoparticle materials. As a new kind of anti-tumor vaccine, the abilities of activation anti-tumor immune response will be studied after DRiPs adsorbed by MMSN. The influence of endoplasmic reticulum stress (ERS), including related signaling pathways of endoplasmic reticulum membrane protein IRE1, PERK and ATF6, and apoptosis of DC will be explored after large amount of tumor protein and nanoparticles swarming into DC. Following the studies, the experimental and the theoretical basis for the clinical application of DRiPs and MMSN will be provided and clarified.

以肿瘤抗原负载树突状细胞（DC）回输恶性肿瘤患者体内为代表的肿瘤生物治疗作为当前肿瘤治疗新模式，具有高效、低毒等特点而广受关注。肿瘤抗原的选择在制备DC疫苗时极为关键，直接关系免疫治疗成败。我们通过抑制肿瘤细胞蛋白酶体和溶酶体功能获得富含缺陷溶酶体产物（DRiPs）的短寿蛋白作为肿瘤相关抗原。利用合成的磁性纳米粒子易于生物标记和MRI成像及介孔氧化硅纳米材料多孔道和大比表面积等特点，合成磁性介孔氧化硅纳米材料。利用氧化硅吸附能力和氧化铁磁性可分离能力获得吸附大量DRiPs肿瘤抗原的纳米复合粒子作为新型肿瘤疫苗，观察其启动免疫反应能力、负载DC后的抗原交叉递呈和抗肿瘤能力。我们还将观察大量肿瘤抗原和纳米粒子进入DC后，DC的内质网应激在抗原交叉递呈和调控凋亡过程中的作用及对内质网跨膜蛋白IRE1、PERK和ATF6相关信号通路的影响，为探索新型肿瘤疫苗抗肿瘤机制和优化抗肿瘤疗效提供理论依据。

树突状细胞是人体最强的抗原提呈细胞，它可以摄取肿瘤抗原进而活化T细胞杀伤肿瘤。抗肿瘤DCs疫苗启动抗肿瘤免疫的有效性依赖于抗原负载。本研究采用 IO-LPMON 负载OVA模式抗原后，验证其 IO-LPMON 的免疫佐剂功能。通过聚乙烯亚胺PEI修饰氢氧化铝纳米颗粒LV@HPA，验证LV@HPA/PEI的免疫佐剂活性得到进一步增强，检测DCs负载LV@HPA/PEI-OVA后的表面共刺激分子变化，及活化特异性T细胞的能力。LV@HPA/PEI吸附肿瘤自噬抗原DRibbles后，可以作为有效的免疫佐剂促进DCs抗肿瘤。.本研究从以下方面进行：. 1、通过材料学检测，检测IO-LPMON的性能特点，并检测IO-LPMON吸附模式抗原OVA的能力，体外观察DCs吞噬IO-LPMON-OVA后对上调DCs表面共刺激分子CD40、CD80、CD86的表达情况。相比于游离的OVA，可以更好地刺激体内特异性T细胞的活化。. 2、通过修饰方式获得LV@HPA/PEI，检测PEI修饰后对抗原蛋白OVA吸附能力的变化。WB与流式检测通过LV@HPA/PEI纳米载体的作用，OVA抗原被DCs摄取的数量。DCs受LV@HPA/PEI刺激成熟的程度通过ELISA检测IL-12的分泌，并通过流式检测表面共刺激分子CD80和CD86的变化，氯酚红-β-D-半乳吡喃糖苷检测(CPRG实验)检测DCs抗原提呈功能的变化。DRibbles与LV@HPA/PEI联合负载DCs，作为DC疫苗免疫小鼠，流式检测其脾脏CD3+CD8+IFN-γ+ T细胞的比例变化。最后我们用LV@HPA/PEI-DRibbles负载DCs作为DC疫苗治疗荷瘤小鼠，观察其肿瘤生长与小鼠生存率的变化。. 本研究明确了新型IO-LPMON纳米材料的特性及其在DCs疫苗作为佐剂的应用。同时LV@HPA/PEI-DRibble-DCs能够特异性抑制肿瘤的生长。