板蓝根多糖对肺气虚证小鼠抗H3N2型猪流感病毒试验研究

发布时间：2018-05-14 23:23

本文选题：板蓝根多糖 + 肺气虚证　；参考：《河南农业大学》2015年硕士论文

【摘要】：肺气虚证导致肺气亏损,体内宗气合成不足,宣发无力,进而表现出免疫功能低下的状态。板蓝根是传统清热药,主入肺经,具有清热解毒、凉血利咽功效。多糖作为板蓝根的主要活性成分,其良好的抗病毒和免疫调节能力受到众多研究者的青睐。猪流感病毒是引起猪呼吸道传染病的重要病原,H3N2型猪流感病毒在猪场广泛存在,可破坏猪的免疫系统进而引起免疫抑制,在规模化猪场难以根除。目前,猪流感无特效药,中药因其资源丰富、毒副作用小、价格低廉等特点有着独特的优势。本试验通过利用MDCK细胞模型和中医症候动物模型分别评价和观察了板蓝根多糖体外和体内抗H3N2型猪流感病毒的作用,丰富了中药抗流感病毒药库,筛选以中医证候模型为基础的抗病毒新药,也为中医证候学研究、中西医结合防治动物疫病提供更多理论依据。现将本文主要研究内容及研究成果归纳如下:一、采用水煮醇沉法提取板蓝根多糖,利用MDCK细胞体外培养系统,通过MTT法检测细胞活性评价板蓝根多糖体外抑制A/Swine/Henan/2010(H3N2)猪流感病毒对细胞的感染作用。结果表明,板蓝根多糖对MDCK细胞表现出一定的毒性作用,半数毒性浓度(TC50)为7.273mg/mL,最大安全浓度(TC0)为2.504mg/mL,板蓝根多糖对猪流感病毒的阻断、抑制和直接杀灭作用治疗指数分别是10.158、19.291、0.038。板蓝根多糖在MDCK细胞上对猪流感病毒的抑制作用和阻断作用效果较好,在此药物安全浓度范围内,板蓝根多糖直接杀灭作用不明显。二、采用A/Swine/Henan/2010(H3N2)猪流感病毒经鼻腔感染6-8周肺气虚证BALB/c小鼠,通过观察和分析感染后小鼠的体重,脏器指数,病理学变化以及免疫学等相关指标,探讨H3N2型猪流感病毒感染肺气虚证BALB/c小鼠动物模型致病性变化。结果显示,感染猪流感病毒肺气虚证小鼠精神状态表现较差,体重下降幅度大,肺脏出现水肿、出血和炎性细胞大量增生、细支气管黏膜上皮严重脱落、血管周围炎性细胞浸润等病变,机体淋巴增值能力、NO、IgG水平降低,促炎因子和抗炎因子水平紊乱且通过抑制IFN-γ与TNF-α水平影响其间接抗病毒能力。此外,从体重、肺部病理变化和机体损伤程度等方面综合分析,肺气虚证小鼠加重了其感染猪流感病毒引起的病变和损伤。三、以猪流感病毒A/Swine/Henan/2010(H3N2)感染肺气虚证BALB/c小鼠为模型,经板蓝根多糖作用后分别在第2,5,9天观察和检测其体征状态、体重增长率、脏器指数、肺脏病理变化、腹腔巨噬细胞活性、淋巴细胞增殖能力、NO和IgG水平变化、细胞因子等免疫学水平。结果显示,经板蓝根多糖干预后可明显改善感染小鼠体征状态,提高体重增长率并提前恢复至感染前水平,减轻或减小了肺脏水肿、出血、炎性细胞浸润、肺泡间质增生和病变区域。板蓝根多糖可能通过促进小鼠促炎细胞因子如IL-1β、IL-6和TNF-α水平,调节免疫应答,增强机体炎性修复能力;通过促进IL-12和IFN-γ的分泌,增强机体抗病毒作用,减轻肺脏炎性反应下调IL-10的表达。此外,不同剂量板蓝根多糖在机体抵抗流感病毒生物学活性调节活动中所起的作用有所差异,其中综合评价灌胃浓度75 mg·kg-1效果最好。综上,猪流感病毒A/Swine/Henan/2010(H3N2)感染肺气虚证BALB/c小鼠可引起机体一系列致病性变化,且肺气虚证小鼠加重了其感染猪流感病毒引起的病变和损伤;板蓝根多糖可在细胞水平和动物机体水平上发挥直接和间接抗猪流感病毒作用。
[Abstract]:The deficiency of the lung qi caused by deficiency of the lung qi, the inadequacy of the body gas synthesis, the weakness of the hair, and the low immune function. The Radix Isatis Root is a traditional antipyretic drug, which is mainly into the lung meridian, and has the effect of clearing heat and detoxifying, cooling blood and taking advantage of the pharynx. The polysaccharide is the main active ingredient of the Radix Isatidis, and its good antiviral and immune regulation ability is subject to many researchers. Swine influenza virus is an important pathogen causing respiratory infectious diseases in pigs. H3N2 swine influenza virus exists widely in pig farms. It can destroy the immune system of pigs and cause immunosuppression and can not be eradicated in large-scale pig farms. At present, there are no special drugs for swine flu. The traditional Chinese medicine has unique characteristics because of its rich resources, small toxic side effects and low price. In this experiment, the effect of MDCK cell model and TCM syndrome model was used to evaluate and observe the anti H3N2 swine influenza virus in vitro and in vivo of Radix Isatidis, enriching the anti influenza virus pharmacy store in Chinese medicine, screening new antiviral drugs based on TCM syndrome model, and also for TCM syndrome study and Chinese and Western medicine. The main research contents and research results of this paper are summarized as follows: first, the extraction and alcohol precipitation method is used to extract the polysaccharide of Radix Isatidis, and the cell activity of MDCK cells in vitro is used to detect the cell activity by MTT method to evaluate the inhibition of A/Swine/Henan/2010 (H3N2) virus to the cells in vitro. The results showed that the polysaccharide of Radix Isatidis showed a certain toxic effect on MDCK cells, half of the toxic concentration (TC50) was 7.273mg/mL, the