PD-1/PD-Ls介导黄芪多糖调控黑色素瘤干细胞MICs免疫逃逸的分子机制研究

Melanoma is one of the malignant cancer diseases frequently happened in young people. The rate of incidence is gradually increasing year by year, and the mortality of the patients with the advanced stage of melanoma is getting to a very high level. Melanoma initiating cells (MICs) are tumor stem cells, specifically express the CSPG4 antigen, have the characteristics of tumor stem cells, and can be induced into various different kinds of melanoma cells because of tumor immunity evasion, therefore can stimulate the growth of tumor. Thus, targeting the regulation of tumor immunity evasion and eliminating MICs is very crucial for eradicating the melanoma cells and preventing the disease recurrence and metastatic spreading. Up to now there is no effective targeting therapeutic method to cure this disease in clinic. Based on the previous research achievements in our laboratory, this proposal aims to investigate the synergic effects of CSPG4-specific monoclonal antibody (CSPG4 mAb) in combination with the extraction of Chinese medical herb, Astragalus polysaccharides (APS), on regulating tumor immunity evasion mediated by costimulatory molecules PD-1/PD-Ls, eliminating MICs, eradicating the melanoma tumors, and on exploring the molecular mechanisms as well. Both the experiments in vitro and in vivo will be performed. Various different kinds of melanoma cells will be cultured and the animal model of melanoma tumor orthotopically implanted in immunodeficient mice will be established. Using the theories and technological methods of modern immunology and molecular biology, the synergic effects of CSPG4 mAb in combination with APS on inhibiting the PD-1/PD-Ls costimulatory molecule signal pathway, which is the negative molecules regulating the immune response, will be observed. Furthermore, the synergic effects on enhancing the expression of the inhibitory molecule PTEN will be also observed. This project will be determining the molecular mechanises of APS in regulating tumor immunity evasion mediated by PD-1/PD-Ls molecules, and exploring the new way to cure the disease of melanoma. It is an absolutely new and innovative project of interdisciplinary research at the forefront in this field. So far there is not any report about it all over the world. It is very important and meaningful for developing the innovative new drugs to cure cancer diseases, and also promoting the modernization of traditional Chinese medicine as well.

黑色素瘤是一种常见的恶性肿瘤，发病率死亡率逐年递增。黑色素瘤干细胞MICs，又称起始细胞，具有肿瘤干细胞的特性，可逃逸免疫监视，分化成不同类型的黑色素瘤细胞，特异性表达CSPG4抗原，诱导黑色素瘤生长。调控黑色素瘤干细胞的免疫逃逸，恢复免疫系统监视和清除功能，是抑制黑色素瘤生长，控制复发和转移的关键。本项目是在前期研究基础上，以介导肿瘤免疫逃逸的协同刺激分子PD-1/PD-Ls为靶点，探讨黄芪多糖清除黑色素瘤干细胞、抑制肿瘤细胞增殖的作用，从细胞、分子及整体等多层次上，探讨其调控黑色素瘤干细胞免疫逃逸的作用机制，并进一步探讨其与CSPG4特异性单克隆抗体的协同作用，藉以探索中药调节肿瘤免疫逃逸的新机制。本项目属跨学科研究的前沿课题，至今在国内外尚无报道，具有鲜明的创新性，为临床有效治疗恶性肿瘤提供新思路，对于促进肿瘤免疫治疗和中医药学现代化发展，具有重要的科学意义。

黑色素瘤干细胞是黑色素瘤复发、转移、产生耐药性并导致最终治疗失败的重要原因之一。黑色素瘤干细胞是具有无限的自我更新及多向分化能力的细胞亚群，可以启动和维持肿瘤生长。黄芪多糖是中药黄芪的重要成分之一，现代药理研究表明黄芪多糖具有调节免疫及抗肿瘤的作用。在实验室前期的研究基础上，本项目研究黄芪多糖对黑色素瘤干细胞的调控作用。首先，体外实验分别采用无血清悬浮培养法富集和免疫磁珠技术分选黑色素瘤干细胞，经黄芪多糖干预后，检测黑色素瘤干细胞的二次球落形成能力，干性因子乙酸脱氢酶ALDH1A3、SOX2、OCT4、Nanog的mRNA及蛋白的表达水平。在体内实验中，建立NOD/SCID小鼠皮下黑色素瘤干细胞移植瘤模型，经黄芪多糖灌胃治疗后观察小鼠肿瘤的生长和肿瘤组织中干性因子CD133、SOX2、OCT4、Nanog的转录水平。其次，在体内实验中，观察黄芪多糖对黑色素瘤小鼠脾脏及肿瘤组织中PD-1、PD-L1/2的表达水平的影响。进一步深入研究分子机制，分别在体外黑色素瘤干细胞和体内肿瘤组织中检测Notch1信号通路分子的表达水平，以研究黄芪多糖调控黑色素瘤干细胞的分子机制。研究结果得到：黄芪多糖可在体外抑制黑色素瘤干细胞的球落形成及黑色素瘤干细胞的干性因子的表达；同时，可在体内抑制肿瘤生长及肿瘤组织中黑色素瘤干细胞的干性因子的表达，表明黄芪多糖在体内外抗肿瘤的作用是通过抑制黑色素瘤干细胞自我更新实现的。其次，在体内实验中，黄芪多糖可抑制黑色素瘤小鼠脾脏及肿瘤组织中PD-1、PD-L1/2的表达，调控肿瘤免疫逃逸的作用，从而抑制小鼠肿瘤生长。进一步研究表明黄芪多糖在体内外调控黑色素瘤干细胞自我更新的作用机制可能与抑制Notch1信号通路有关。本研究综合了肿瘤学、药理学及分子生物学的研究方法，从分子、细胞、动物水平探讨黄芪多糖对黑色素瘤干细胞自我更新的作用及机制，为临床用药提供更充分的科学依据。

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