【作者】 钟菊迎；

【导师】 曹洪欣；

【作者基本信息】 中国中医科学院 ， 中医基础理论， 2010， 博士

【摘要】 本课题在深入分析流感病毒致病机理基础上,采用目前通用的流感动物模型、细胞模型,以肺指数、病死率为评价指标,证实金柴抗病毒胶囊对不同免疫状态流感动物模型的防治作用,以病毒-细胞、病毒-机体-免疫系统的相互作用作为研究对象,运用激光扫描共聚焦、实时荧光定量RT-PCR以及免疫印记Western-Blot等方法,从细胞、分子水平明确金柴抗病毒胶囊抗流感病毒的作用靶点,阐明抗流感病毒作用机制。本研究分三部分。文献综述：主要对流感病毒致病机理以及中医药防治流行性感冒研究进展进行总结；实验研究：在明确金柴抗病毒胶囊防治流感作用基础上,从病毒-细胞、病毒-机体-免疫两方面对其抗病毒机制进行研究。理论探讨：结合流行性感冒的特点,探讨透邪解毒法防治流行性感冒的理论依据和优势。1透邪解毒法治疗流行性感冒的理论探讨流行性感冒属于中医“时行感冒”时行伤寒”时行疫病”等范畴,其病因为“非时之气”挟“时行疫毒”侵袭人体所致。由于社会发展、环境变迁以及人们生活、就医习惯的影响,流行性感冒的病邪性质和病机变化不再遵循固有的规律,探索其病机特点、证候演变规律,并以此确立新的治则治法是中医药行业的重要任务。透邪解毒法是针对流行性感冒“在表之邪犹存,趋里之邪易郁化热”的病机特点而设,以此法为组方依据的金柴抗病毒胶囊由金银花、柴胡、蝉蜕等药组成,全方配伍轻清宣透、引邪外出,清热解毒祛除病因,参以培补正气抵御外邪,体现了“早期治疗,截断病势,防止传变”的立法宗旨。2实验研究2.1金柴抗病毒胶囊防治流感的作用研究采用甲型H1N1流感病毒鼠肺适应株(FM/1/47株和PR/8/34株)滴鼻感染正常免疫状态和免疫低下小鼠造成肺炎模型,以肺指数和病死率为评价指标,评价金柴抗病毒胶囊对不同免疫状态小鼠甲型流感的防治作用,并与目前世界卫生组织推荐治疗流感主要药物达菲进行比较。实验结果显示：金柴抗病毒胶囊对正常免疫状态和免疫低下小鼠肺部炎症均有明显的治疗和预防作用,并在减轻肺指数、降低小鼠死亡率、延长小鼠生存时间上,与达菲比较无显著性差异(P>0.05)。2.2金柴抗病毒胶囊抗流感病毒作用机制研究2.2.1对流感病毒吸附环节的影响通过药物先与细胞作用1小时后,加入流感病毒再作用1小时的方法,采用实时荧光定量RT-PCR,检测药物作用细胞后细胞中流感病毒HA-mRNA表达含量。实验结果显示：金柴抗病毒胶囊能显著减少细胞中流感病毒HA-mRNA的表达含量,提示金柴抗病毒胶囊可通过干预病毒对细胞吸附环节发挥抗流感病毒作用。2.2.2对流感病毒-细胞膜融合的影响通过药物与流感病毒同时作用细胞的方法,采用激光扫描共聚焦结合PH值敏感荧光染料,观察金柴抗病毒胶囊对流感病毒感染细胞后胞内PH值变化的影响。实验结果显示：金柴抗病毒胶囊能显著降低流感病毒感染细胞后引起胞内PH酸化的程度,提示金柴抗病毒胶囊可干预病毒-细胞膜融合环节发挥抗流感病毒的作用。2.2.3对流感病毒转录与复制的影响利用病毒感染细胞1小时后,加入相应稀释浓度的金柴抗病毒胶囊,作用48-72小时后,采用实时荧光定量RT-PCR法,检测细胞中流感病毒多聚酶PA亚基mRNA表达含量,观察金柴抗病毒胶囊对流感病毒多聚酶PA亚基mRNA表达的影响,进一步探讨其抗流感病毒作用靶点。实验结果显示：金柴抗病毒胶囊能显著降低细胞中流感病毒多聚酶PA亚基mRNA表达含量,提示金柴抗病毒胶囊抗病毒的作用机制与干预流感病毒的转录和复制有关。2.2.4对流感病毒释放环节的影响利用病毒感染细胞1小时后,加入相应稀释浓度的金柴抗病毒胶囊,作用72小时,采用荧光Elisa的方法,观察金柴抗病毒胶囊对流感病毒NA活性的影响。实验结果显示：金柴抗病毒胶囊对流感病毒NA活性没有明显的抑制作用,提示金柴抗病毒胶囊对流感病毒的释放环节没有影响作用。2.2.5对病毒载量及γ-IFNmRNA表达的影响通过甲型H1N1流感病毒鼠肺适应株(FM/1/47株)感染正常小鼠造成肺炎模型的方法,检测流感病毒感染小鼠后第1天、第3天、第5天以及第7天小鼠肺组织中病毒载量、γ-IFNmRNA的动态变化情况,探讨金柴抗病毒胶囊调节机体免疫机制抗病毒作用机理。实验结果显示：金柴抗病毒胶囊大、中、小3个剂量组对不同时间点肺组织中病毒载量的表达均有明显的降低作用,对相应时间点小鼠肺组织中γ-IFNmRNA的表达含量均有明显的升高作用,提示金柴抗病毒胶囊通过上调γ-IFNmRNA的表达发挥抗病毒作用。2.2.6对干扰素诱导跨膜蛋白表达的影响采用免疫印记Western-Blot的方法,观察流感病毒感染小鼠后第1天、第3天、第5天以及第7天肺组织中天然抗流感病毒蛋白-干扰素诱导跨膜蛋白(IFITM3)的动态变化情况,探讨金柴抗病毒胶囊抗流感病毒的作用靶点。实验结果显示：金柴抗病毒胶囊在流感病毒感染早期即可调节机体产生IFITM3发挥抗病毒作用,并且在其后几天内与模型组比较,其含量一直维持在一个较高水平,提示金柴抗病毒胶囊可调节机体免疫机制产生更多的IFITM3发挥抗流感病毒作用。更多还原

