定喘汤"宣降清三法"干预合胞病毒感染T-bet哮喘易感基因及代谢组学研究

Respiratory syncytial virus (RSV) is the common cause of pneumonia, bronchiolitis and inducing asthma. In our primary research we discussed that Dingchuantang decoction can intervene the therapeutical basis of RSV pneumonia and we also found out the biological basis of symptoms in RSV pneumonia. Whether this result apply to "treating same disease with different methods" has yet to be further study and validation. These observations have led us to posit that Dingchuantang decoction can improve immune injury of RSV infection and regulate the imbalance of Th1/Th2. Using randomized controlled trial, we collected the information of symptoms, metabonomics and disease to establish of the dynamic association of symptoms and metabonomics by individual evaluation, population analysis and metabolic network. We explored that the relationship between common metabolic biomarkers in the three diseases and single nucleotide polymorphism of T-bet gene which is genetic susceptibility to asthma. Combination of variation of metabolic network and clinic research, we discussed the biomarker which is accordance with the symptoms of traditional Chinese medicine. Further more, we clarified the important value of the aspect of macroscopic syndrome differentiation of traditional Chinese medical entireties and microcosmic diagnosis differentiation of metabolomics after ingesting Dingchuandang decoction to RSV rat model. Combining the above researching, we provided a scientific basis of using traditional Chinese medicine to prevention of RSV infection and the subsequent development of asthma later in clinical practice.

呼吸道合胞病毒（RSV）感染可导致毛细支气管炎(肺炎)、诱发哮喘。项目前期研究初步探讨定喘汤干预RSV肺炎药效物质基础，发现该病机证候群的生物学基础，但该结果是否适用于"异病同治"还有待于深入研究与验证。本研究提出定喘汤"宣升与清降并用改善RSV感染的免疫损伤，发散与收敛兼施调节Th1/Th2的免疫失衡"的假说。针对基本证型变化，动态采集受试者证候、代谢组学和疾病信息，通过个体评价、群体分析，代谢网络构建相关分析研究证候代谢组学之间的动态关联。探求该证在三种疾病中的共性标志物、代谢网络及哮喘易感基因位点的表达。力图通过临床和实验研究，明确RSV感染前后T-bet基因单核苷酸多态性的变化及Th1/Th2免疫失衡状态。继而用"定喘汤"干预RSV模型鼠，考察RSV鼠体内整体代谢网络的变化，并结合临床试验结果，探讨符合中医症候群的生物标记物群,进一步阐明中医整体宏观辨证与生物组学微观辨病的重要性。

呼吸道合胞病毒（Respiratory Syncytial Virus，RSV）感染可导致小儿毛细支气管炎和（或）肺炎，并在哮喘的发生、发展和加重中扮演重要角色，临床实践证实中医药防治小儿肺系病证有一定优势，定喘汤是临床防治RSV感染所致喘息性疾病的有效方剂，深入探讨其抗病毒作用靶点及其防治机制对中药研究具有重大而深远的意义。本研究分为动物实验和临床分析两大部分。首先完成了RSV诱导大鼠病毒性肺炎模型的建立；应用流式细胞术检测RSV感染后大鼠机体整体免疫状态，应用免疫组化、westen-blot法、RT-PCR技术检测RSV感染后大鼠肺组织中胸腺基质淋巴细胞生成素（Thymic stromal lymphopoietin,TSLP）、T盒转录因子（T-box expressed in T cells,T-bet）、转录因子 GATA 结合蛋白 3（GATA-binding protein-3,GATA-3）蛋白及基因的表达情况，采用ELISA法检测RSV肺炎大鼠血清中IFN-γ、IL-4、IL-10、IL-13、IL-17、IL-33炎症因子的含量，探讨定喘汤宣降清三法对RSV诱导大鼠肺炎后机体Th1/Th2免疫平衡的调控机制和作用靶点；采用液相色谱-质谱联用（LC-MS）技术检测RSV肺炎大鼠血清中代谢产物的变化，阐明定喘汤宣降清三法对RSV诱导后大鼠内源性代谢网络的影响；该研究基础实验部分从基因层面、蛋白表达层面及代谢组学等全方位多角度阐述了定喘汤宣降清三法对RSV诱导肺炎大鼠机体的良性调节作用。其次通过临床分析RSV毛细支气管炎和（或）肺炎及哮喘患儿不同证型变化和疾病信息，采用LC-MS法检测患儿血清中代谢产物，筛选出三种疾病及治疗后的差异性代谢产物；应用代谢通路分析，明确了RSV毛细及哮喘患儿在磷脂代谢及氨基酸代谢均发生改变，阐述了两种疾病中共性代谢网络变化。本研究通过基础实验和临床试验的结合，探讨了符合中医症候群的生物标记物群，也进一步阐明了中医整体宏观辩证与生物组学微观辨病的重要性。