肿瘤相关MUC1-氟修饰STn糖型胞外微囊泡激活免疫应答的分子机制

Cell surface glycoprotein plays a vital role in the malignant transformation of cell and the development of cancer. The aberrant glycosylation has been regarded as a hallmark of cancer. Aberrant glycosylation of MUC1 is associated with a wide variety of cancers. In the healthy scenario, MUC1 is O-glycosylated with long glycans. In the cancerous scenario, MUC1 carries truncated O-linked glycans, the most common forms being sialyl. STn is the most tumor-associated carbohydrate antigen on MUC1. MUC1 is the major glycoproteins that can express STn. They are both ideal targets for immunotherapeutic strategies. STn can hardly be expressed in normal tissues, while it is highly expressed in tumors. Fluorine-modified STn has stronger immunogenicity. Microparticles released by cells in response to stress contain specific antigens and a portion of active components of the parental cells. So they have the similar immunogenicity to parental cells. We expect to establish a kind of tumor cell with high expression of MUC1 and fluorine-modified STn, isolate microparticles of the cell, evaluate the function of microparticles in tumor immunoprophylaxis and immunotherapy and elucidate the mechanisms. We hope that we could find a new pathway and theoretical basis for designing anticancer vaccines and broadening the field of tumor immunotherapy.

细胞表面糖蛋白在细胞恶性转化及肿瘤疾病进程中具有至关重要的作用，异常的糖基化修饰被认为是肿瘤发生发展的标志。肿瘤细胞中MUC1糖基化修饰的糖链结构异常，由正常细胞的具有分支的长链变为末端高度唾液酸化的短链。STn抗原是MUC1中最常见肿瘤相关糖抗原，MUC1是STn抗原最主要的糖蛋白载体，二者均被视为肿瘤免疫治疗的靶标。STn抗原在正常组织中几乎检测不到，而在肿瘤组织中表达量却很高，是理想的肿瘤疫苗候选物，氟修饰STn抗原具有更高的免疫原性。细胞在应激状态下形成的胞外微囊泡携带母体细胞表面抗原及胞内的部分活性成分，具有与母体细胞类似的免疫原性。本申请项目拟得到高表达MUC1-氟修饰STn抗原的肿瘤细胞，分离细胞产生的胞外微囊泡，评价其所引起的肿瘤免疫预防和治疗效果，并阐明免疫应答各环节的具体机制，为设计特异有效的肿瘤糖疫苗、拓宽肿瘤免疫治疗的领域提供新的思路和理论依据。

胞外微囊泡（cell-derived microparticles，MP）是细胞在应激状态下，细胞膜包裹细胞内容物形成并释放到胞外的直径为100-1000nm的一类微小囊泡。微囊泡保留母体细胞特异的免疫原性以及胞内部分活性物质，并将这些信号传递给相邻或远端的靶细胞，在维持生理平衡与稳态以及疾病进程中均发挥重要作用。.由于天然的胞外微囊泡具有弱免疫原性、弱生物稳定性以及在体内半衰期过短等不足之处，我们构建了高表达糖蛋白MUC1和肿瘤相关糖抗原的稳转细胞株，提高糖抗原的表达量，结合代谢寡糖工程（metabolic oligosaccharide engineering，MOE）策略，将化学合成的氟修饰寡糖底物用于细胞膜表面糖抗原的合成，使细胞表达含氟修饰基团的非天然糖抗原，进而得到含有糖蛋白MUC1和氟修饰糖抗原的胞外微囊泡。氟修饰基团具有增加化合物在体内的吸收、分布和利用，增强稳定性和延长半衰期的作用。本课题中氟修饰STn抗原与MHCⅠ分子的相互作用明显增强，含氟修饰基团的胞外微囊泡F-MP能够更显著地促进DCs成熟及细胞因子的释放，促进T细胞增殖与分化，增加Th1细胞及其细胞因子IFN-γ的释放，提高CTLs的比例，最终提高对靶细胞的杀伤效率，发挥更强的抗肿瘤免疫预防与治疗作用。.本研究不仅对胞外微囊泡促进DCs成熟及效应T细胞活化的过程和机制进行了阐释，还获得了含有氟修饰基团的非天然胞外微囊泡，用于抗肿瘤免疫预防与治疗，取得了理想的效果，为设计更加有效的抗肿瘤糖疫苗候选物，拓宽肿瘤免疫预防及治疗的研究领域提供了新思路。