【Abstract】 Based on the analysis of the influenza virus pathogenesis,the article uses the animal models of influenza and the models of cell and evaluates the lung index and mortality to indentify the effects of Jin Chai anti-vital capsule of animal models under differently immune status.Through the studies of the interaction of virus-cell and virus-body-immunity system and the uses of the methods of laser scanning confocal microscopy real-time fluorescent RT-PCR and Western-Blot,this experiment reveals the mechanism of Jin Chai antivital drugs on the molecular level and the effiency of the Tou Xie Jie Du to treat the influenza and provides the scientific proofs.The article consists of three parts:The first part reveals the machanism of influenza virus pathogenesis and the effects of traditional chinese medicine.The second part reveals the theoretical basis and the advantages of Tou Xie Jie Du.The third part reveals the machanism from two facets:virus-cell and virus-body-immunity system.1. Theoretical DiscussionThe influenza belongs to the Chinese medical syndrome of Shi Xing Gan Mao,Shi Sing Shang San and Shi Xing Yi Bing Because of the influences of the social development and environmental change and the effects of the people’s habits and medical practices, the nature of pathogenic factors and the mechanism of influenza pathology no longer adhere to inherent rules.The explosion of the character of pathogenesis and the regulation of syndrome is an important task of treaditional Chinese medicine. 2 Experimental Study2.1 The role of prevention and treatment of influenzaUse the nose dropping method with influenza virus (FM/1/47 and PR/8/34 strains) to establish immunocompromised mouse model. Respectively do the theraputical administration and prophylactic administration to observe the lung weight and calculate the lung index and mortality.①Therapeutical effect:Jin Chai antivital capsules markedly decrease the lung index and mortality of mouse model with infuluenza virus and prolong the live time of the mouse model with infuluenza virus.③The antivital efficacy is not statistically significant between Jin Chai antivital capsules and Tamiflu.2.2 Mechanism of anti-influenza virus②Use Real-time PCR to observe the levels of HA-mRNA expression of influenza. The results show that Jin Chai antivital capsule can significantly reduce the levels of HA-mRNA expression of influenza and suggest the efficacy of Jin Chai antivital capsules through the intervention of the virus adhersion.②Use the combination of laser scanning confocal fluorescencent dye which is sensitive to PH value, to observe Virus-cell membrane fusion, The results show that Jin Chai antivital capsule can improve the levels of PH value, and suggest the efficacy of Jin Chai antivital capsules through the intervention of the Virus-cell membrane fusion.③Use Real-time PCR to observe the levels of PA-mRNA expression of influenza. The results show that Jin Chai antivital capsule can significantly reduce the levels of PA-mRNA expression of influenza and suggest Jin Chai antivital capsules effect on the Transcription and Replication.④Use the Elisa method of fluorescence to observe the Jin Chai capsules on the NA, the results show that Jin Chai anti-viral capsules do not inhibit the efficacy of NA activity.⑤Use Real-time PCR and Western-Blot method to observe the Jin Chai anti-virus Capsule effect on the interferon-y and natural anti-virus protein-interferon induces transmembrane protein,.the results show that Jin Chai anti-virus Capsule can improve the expression.of the interferon-yand interferon induced transmembrane protein expression.更多还原