三七多糖抗肿瘤药效物质基础及痊愈小鼠再次免疫应答的抗肿瘤分子机制研究

Traditional Chinese medicine polysaccharide is a kind of widely used immunomodulator. Science Magazine and other magazines Name Cancer Immunotherapy as Scientific Breakthrough of the Year. The preliminary study of this research group found: Polysaccharide from Panax notoginseng was extracted from the residue of Panax notoginseng, and the content of polysaccharides was 56.70%. It was confirmed that polysaccharide from Panax notoginseng had the effect of enhancing the immune function of mice; In collaboration with Professor Robert W. Sobol, Jr. of the Mitchell Cancer Institute at the University of South Alabama in the United States, it was found that the polysaccharide from Panax notoginseng could not only prolong the survival of H22 tumor mice, but also in the middle dose of the polysaccharide from Panax notoginseng 20% of the tumor-bearing mice were cured, the mice were again subcutaneously inoculated with H22 cancer cell suspension, no tumor growth was found and this part of the research content has been published by（OncoTargets and Therapy,2016:9 3433–3441.）. But its mechanism of action has not been elucidated. Therefore, in this topic, based on previous studies, the separation and purification of polysaccharide from Panax notoginseng was studied. The molecular basis of the anti-tumor effect of Polysaccharide from Panax notoginseng was determined by molecular, cellular and animal experiments. By re-vaccination of the recovered mice, the immune cells, immune cytokines and IgG were measured to explore the mechanism of anti - tumor immune response in mice.

中药多糖是一种研究应用广泛的免疫调节剂，Science等杂志将肿瘤的免疫治疗列于十大科学突破的首位。本课题组与美国南阿拉巴马大学米切尔癌症研究所Robert W. Sobol, Jr.教授合作，从提取过三七皂苷的废渣中提取含量为56.70%三七粗多糖，并发现三七粗多糖不仅能延长荷H22瘤小鼠的生存期，而且中剂量三七粗多糖组有20%的荷瘤小鼠痊愈，痊愈小鼠再次皮下接种H22癌细胞混悬液，未发现肿瘤生长，此部分研究内容已发表SCI论文（OncoTargets and Therapy,2016:9 3433–3441.），但其作用机理尚未阐明。因此，本课题在前期研究基础上，进行三七粗多糖的分离纯化、结构确定；通过分子、细胞、动物水平实验，确定三七多糖抗肿瘤的药效物质基础；通过对痊愈小鼠再次接种，测定免疫细胞、免疫细胞因子及IgG含量，探究痊愈小鼠抗肿瘤的再次免疫应答机制。

多糖是构成生命的四大基本物质之一，具有多靶点、多个作用部位及多种药理活性，且几乎无毒，因此，多糖研究一直是热点，但多糖是大分子物质，结构复杂，结构解析难度大，制约了多糖构效关系及作用机制研究。.本课题以水提醇沉法从工业三七药渣中提取三七粗多糖，采用树脂分离技术对三七粗多糖进行纯化，通过硫酸蒽酮法、间羟基联苯法、HPGPC法、HPAEC法、甲基化反应、ATR FT-IR法和SEM对各三七多糖组分理化性质及结构进行分析。.MTT法测定各三七多糖组分对7种不同肿瘤细胞的体外增殖活性的影响，实验发现：中性三七多糖对各细胞株的生长均有抑制作用，因此，选用中性三七多糖对体内抗肿瘤活性进行研究。.构建H22实体瘤模型，通过测定免疫器官指数、免疫细胞活性及百分比、免疫细胞因子、免疫球蛋白IgG的的血清水平、肿瘤组织病理学切片、血液指标、抑瘤率等，考察中性三七多糖以及中性三七多糖联合环磷酰胺对肝癌H22细胞肿瘤模型的治疗作用。结果证实：中性三七多糖通过提高肝癌H22细胞荷瘤小鼠的细胞免疫和体液免疫，实现抗肿瘤的目的，中性三七多糖联合环磷酰胺可提高抑瘤率。.骨髓是造血和免疫器官，课题进一步探究中性三七多糖对骨髓抑制小鼠造血功能的保护作用。结果发现：中性三七多糖通过解除骨髓有核细胞细胞周期S期阻滞，拮抗环磷酰胺所致细胞异常凋亡，提高血清造血生长因子GM-CSF、TPO、EPO含量，促进造血干细胞分化及成熟，提高外周血中红细胞数、血红蛋白浓度、白细胞数、中性粒细胞数、红细胞压积、血小板数，对骨髓抑制小鼠造血功能发挥保护作用。.痊愈小鼠再次接种H22肿瘤细胞后，肿瘤不生长，但接种14天后接种部位肿瘤细胞仍然存在，CD49b+NK细胞数量均升高，提示痊愈小鼠再次接种肿瘤后肿瘤不生长可能与记忆NK细胞有关。.此外，三七多糖还具有良好的吸湿、保湿性能、抗氧化及延缓衰老，可用于日化用品、保健食品及药品的研发。