甘露糖-6-磷酸受体在恶性肿瘤免疫治疗联合化疗中的作用研究

Cancer immunotherapy in combination with chemotherapy can result in synergistic antitumor effects, however the mechanism remains unknown. Mannose-6-phosphate receptor (M6PR) is an integral membrane protein which mediates biosynthetic sorting and endocytosis of various molecules. Our previous studies showed that M6PR on tumor cells was up regulated by chemotherapy and stronger cytotoxic effect was also observed because of increased granzyme B entrance into tumor cells. The role of M6PR in combined immunochemotherapy is not clear. In this study, the interaction between M6PR on tumor cells and granzyme B released by CTL will be investigated to test the first hypothesis that chemotherapy enhances the CTL-mediated killing through the up regulated M6PR on tumor cells. Our previous studies also showed that activated CTLs through antigen recognition were able to kill not only tumor cells that expressed specific antigen but also those bystander tumor cells that did not express antigen. The mechanism of this phenomenon has not been elucidated. In this study, tumor cells loaded with specific peptide/ control peptide will be used and tumor model in mice containing wild type mixed with MHC I negative (H2Kb-) tumor cells will be established in order to test the second hypothesis that the up regulated M6PR induced by chemotherapy mediates a bystander effect of combination therapy. This study will evaluate the IGF-II (one of the main ligands of M6PR) protein level in tumor cells treated with chemotherapy, detect the distribution of M6PR in tumor cells after chemotherapy, and investigate the mechanism of the up regulation of M6PR on tumor cells which will provide scientific evidence for the molecular mechanism of synergistic antitumor effect of immunotherapy and chemotherapy.

肿瘤免疫治疗联合化疗能产生协同效应，其分子机制仍有很多盲点。M6PR是一种膜整合蛋白,其在大分子物质的降解过程中发挥作用。前期研究发现化疗可上调肿瘤细胞表面M6PR，同时CTL所释放的颗粒酶B也被更多摄入肿瘤细胞内而发挥细胞毒效应，但M6PR在CTL联合化疗中的作用尚无报道。本课题拟研究M6PR与颗粒酶B之间的相互作用，阐明M6PR在联合治疗中的作用。 前期研究还发现激活的CTL不仅杀死表达抗原的肿瘤细胞，也可杀伤那些"旁观"的缺乏抗原表达的细胞，该现象机制尚不清楚，本研究拟构建负载特异性肽/对照肽的肿瘤细胞，构建野生型与MHC I类分子阴性（H2Kb-）的混合肿瘤小鼠模型，阐明化疗诱导的M6PR变化是否介导了联合治疗中的"旁观者效应"。 本课题拟分析化疗后M6PR主要配体IGF-II的变化，探索化疗上调肿瘤细胞表面M6PR的机制，为阐明免疫治疗联合化疗产生协同效应的分子机制提供理论依据。

肿瘤免疫治疗联合化疗能产生协同效应，其分子机制仍有很多盲点。M6PR是一种膜整合蛋白，前期研究发现化疗可上调肿瘤细胞表面M6PR，同时CTL所释放的颗粒酶B也被更多摄入肿瘤细胞内而发挥细胞毒效应，但M6PR在CTL联合化疗中的作用尚不明确。.本研究发现，在转染了control shRNA的B16F10 荷瘤小鼠中，与单一治疗相比，TAX联合Pmel-1 CTL治疗抗肿瘤效应显著增强。在转染了M6PR shRNA的荷瘤小鼠中，联合治疗未显示出增强作用。针对B16F10-Luc肿瘤，联合治疗比单一免疫治疗或化疗显示出增强的抗肿瘤效应；针对control shRNA Kb-肿瘤，联合治疗未显示出增强作用。在B16-Luc与control shRNA Kb-混合肿瘤模型中，与单一治疗相比，联合治疗导致肿瘤总体积显著缩小。 .本研究还发现，用TAX处理B16F10细胞后，M6PR总蛋白表达水平没有明显变化，M6PR膜蛋白水平显著增加（Western blotting）。激光共聚焦显微镜结果显示，在U266肿瘤细胞内化疗前M6PR集中在细胞浆内，而化疗后其主要定位于细胞膜；在多发性骨髓瘤患者骨髓切片中，治疗前只有20%的肿瘤细胞出现M6PR在细胞膜上的聚集现象，化疗后3天该比例增至50%以上。.此外，本研究结果显示，化疗药物体外对4T1、EL4、B16F10三种肿瘤细胞内IGF-II表达水平没有显著作用（Western blotting）。激光共聚焦显微镜结果显示，应用TAX、CIS、DOX处理肿瘤细胞后，自噬被迅速诱导。应用3-MA抑制自噬，DOX原先诱导的U266细胞表面M6PR的上调表达能力明显下降；经过TAX处理后的B16F10细胞也出现此现象。应用atg5 siRNA转染B16F10细胞下调自噬，TAX原先诱导的细胞膜M6PR的上调性表达现象显著下降。应用Rapamycin诱导自噬，肿瘤细胞表面的M6PR明显增加。.综上，化疗可以增强CTL免疫治疗的效果，而该效应与M6PR介导的细胞内吞相关。化疗后肿瘤细胞表面M6PR的短暂诱导介导了化疗联合免疫治疗的旁观者效应，有利于二者产生协同作用。该作用与化疗诱导的自噬相关，而与肿瘤细胞内的IGF-II水平无关。因此，本研究探索了化疗上调肿瘤细胞表面M6PR的机制，为阐明免疫治疗联合化疗产生协同效应的分子机制提供理论依据。