maximum safety concentration (TC0) was 2.504mg/mL, the blocking, inhibition and direct killing effect of Isatis polysaccharide on swine influenza virus was 10.158,19.291,0.038. Isatis polysaccharide in MDCK thin, respectively. The inhibitory effect and blocking effect of the cell on the swine influenza virus was better. In the range of the drug safety, the direct killing effect of Radix Isatidis polysaccharide was not obvious. Two, the A/Swine/Henan/2010 (H3N2) swine influenza virus was used to observe and analyze the body weight and organ index of the mice after the infection of the BALB/c mice after the nasal infection for 6-8 weeks. The pathogenicity of H3N2 swine influenza virus infected BALB/c mice was investigated by pathological changes and immunology. The results showed that the mental state of the mice infected with swine influenza virus lung qi deficiency syndrome was poor, the weight loss was large, the lungs were swollen, bleeding and inflammatory cells proliferated, and the bronchioles were sticky. The membrane epithelium is severely shedding, perivascular inflammatory cell infiltration and other lesions, the body lymphatic increment ability, NO, IgG level, the level of proinflammatory and anti-inflammatory factors are disorganized and the ability to resist the anti viral activity between IFN- gamma and TNF- alpha is influenced by the comprehensive analysis of the body weight, the pathological changes of the lungs and the degree of injury of the body. The mice aggravated the disease and damage caused by the infection of the swine influenza virus. Three, the BALB/c mice infected with the lung qi deficiency syndrome of the swine influenza virus A/Swine/Henan/2010 (H3N2) were used to observe and detect the physical signs, the weight growth rate, the organ index, the pathological changes of the lungs and the peritoneal macrophages on day 2,5,9 after the action of the Radix Isatidis polysaccharides. The activity, lymphocyte proliferation, NO and IgG levels, cytokines and other immunological levels. The results showed that the signs of infection in mice can be significantly improved by the prognosis of Radix Isatidis polysaccharides, increase the rate of weight growth and advance to the pre infection level, reduce or reduce the pulmonary edema, bleeding, inflammatory cell infiltration, alveolar interstitial hyperplasia. The diseased area. The polysaccharide of the Radix Isatidis may be promoted by promoting the level of IL-1 beta, IL-6 and TNF- alpha in mice, regulating the immune response and enhancing the ability of inflammatory repair. By promoting the secretion of IL-12 and IFN- gamma, the antiviral effect of the body is enhanced and the expression of IL-10 under the inflammatory response of the lung is reduced. The effect of body resistance to the biological activity regulation of influenza virus was different, in which the comprehensive evaluation of the concentration of 75 mg. Kg-1 was best. To sum up, the swine influenza virus A/Swine/Henan/2010 (H3N2) infection of the lung qi deficiency syndrome BALB/c mice could cause a series of pathogenic changes in the body, and the lung qi deficiency mice aggravated the infection of the swine influenza. Virus induced lesions and injuries; isatis root polysaccharides can play a direct and indirect role against swine influenza virus at cellular level and animal level.

【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7

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