黄芪、莪术配伍重构肿瘤免疫微环境的作用机制及物质基础研究

As the main immune cells in the tumor microenvironment, tumor associated macrophages（TAM）and regulatory T cells (Treg) play an important role in the growth and metastasis of tumor. Under the support of past two national natural science foundation of china , Research Group found that compatibility of Astragalus and Zedoary has obvious inhibitory effect on the proliferation and metastasis of human ovarian cancer HO-8910 and human hepatocellular carcinoma HepG2 in nude mice orthotopic transplantation tumor. Besides,it downregulated the expression of MMP-2, VEGF, FGF-2, BCL-2. This study intends to establish the co-culture system of tumor cells, tumor associated macrophages and regulatory T cells to simulate tumor immune microenvironment. Using molecular biology, cell biology, epigenetic, we will study the intervention effect of compatibility of Astragalus and Zedoary or single medicine or monomer on Immune activity of tumor associated macrophages and regulatory T cells in the co-culture system and the effect on the biological behavior of tumor cells. The active site and monomer may be tracked and separated closely as the application of analytic technology, such as modern chromatographic spectrum. Meanwhile, this technology can identify the structure. Assisted the computer simulation technology, we will reveal the mechanism and the material basis of drug efficacy of immune microenvironment interposed by the compatibility of chinese medicine. Besides, we will futher explore the antitumor mechanism of the compatibility of Astragalus and Zedoary, which can provide the experimental basis to clinic.

肿瘤相关巨噬细胞(TAM)及调节性T细胞（Treg）作为肿瘤微环境中的主要免疫细胞，在肿瘤生长转移中起着重要的促进作用。课题组在以往两个国家自然基金项目的资助下，发现黄芪、莪术配伍使用对裸鼠人卵巢癌HO-8910和肝癌HepG2原位移植瘤增殖和转移具有明显抑制作用，且能下调MMP-2、VEGF、FGF-2、BCL-2等因子的表达。本研究拟建立肿瘤细胞与肿瘤相关巨噬细胞、调节性T细胞共培养体系模拟肿瘤免疫微环境，采用分子生物学、细胞生物学、表观遗传学等手段，研究黄芪、莪术配伍、单味药及单体对共培养体系中巨噬细胞和调节性T细胞免疫活性干预作用和肿瘤细胞生物学行为的影响，应用现代色谱波谱等分析技术，密切追踪分离活性部位、鉴定结构，辅以计算机分子模拟技术，研究揭示中药配伍干预免疫微环境的作用机制及药效物质基础，进一步探讨黄芪、莪术抗肿瘤作用机理，为临床合理使用中药提供实验依据。

（1）本课题采用分子生物学、细胞生物学等手段，研究了黄芪、莪术配伍分别对共培养体系中巨噬细胞免疫活性干预作用和肿瘤细胞生物学行为的影响；结果显示IL-4能够诱导人巨噬细胞向M2型巨噬细胞分化，同时也发现黄芪及黄芪莪术配伍能够较好的抑制CD206，初步证明黄芪及黄芪、莪术配伍组能够抑制IL-4诱导M2型巨噬细胞分化。 .（2）采用高效液相色谱法，系统分析和比较黄芪、莪术单煎及共煎后有效成分含量两变化，研究发现黄芪甲苷、黄芪皂苷Ⅰ、黄芪皂苷Ⅱ、毛蕊异黄酮、芒柄花素、莪术醇、莪术二酮、吉马酮等成分在共煎液中含量均高于单煎及合并液。采用超高效液相色谱-三重四极串联质谱法同时测定黄芪、莪术中17种主要生物活性成分在黄芪、莪术单煎和共煎中含量的变化，结果显示与单味药相比，黄芪、莪术配伍混合制剂中莪术醇，莪术二酮，异莪术醇，莪术呋喃二烯酮和吉马酮等脂溶性成分含量明显增加，而脂溶性成分多具有抗肿瘤的作用，为黄芪、莪术两药配伍抗肿瘤协同增效作用提供了实验依据。.（3）槲皮素是一种黄酮类化合物，是黄芪中抗肿瘤的重要成分，本研究通过建立DMBA诱导的金黄仓鼠颊囊癌模型，观察槲皮素抗肿瘤的效果及作用机制。实验采用TUNEL，RT-PCR 和Western Blot 、蛋白分子对接技术等方法对相关因子进行检测和分子虚拟对接，研究发现，槲皮素能够有效促进DMBA诱导的金黄仓鼠颊囊癌细胞的凋亡，抑制颊囊癌的发生、发展。作用机制可能是通过抑制IKKβ诱导IκBα磷酸化进而阻碍NF-κB信号通路激活来实现的。