"健脾益气、消癥散结"法调控miR-155介导的肿瘤相关巨噬细胞极化抑制胃癌上皮间质转化的机制研究

One of the key mechanisms involved in gastric cancer metastasis is that tumor-associated macrophages (TAMs) with M2 polarization phenotype could promote epithelial mesenchymal transition (EMT) in gastric cancer cells. M1 polarization of TAMs induced by miR-155 can reduce EMT in gastric cancer and finally inhibit the metastasis, which now is a research focus in cancer immunology. Our previous study revealed that traditional Chinese medicines formula with effects of "spleen-strengthening, Qi-invigorating and mass-eliminating" could effectively reduce the recurrence of gastric cancer in patients. In the animal models with tumor xenografts, we reported that the EMT in gastric cancer decreased after the inhibition of inflammatory pathways by improving the activity of immune system which has the macrophages. Therefore, we suppose the formula could inhibit gastric cancer EMT and metastasis by promoting TAM M1 polarization through miR-155. This proposal will discover the intervention of this formula in mediating TAM polarization by regulating miR-155. Further,how TAM polarization influences gastric cancer EMT and its key factors involved in this process in animal models with gastric cancer orthotopic transplanted and co-cultured cell models will also be investigated. This study will help to specify some mechanisms in the gastric cancer metastasis inhibited by the formula, and reveal the significance of traditional Chinese medicine theory in gastric cancer relapse and metastasis. This study will help to specify some mechanisms in the gastric cancer metastasis inhibited by the formula, and reveal the significance of traditional Chinese medicine theory in gastric cancer relapse and metastasis.

肿瘤相关巨噬细胞（TAM）类M2极化表型促进胃癌细胞上皮间质转化（EMT）是胃癌转移重要机制；通过miR-155调控TAM向M1极化，减少胃癌EMT，抑制胃癌转移是肿瘤免疫学研究热点。前期研究表明以"健脾益气、消癥散结"为治法的中药组方能有效抑制胃癌患者的复发转移；在移植瘤动物模型中通过提高巨噬细胞在内的免疫系统活性而抑制炎性通路，减少胃癌EMT。我们基于前期工作基础提出假说：以"健脾益气、消癥散结"为指导的中药组方通过调控miR-155及其靶基因促进TAM的M1型极化，抑制胃癌细胞EMT从而抗胃癌转移。本课题拟采用胃癌皮下移植瘤动物及共培养细胞模型，研究该治法下的中药组方引起的miR-155调控TAM极化的干预环节；进一步研究TAM极化改变对胃癌EMT的影响及关键分子的变化。该研究有利于明确"健脾益气、消癥散结"法抑制胃癌复发转移机制，诠释中医理论指导下治疗胃癌复发转移干预策略的科学性。

本项目通过与胃癌细胞共培养构建的TAM细胞模型及胃癌皮下移植瘤动物模型，研究“健脾益气、消癥散结”法指导下的中药组方对TAM极化方向的调控及机制，并进一步研究TAM极化改变对胃癌EMT的影响及关键分子的变化。本实验结合流式细胞仪、实时定量PCR、免疫组化、wsternblot、CBA、ELISA等技术，研究证实：1.“健脾益气、消癥散结”法代表方剂健脾养正消癥方及健脾养正拆方能较好的减少TAM的M2表型；同时健脾养正方在减少TAM从M2表型向M1表型转化中，优于全复方及消癥散结拆方。其作用机制可能与抑制TAM上p-CEBP/β表达、增加NF-κB表达有关，但其对miR155无显著调节作用。2.健脾养正消癥方及健脾养正拆方有通过TAM表型改变对胃癌生长转移的趋势，但与对照组无显著统计学差异。3. 健脾养正消癥方能通过改善TAM表型及减轻胃癌EMT两种途径，抑制胃癌生长，总体效果优于拆方组。在此基础上，我们增加了“健脾益气、消癥散结”法中“健脾益气”的药物，结合临床实践，创建了新的复方“益气健脾化瘀方”。细胞实验证实，益气健脾化瘀方能通过促进TAM从M2表型向M1表型转化，减轻肿瘤微环境的免疫抑制，减轻胃癌EMT，抑制胃癌细胞的生长转移。同时，我们又选取了扶正药物中常见的单体甘草查尔酮A进行了研究，研究证实，该单体能通过抑制胃癌细胞中PI3K/AKT通路，下调HK2表达，减轻胃癌糖酵解，减少酸性微环境，促进胃癌细胞凋亡。