肠道菌群介导TAMs在脾虚湿阻型结肠癌发展中的作用机制

Colon cancer is one of the most common malignance in the digestive system with high incidence and mortality. gut microbiota disorder play an important role in the development and prognosis of colon cancer. We identified a specific feature of intestine bacterial disorder in the colon cancer patients under the status of spleen deficiency, which is correlated with epithelial immunity after polarization of macrophage and the activation of TLR4 signaling pathway. We predict that intestine bacterial promote the status of dampness encumbering spleen due to asthenia after the activation of macrophage which regulate TLR4/TGF-beta/CXCR4 signaling pathway, resulting the development of colon cancer. To verify our hypothesis, animal model and molecular technique will be applied in this project. 1, To clarify the specific feature and the biological characteristics of intestine of intestine bacterial in colon cancer induced by dampness encumbering spleen due to asthenia. 2, To demonstrate that the intestine bacterial in colon cancer induced by dampness encumbering spleen due to asthenia regulate the activation of macrophage and TLR4/TGF-beta/CXCR4 signaling pathway, which leads to the development of colon cancer. Our project is based on the syndrome differentiation and treatment. Through examination of the intestine bacterial, we will explore the mechanism of dampness encumbering spleen due to asthenia in the development of colon cancer. Metagenome, Macrotranscriptome and Macrometabolome will also be applied to demonstrate the progress from dampness and intestine bacterial to the cancer. We try to provide a precise Chinese medical treatment and theory for the colon cancer.

结肠癌是具有高致病率和死亡率的恶性肿瘤。肠道菌群失调是影响结肠癌发展和预后的重要因素。课题组前期研究发现脾虚湿阻状态下的结肠癌肠道菌群具有特异性，并与巨噬细胞极化后的黏膜免疫及肠上皮中活化的TLR4、TGF-β、CXCR4蛋白密切相关，由此我们推测脾虚湿阻型结肠癌患者中，特定肠道菌群导致免疫环境中的巨噬细胞极化形成肿瘤相关巨噬细胞（TAMs）从而促使结肠癌发展，其作用机制与激活肠黏膜免疫反应调控下的结肠癌上皮TLR4/TGF-β/CXCR4信号通路有关。为验证该假说，本研究立足于病证结合，以肠道菌群为切入点，基于模式动物及分子生物学等技术，探究脾虚湿阻型结肠癌特异性菌群的生物学特征，阐明脾虚湿阻状态下的结肠癌菌群通过影响巨噬细胞极化形成TAMs，调控结肠癌TLR4/TGF-β/CXCR4信号促进其发展的机制，以期拓展脾虚湿阻证候及“湿-菌-瘤”发展的微观机制，为中医药精准治结肠癌奠定基础。

脾虚湿阻证候是结肠癌常见的中医证候，与结肠癌不良预后密切相关。因此寻找到脾虚湿阻证候的分子生物学特征并探索其促进结肠癌进展的生物学机制是结肠癌防治研究的重点。本研究通过结肠癌细胞实验及结肠癌自发成瘤模型实验，并联合转基因动物模型，挖掘出脾虚湿阻证候下结肠癌肠道菌群的特异性，并证实该类肠道菌群能够诱导巨噬细胞募集促进结肠癌组织增生以及上皮间质转化进程，其主要机理与肿瘤中TLR4/TGF-β/CXCR4信号的活化相关。在此基础上，通过16sRNA测序技术、流式细胞技术、荧光免疫组化技术、蛋白质免疫印迹法（WB）及实时荧光定量PCR（RT-PCR）分别中细胞免疫检测，蛋白及基因分析的层面证实脾虚湿阻证候下特定的肠道菌群诱导巨噬细胞极化形成肿瘤相关巨噬细胞，并具有M2型特征，其诱导结肠癌中TLR4受体的活化，从而激活细胞中TGF-β信号，最终促使结肠癌高表达CXCR4，具有高侵袭特征。通过慢病毒转染TLR4、TLR4条件性敲除的转基因动物模型或巨噬细胞清除等手段后，本研究从上而下分别验证了巨噬细胞与结肠癌的相关性，以及结肠癌TLR4/TGF-β/CXCR4信号与肿瘤侵袭的相关性。在本研究中，进一步深化了脾虚湿阻证候动物模型的创新性构建，通过粪菌移植技术复现了脾虚湿阻中医证候的临床表现，由此可知肠道菌群能够介导中医证候的发展。综上所述，本研究证实了脾虚湿阻证候下结肠癌肠道菌群的特征性改变，着眼于巨噬细胞极化，从极化后的巨噬细胞所形成的“内湿”的微环境入手，丰富了中医脾虚湿阻证候的免疫微环境的机制。本项目不仅从全新的角度诠释了中医脾虚湿阻证候的本质，探索将中医证候转化成具体的临床生物学指标的新方法，更为肿瘤的防治提供新的途径，进而为中医药防癌抗癌以及中医药的精准治疗打下坚实的理论和实验基础。