乌头煎剂调节NK细胞受体/配体群谱偏移干预肝癌免疫逃逸的分子机制研究

It is no doubt that TCM plays an important role in comprehensive prevention and control on liver cancer, and the therapeutic principle of TCM is syndrome differentiation and treatment administration. However, many doctors of TCM always treat liver cancer with heat-clearing and detoxifying herbs, rarely think about different syndromes, like yang deficiency syndrome. Aconite, which is the main one of the herbs with effect of warming Yang and eliminating cold, always is used to treat liver cancer. What’s more, our previous experiments have established it’s curative effect on liver cancer, but it was hard to inhibit proliferation of cancer cells directly. Tumor microenvironment is the material basis for the growth of liver cancer, in which the inflammatory microenvironment is closely related to its development. It is the key of escaping from immune function to the growth of tumors. It has been verified that Aconite has the prominent anti-inflammatory and immune regulation function. What’s the important, our previous experiments verified that a certain concentration of aconite decoction by adjusting certain inflammatory factor, and the different receptor/ligand ratio of Natural Killer cells especially, to improve the immune function of mice with liver cancer. With the view of pathogenesis in TCM, the conflict between the vital - qi and pathogens, we put forward the hypothesis that controlling inflammatory microenvironment surrounding liver cancer cells (strengthen vital qi) plays a role of the mechanisms of Aconite on tumor (against pathogens). This project is proposed to confirm the anti-tumor effect of Aconite on different models of liver cancer and on inflammation factors and immune function in the different stages of hepatocellular carcinoma. Through modern technology such as gene chip to screen signaling pathways related and clarify it by in vitro and in vivo experiment. It aim to clarify that changing the liver immune cells ,especially the natural killer cells and the receptor/ligand ratio of them in different stages of liver cancer is the one of the mechanism of anti-cancer for aconitum, and explored the effect of warming Yang and eliminating cold herb in the hepatocellular carcinoma, and provide scientific experimental basis for better clinical application.

中医药在肝癌综合防治中的作用毋庸置疑，而辨证论治则是中医临床诊疗的核心。近些年，众多临床工作者动辄选用具有清热解毒抗癌作用的中药，全然不顾许多晚期患者诸多明显的阳虚之征，摒弃了辨证论治的思维。乌头自古以来就是临床治疗寒性癥积的基本用药，当代更是被制成注射剂用于肝癌的临床。我们前期动物实验证实，乌头煎剂治疗肝癌疗效确切，但却不是表现为对肿瘤的直接杀伤作用，而具有明显的抗炎和免疫调控作用，尤其是能影响NK细胞受体/配体群谱偏移。肿瘤微环境下的“免疫逃逸”是其赖以生长的关键。结合中医药“邪正交争”的发病观，我们提出“调控肝癌微环境的免疫功能（扶正）是乌头发挥抗肝癌作用（抗邪）的主要机制”的假说。本课题拟通过体内外实验，进一步验证乌头对肝癌的治疗作用，并从调节NK细胞受体/配体群谱偏移干预肿瘤免疫逃逸角度，探讨其抗癌作用的可能机制，从而为更好的用于肝癌临床及温阳散寒类抗肝癌药物的开发提供依据。

中医药在肝癌综合防治中的作用毋庸置疑，目前众多医家多采用清热解毒药物治疗肝癌，而许多晚期患者多呈现明显的阳虚之征，因此“温阳散寒”也是治疗肝癌的重要原则。乌头自古以来就是临床治疗寒性癥积的基本用药，当代更被制成注射剂用于肝癌的临床。我们前期动物实验证实，乌头煎剂治疗肝癌疗效确切，但却不是表现为对肿瘤的直接杀伤作用，而具有明显的抗炎和免疫调控作用。.本课题经研究确认了温阳药物乌头煎剂在一定浓度（按照人9-30g/60kg）、一定的治疗窗口（预防/干预）对不同的小鼠肝癌模型（H22腹水瘤种植BALB/c荷瘤小鼠、Hep1-6种植的C57荷瘤小鼠）的肿瘤均具有显著的抑制作用；同时证实乌头煎剂冻干粉在一定浓度下（>250ug/ml）对多种肝癌细胞株（97H、7402、7721、Hep3B、HepG2、Huh 7和LM3）具有一定的抑制增殖、诱导凋亡及迁移的作用；进一步观察到乌头煎剂对肝癌模型小鼠的免疫功能在不同阶段有不同的调节作用，增加外周血T细胞、NK细胞的数量，增强血清中TNF-α、IL-1β、IFN-γ细胞因子的分泌、提高肿瘤组织内IL-6、IL-1β、TNF-α蛋白及mRNA表达；增加肿瘤组织内NK细胞的浸润，CD107a表达增强，NK细胞活性增强，降低外周血中NKp46、NKG2D和TIGIT受体，升高脾脏抑制性受体TIGIT和TACTILE表达的作用；进一步经细胞因子PCR芯片和信号通路筛选发现，乌头煎剂增强肿瘤组织内多种细胞趋化因子的分泌，CCL2升高最明显，经NK细胞抗体阻断检验，发现趋化因子CCL2分泌因子的增加是通过MAPK/JNK信号通路及其关键因子来实现，这也是乌头煎剂抗肝癌的主要机制。.本课题提出“温阳散寒”治疗肝癌的思路，着眼于肿瘤微环境中的NK细胞，从调节NK上下游信号通路角度探讨了其抗癌作用的机制，为乌头用于肝癌临床及温阳散寒类抗肝癌药物的开发提供数据支持。