基于恶性胸腔积液微环境Th17细胞调控探讨中药消水方的分子机制

Malignant pleural effusion ( MPE ) is one of the common complications in advanced tumors, whose development is associated with tumor cells and local environment of inflammation as well as immune cells. Research shows that Th17 cells as pro-inflammatory cells, are increasing in a variety of tumor microenvironment. It's same with MPE,the quantity is also higher than in peripheral blood, mediates pleural effusion information and development. Chinese traditional medicine" Xiaoshui Decoction" has been traditional clinical MPE treatment prescription of our department for more than 30 years. Clinical studies showes that it can significantly improve the patients' life quality, relieve symptoms and improve immune function. We speculate that its active mechanism is the adjustment to MPE microenvironment Th17 cells and cytokine. This project plans to establish MPE model of lung cancer in mice , adopt technique of flow cytometric detection, gene chip, Western blot, RT-PCR ect. , analysize relative features of MPE Th17 cellular pathways and explore the effection of " Xiaoshui Decoction" on MPE and Th17 cell path, discuss Th17 cell migration mechanisms with CCRs/CCLs chemotactic axes and Th17 cell differentiation mechanisms with ERK1/2-STAT3 signaling pathways. The reseach will provide experimental basis for further drug development and promote the application of Chinese Traditional Medicine in MPE treatment.

恶性胸腔积液（MPE）是晚期肿瘤常见并发症之一，其产生发展跟肿瘤细胞与局部的炎症环境、免疫细胞有关。研究表明：Th17细胞作为促炎细胞，在多种肿瘤微环境中均增多，MPE中数量也高于外周血，介导胸腔积液的形成发展。中药"消水方"为我科临床治疗MPE经验用方，临床应用30余年。临床研究发现能明显改善患者生存质量，缓解症状，提高免疫功能。我们推测消水方的作用机制在于对MPE微环境Th17细胞、细胞因子的调控。本项目拟通过建立小鼠肺癌MPE模型，采用流式细胞检测、基因芯片、Western blot、RT-PCR等技术分析MPE中Th17细胞通路相关特征，探讨"消水方"对MPE的作用及对Th17细胞通路的影响，从CCRs/CCLs趋化轴探讨Th17细胞的迁移机制，从ERK1/2-STAT3信号通路探讨药物作用下Th17细胞的分化机制，为药物的进一步开发提供实验依据，推动中医药在治疗MPE中的应用。

本研究通过建立C57BL/6小鼠Lewis 肺癌胸腔积液模型，选取MPE微环境中与Th17细胞相关的免疫调控网络为切入点，主要通过动物实验来研究中药消水方对模型小鼠MPE的抑制作用，及调控MPE微环境中Th17细胞相关细胞因子和蛋白表达来增强机体对肿瘤免疫功能的作用机制。研究结果显示：1、运用C57BL/6小鼠胸腔内注射Lewis细胞建立小鼠肺癌MPE模型成胸水率较好。2、小动物光学成像系统观察消水方具有一定的抑制小鼠Lewis肺癌MPE和胸膜转移瘤的作用，而与化疗联合抑制作用更加明显。3、消水方可明显减轻小鼠体重下降，防止恶液质的发生；同时降低化疗对于小鼠体重的影响，增加化疗耐受性。4、消水方可明显延长小鼠总生存时间，其生命延长率为15.79%，明显优于联合用药(10.51%)和单纯化疗(0%)。5、消水方具有抑制MPE生成，减少MPE体积，降低肺癌胸膜转移作用，而与化疗联合时抑制MPE和减少胸膜转移作用更强，表现出协同效应。6、流式细胞技术检测MPE和外周血Th17、Treg细胞含量，发现MPE微环境Th17、Treg 细胞含量明显高于外周血。模型组小鼠外周血Th17、Treg 细胞含量明显高于正常小鼠。7、消水方可明显增加MPE微环境和外周血中Th17 细胞含量，增强MPE微环境和循环微环境对肿瘤细胞的免疫作用，减少血行转移。而与化疗联合并不能提高这种增高趋势；与单纯化疗相比则可显著提高这种增强趋势。8、消水方可明显降低MPE微环境和外周血中Treg细胞含量，减轻MPE微环境和循环微环境对肿瘤细胞的免疫抑制作用，同时减少血行转移。而与化疗联合并不能增强MPE微环境中Treg细胞这种减轻趋势；与化疗联合则可以显著增强外周血中Treg细胞这种减轻趋势。9、CBA法和Elisa法检测MPE和外周血中细胞因子，发现消水方单独应用或者联合化疗均可上调MPE微环境和外周血中IL-6、IL-17A、IL-23表达，下调IL-21、以及TGF-β1表达，从而从局部和全身诱导初始Th0细胞向Th17细胞分化，促进肿瘤免疫作用；减少向Treg细胞分化，减少肿瘤免疫抑制作用。10、Western Blot实验检测瘤组织中STAT-3蛋白的表达，发现消水方单独应用或者联合化疗均显著提高模型小鼠实体瘤中STAT-3蛋白的表达，促进CD4+T细胞向Th17 的分化，增强对肿瘤的免疫作用。