【摘要】：豬流感(Swine Influenza,SI)是由豬流感病毒(Swine Influenza Virus,SIV)引起的一種急性、熱性、高度接觸性呼吸道傳染病,是規(guī)�；B(yǎng)豬場的常發(fā)病之一,以高致病率、低死亡率為特征。本病極易與其他呼吸道病原混合感染,使其死亡率急劇升高,給養(yǎng)豬業(yè)造成巨大的經(jīng)濟(jì)損失。豬是豬流感、人流感及禽流感等多種流感病毒共同的易感宿主,流感病毒易在其體內(nèi)重組而產(chǎn)生新的流感病毒株。同時,豬流感病毒不僅能感染豬,也能感染其他動物。因此,對豬流感病毒的研究不但有重要的獸醫(yī)傳染病學(xué)意義,更有深遠(yuǎn)的公共衛(wèi)生意義。流感病毒因血清型多、易變異以及易產(chǎn)生耐藥性等,疫苗接種及抗流感西藥的療效甚微,因此當(dāng)前尚無豬流感特效藥。所以,研究及開發(fā)抗豬流感病毒天然藥物及其有效成分迫在眉睫。目的:本研究旨在細(xì)胞水平上探討苦參主要成分的抗H3N2亞型豬流感病毒效果,并對其作用機(jī)制進(jìn)行初步探索。研究結(jié)果將為抗流感機(jī)制的深入研究提供基礎(chǔ),為獸醫(yī)臨床及醫(yī)學(xué)臨床用藥提供科學(xué)依據(jù)和參考,更為抗流感病毒天然藥物的開發(fā)及研制奠定基礎(chǔ)。方法:采用傳統(tǒng)有機(jī)溶劑回流提取法、傳統(tǒng)水提醇沉法分別制備苦參生物堿、黃酮、皂苷和粗多糖成分,以利巴韋林作為陽性對照藥,選擇犬腎上皮細(xì)胞株(MDCK細(xì)胞)作為H3N2豬流感病毒作用的細(xì)胞體系,通過細(xì)胞病變(CPE)觀察法和噻唑藍(lán)鹽微量酶反應(yīng)比色法(MTT法)定性、定量測定細(xì)胞狀態(tài),從而對苦參生物堿、黃酮、皂苷、粗多糖成分抗H3N2豬流感病毒作用加以比較,然后通過改變給藥方式等條件對其作用機(jī)制進(jìn)行初步探索。結(jié)果顯示:高濃度的苦參生物堿、黃酮、皂苷、粗多糖均會影響MDCK細(xì)胞的生長狀態(tài),其最大安全濃度分布在6.9μg/ml~1.5138 mg/ml之間,其中皂苷成分的毒性作用最小,濃度為1.5138 mg/ml時仍不表現(xiàn)細(xì)胞毒性作用。對四種成分體外抗SIV作用的研究發(fā)現(xiàn),四種成分都能顯著性抑制H3N2豬流感病毒,其中生物堿和粗多糖成分的效果優(yōu)于利巴韋林。此外,⑴四種成分均能明顯抵抗SIV的吸附,皂苷效果優(yōu)于利巴韋林;⑵四種成分均能干擾病毒復(fù)制,生物堿的作用優(yōu)于利巴韋林;⑶四種成分與病毒同時接觸細(xì)胞時,均有顯著抑制SIV作用,皂苷的效果優(yōu)于利巴韋林;⑷四種成分均有體外直接殺滅病毒作用,生物堿的作用優(yōu)于利巴韋林�？鄥⑺姆N成分的最小有效劑量分布在0.1078μg/ml~19.2μg/ml之間,其中苦參黃酮的最小有效濃度最低;治療指數(shù)在32~128之間,皂苷成分的治療指數(shù)最高�？鄥⒊煞峙c病毒同時接觸細(xì)胞時,各成分均呈現(xiàn)出明顯的劑量效應(yīng)關(guān)系。結(jié)論:苦參生物堿、黃酮、皂苷、粗多糖成分均有抗H3N2豬流感病毒作用,其中皂苷干擾病毒吸附的作用最強(qiáng)且能有效阻止病毒與細(xì)胞的結(jié)合,生物堿干擾病毒復(fù)制和直接殺病毒的作用最強(qiáng)。
[Abstract]:Swine flu (Swine Influenza,SI) is an acute, feverish and highly contact respiratory infectious disease caused by swine flu virus (Swine Influenza Virus,SIV. It is one of the common diseases in large-scale pig farms, characterized by high pathogenicity and low mortality. This disease is easy to be mixed with other respiratory pathogens, resulting in a sharp increase in mortality and a huge economic loss to the pig industry. Pigs are the common susceptible hosts of swine flu, human influenza and avian influenza. Influenza viruses are easy to recombine in their bodies to produce new strains of influenza viruses. At the same time, swine flu virus can infect not only pigs, but also other animals. Therefore, the study of swine flu virus not only has important veterinary infectious disease significance, but also has far-reaching public health significance. Due to the variety of serotypes, variability and drug resistance of influenza viruses, vaccination and anti-influenza drugs have little effect, so there is no specific swine flu drug. Therefore, the research and development of anti-swine flu virus natural drugs and their effective components are urgent. Objective: to investigate the effect of main components of Sophora flavescens against H3N2 subtype swine flu virus at cell level and to explore its mechanism. The results will provide a basis for the further study of the mechanism of anti-influenza, provide scientific basis and reference for the clinical use of veterinary medicine and medicine, and lay a foundation for the development and development of natural anti-influenza drugs. Methods: the alkaloids, flavonoids, saponins and crude polysaccharides of Sophora flavescens were prepared by traditional organic solvent reflux extraction method and traditional water extraction and alcohol precipitation method. Ribavirin was used as positive control drug. Canine renal epithelial cell line (MDCK cell) was selected as the cell system of H3N2 swine flu virus. The cytopathic (CPE) assay and MTT assay were used to determine the cell status quantitatively. The effects of alkaloids, flavonoids, saponins and crude polysaccharides of Sophora flavescens against H3N2 swine flu virus were compared. The results showed that high concentration of alkaloids, flavonoids, saponins and crude polysaccharides of Sophora flavescens affected the growth of MDCK cells. The maximum safe concentration was between 6.9 渭 g/ml~1.5138 mg/ml and the toxicity of saponins was the least. The concentration of 1.5138 mg/ml still showed no cytotoxicity. It was found that the four components could significantly inhibit H3N2 swine flu virus in vitro, and the effects of alkaloids and crude polysaccharides were superior to ribavirin. In addition, all the four components could resist the adsorption of SIV, and the effect of saponins was better than ribavirin, and the effect of alkaloids was superior to ribavirin. (3) when the four components were in contact with the virus at the same time, they all had significant inhibitory effects on SIV, and the saponins were superior to ribavirin in killing virus directly in vitro, and alkaloids were superior to ribavirin. The minimum effective dose of four components of Sophora flavescens was distributed in the range of 0.1078 渭 g/ml~19.2 渭 g/ml, in which the minimum effective concentration of flavone of Sophora flavone was the lowest, and the therapeutic index was between 32 ~ 128.The therapeutic index of saponins was the highest. When the components of Sophora flavescens and the virus were in contact with the cells at the same time, there was a significant dose-effect relationship between the components. Conclusion: the alkaloids, flavonoids, saponins and crude polysaccharides of Sophora flavone have the effect of resisting H3N2 swine flu virus, and the saponins have the strongest effect on interfering the virus adsorption and can effectively prevent the virus from binding to the cells. Alkaloids have the strongest effect on virus replication and direct virus killing.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S853.7

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本文編號：2379117