基于"补肾健脾方"调控树突细胞TLRs/MyD88/TRIF通路探讨"扶正"治则在慢乙肝治疗中的作用机制

"Strengthening healthy energy" is the important rule of traditional chinese medicine.The defense of "healthy energy" is very similar to the immune system of modern medicine. Now there is shot of investigation whcih elaborated "Strengthening healthy energy"theoreory by immunoregulation. Immune tolerance is an important cause of the chronic hepatitis B. Dendritic cells (DCs) are important antigen-presenting cells, its function and maturation defects caused immune tolerance of chronic hepatitis B. Studies have shown that patients with hepatitis B of immune tolerant phase are visible to varying degrees of weakened body resistance, the function and maturation of peripheral blood DCs subgroup are defected. Preliminary experiments confirmed "reinforcing the kidney and invigorating the spleen prescription"guided by "Strengthening healthy energy" can improve the function and phenotype of DCs. Mature of DCs mediated by TLRs/MyD88/TRIF pathway and closely related to extracellular cytokines, but HBV infection can break this balance. This project is continuation of previous work, that aims to study the regulation of "reinforcing the kidney and invigorating the spleen prescription" on TLRs/MyD88/TRIF signaling pathway and DCs differentiation associated cytokines when DCs infected by HBV through modern molecular biology techniques, and clarify the mechanism of "Strengthening healthy energy", finally enrich the material basis of "Strengthening healthy energy" therapeutical principle for the treatment of chronic infectious diseases.

"扶正"是中医的重要治则。"正气"所包含的防御、抗邪功能与现代医学免疫系统极为相似。目前从免疫调节阐述"扶正"理论内涵尚缺乏深入研究。免疫耐受是乙肝慢性化的重要原因，树突状细胞（DCs）是重要的抗原提呈细胞，其功能和成熟缺陷是造成乙肝免疫耐受的主要因素。研究表明免疫耐受期乙肝患者均可见不同程度的正虚，且存在外周血DCs各亚群的功能和成熟缺陷。前期实验证实以"扶正"为理论指导的"补肾健脾方"可改善DCs的功能和表型。DCs成熟受TLRs介导的MyD88/TRIF通路的调控，同时与胞外细胞因子密切相关，而HBV感染可打破这一平衡。本项目是在前期工作基础上，通过现代分子生物学技术，研究"补肾健脾方"对HBV感染后TLRs/MyD88/TRIF信号通路及DCs分化相关细胞因子的调控；以免疫调节为靶点阐明中医"扶正"理论的作用机制，充实中医"扶正"治则在慢性感染性疾病治疗中的物质基础。

乙型肝炎是严重危害人类健康的传染病，现已证实细胞免疫缺陷是造成乙肝慢性化、难治化的重要原因。DCs是人体内功能最强的抗原提呈细胞是机体抗病毒免疫应答的重要功能细胞，它的成熟、功能缺陷是造成乙肝免疫耐受的重要原因。慢性乙型肝炎患者外周血DCs 各亚群存在明显的成熟障碍和功能缺陷，而HBV 感染是诱发DCs 成熟障碍的首要因素。. 中医认为“正气存内，邪不可干” ，正气不足在发病过程中的决定作用，脾肾不足是正气不足的关键，因此，我们选用了补肾健脾为主的“六味地黄汤”和“四君子汤”拟“补肾健脾方”探讨其对DCs成熟的影响。本研究通过分离健康人外周血PBMC，同时予“补肾健脾方”进行干预，检测HBV 病毒颗粒刺激后不同时段各实验组培养上清中细胞因子含量；比较刺激前后mDCs和pDCs 成熟差异。应用免疫磁珠分离外周血单核细胞和pDCs，单核细胞以 rhGM-CSF、IL-4、TNF-α 诱导mDCs 成熟，CpG ODN 诱导pDCs，诱导同时予HBV 病毒颗粒共培养及“补肾健脾方”进行干预，检测各实验组DCs 的TLRs/MyD88/TRIF信号通路TLR3、TLR9、MyD88、TRIF、NF-κB、 AP-l（C-JUN）、IRF3、IRF7 蛋白及基因水平。此外，增加了临床部分研究，观察了脾肾两虚慢性乙型肝炎患者DC亚群与健康人和非脾肾两虚患者的差异。. 研究结果证实了慢性乙型肝炎患者DC亚群数量均低于健康人，而且脾肾两虚慢乙肝患者患者MDCs、PDCs数量均低于非脾肾两虚患者。发现补肾健脾方可提高HBV干预后外周血PBMC中PDC、MDC数量。部分阐明补肾健脾方对DC亚群的调控可能是通过上调PDC中MyD88依赖通路的TLR9、MYD88、IRF7，MDC中TLR3、IRF3、NFκB实现的。