莪术多糖CKAP-2逆转髓源抑制细胞（MDSC）介导免疫功能抑制的作用机制研究

Immunosuppression induced by myeloid-derived suppressor cells (MDSCs) is a key obstacle to hinder cancer immunotherapy. It is a novel strategy for tumor immunotherapy to eliminate the immunologic negative regulation status. In our previous study, a purified polysaccharide designated CKAP-2 was isolated from Curcuma kwangsiensis, a traditional Chinese medicine used for cancer therapy. It is structurally characterized to be a highly methyl-esterified pectin-type polysaccharide. Bioactivity test showed that CKAP-2 could recover the MSC2 (MDSC cell line) supressed proliferation of T cells. This finding suggested that CKAP-2 can reverse MDSC-mediated T-cell suppression. To explore the molecular mechanism of CKAP-2 on the immunosuppression mediated by MDSCs derived from Hepa1-6 tumor-bearing mice, matures and differentiation result of MDSCs after treatment with CKAP-2 will be investigated firstly. Additionally, the recognition receptor of CKAP-2 on MDSCs will be screen by proteomics and transcriptomics techniques, and its signal transcription pathway will be explored. The obtained recognition receptor and signaling pathway will be verified in vitro and in vivo through receptor antagonist and knockout mice model. Furthermore, the in vivo effect of immunosuppression mediated by MDSCs will be evaluated in model mice after treatment with CKAP-2. This project provides a new-type candidate molecules witn antitumor activity in Curcuma kwangsiensis. It also will provide a scientific basis for the clinical research and development and application of Curcuma kwangsiensis polysaccharides.

髓源抑制性细胞（MDSC）诱导的免疫功能抑制是癌免疫治疗的最大障碍，解除其介导的免疫负调控状态是癌免疫治疗的重要策略。在前期工作中，我们从传统抗癌中药莪术中分离得到一个纯化的多糖CKAP-2，并对其进行了结构全解析，生物活性研究发现CKAP-2能够体外逆转MSC2（MDSC细胞系）介导的T细胞增殖抑制，提示CKAP-2具有逆转MDSC介导的T细胞免疫抑制的功能。为进一步探讨CKAP-2逆转MDSC介导的免疫功能抑制的作用及机制，本项目以Hepa1-6皮下移植瘤肝癌小鼠模型为载体，首先拟明确CKAP-2促进MDSC成熟分化的方向；其次以蛋白组学、转录组学、基因敲除等技术筛选并验证MDSC细胞表面能够识别CKAP-2的受体类型和信号通路；最后评价体内CKAP-2对MDSC介导的免疫抑制功能的调控。本项目预期结果将丰富莪术抗癌的药效物质基础，为莪术多糖的临床研究和应用开发提供科学依据。

髓源抑制性细胞（MDSC）诱导的免疫功能抑制是癌免疫治疗的最大障碍，解除其介导的免疫负调控状态是癌免疫治疗的重要策略。广西莪术为临床常用的抗癌中药，根据其传统的水煎应用，我们推测其水煎剂中所含的大分子多糖类成分亦可能为除莪术油之外的另外一种抗癌有效成分。前期研究结果显示一个纯化的莪术多糖CKAP-2能够逆转MDSC介导的T细胞免疫抑制的功能，提示莪术多糖可能为通过拮抗MDSCs介导的免疫负调控状态其抗肿瘤作用。基于此，本项目从广西莪术中提取制备了的纯化的莪术多糖nCKAP-2，并通过高效凝胶渗透色谱、化学衍生物分析、GC-MS、FT-IR和NMR对多糖进行了表征。结果表明，CKAP-2的主链为高度支化的(1→3)-α-Arabinan，在O-5位有单取代（17.07%)，在O-2和O-5位有双取代(16.67%)，侧链组成比较复杂。活性研究结果显示，nCKAP-2对小鼠来源的脾细胞无毒副作用，而对MDSC细胞具有细胞毒性，且呈剂量依赖性。进一步的研究表明nCKAP-2能够诱导MDSCs细胞周期阻滞在G0/G1期，提高Cleaved Caspase3、Cleaved Caspase9和Cleaved PARP的表达水平，从而诱导MDSCs内源性凋亡，消除其对效应T细胞的增殖抑制作用。机制研究表明，nCKAP-2可以通过MDSC细胞表面的TLR4受体介导的NF-κB信号通路诱发MDSCs细胞凋亡；同时AKT（蛋白激酶B）亦参与了这一过程。另外，nCKAP-2作用后，MDSCs的活性氧ROS水平也显著下调，从而降低MDSCs 对T细胞的免疫抑制功能。体内实验表明nCKAP-2可以显著下调荷瘤小鼠脾脏中MDSCs比例，提示nCKAP-2至少能够通过诱导MDSCs的凋亡降低其介导的免疫功能抑制。本项目的研究阐明了莪术多糖nCKAP-2拮抗MDSCs诱导的T细胞增殖抑制的作用机制，为莪术多糖的临床研究和应用开发提供了科学依据。