抗A型流感病毒和肠道病毒71型天然化合物的筛选及其抗病毒分子机制研究

发布时间：2018-03-20 21:58

本文选题：H5N1A型流感病毒　切入点：肠道病毒71型(EV71)　出处：《武汉大学》2012年博士论文　论文类型：学位论文

【摘要】：流行性传染病会规律性或者非规律性地在全世界范围内引起大范围的感染疫情,从而威胁到人类的健康甚至生命。其中A型流感病毒和肠道病毒71型(EV71)是近年来亚太地区爆发频率较高,并且疫情影响范围较广的两种急性流行病病毒。本研究在药用植物提取的天然化合物库中,体外筛选获得分别对这两种病毒具有较强抗病毒活性的化合物,并且对这些有效化合物的抗病毒分子机制做了进一步的研究。众所周知,A型流感病毒每年都会引起大范围的传染病疫情,并且造成全球数百万人的健康和财产受到损害。但是由于流感病毒的高度变异性和耐药性的不断出现,新型抗病毒的药物的研发显得尤为重要。本研究中,我们选用了高致病性禽流感病毒H5N1亚型(HPAI H5N1)毒株,以MDCK为筛选用细胞模型,在三百多种药用植物提取的纯化合物中,用MTT筛选法筛选出Berberine(BR)和Usnic Acid (UA)有明显的抗HPAI H5N1的抗病毒活性,并且具有很低的细胞毒性。进一步的研究中,我们运用了血凝抑制实验(HI实验)和HA免疫荧光验证了这两种化合物抑制病毒复制的能力。并且,利用荧光定量PCR验证了这两种化合物可以抑制病毒cRNA和mRNA的复制。另外,通过哺乳动物双杂交实验证明了BR可以干扰NP和PB2两种病毒蛋白之间的相互作用,这个相互作用可能会影响病毒RNP复合体的功能。总结以上发现,BR和UA具有良好的A型抗流感病毒活性,可能成为未来流感治疗中重要的新药候选。而对于肠道病毒71型(EV71),近年来,由于多次的疫情爆发,影响的地理范围越来越广,受感人群数量也逐渐增多,使得它已成为亚太地区甚至更广范围的主要公共健康问题。严重的EV71感染会导致神经性疾病、休克并伴有很高的死亡率。但是到目前为止EV71的治病机理尚不明确,也没有非常有效的特异性药物可以预防和治疗。这也更增加了新药开发的必要性和紧迫感。在本研究中,我们选用了实验室自己分离的EV71毒株,以RD细胞作为筛选用细胞模型,在两百多种药用植物提取的纯化合物库中,体外筛选获得Matrine(Mat),灵芝酸L13A和灵芝酸L13B具有明显的抗EV71病毒活性。另外,细胞毒性试验证明了在实验浓度范围内,这三种药用化合物还具有促进宿主细胞存活和生长的作用。进一步的研究中,我们利用药物作用模型实验初步判断了三种药用化合物的作用时期,并且利用生物信息学模拟分子对接实验,从结构和能量变化的角度证实了三种化合物可以直接与病毒粒子相互作用,通过阻碍病毒脱壳作用起到明显的看病毒效果。综上所述,我们的研究证实了体外筛选出的天然化合物对HPAI H5N1和EV71有着良好的抗病毒活性,极低的细胞毒性,并且揭示了它们的抗病毒分子机制；为两种流行性传染疾病的预防及治疗提供了新的候选药物、理论和实验基础,同时也为两种病毒的感染病人的临床治疗提供了更多的理论支持。
[Abstract]:The epidemic of infectious diseases will be regular or non regular in the worldwide epidemic caused by infection in large scale, and thus a threat to human health and even life. The influenza A virus and enterovirus 71 (EV71) is the Asia Pacific region in recent years the outbreak of the epidemic situation of high frequency, and the effect of two kinds of acute virus epidemic a wide range of this study. In the extraction of medicinal plants of natural compounds, compounds were screened in vitro with strong antiviral activity against the two viruses, and the antiviral molecular mechanism of these compounds effectively do a further study.
As everyone knows, A type A influenza virus every year will cause the epidemic situation of infectious diseases in large scale, and caused millions of global health and property damage. But due to high variability of influenza virus and drug resistance appear constantly, the new antiviral drug development is particularly important. In this study, we selected a high pathogenicity avian influenza virus subtype H5N1 (HPAI H5N1) strains, screening cell model with MDCK as pure compounds extracted in more than 300 species of medicinal plants in Berberine were screened with MTT screening method (BR) and Usnic Acid (UA) has obvious anti HPAI antiviral activity of H5N1, and has a very low cytotoxicity. The further research, we use the hemagglutination inhibition test (HI test) and HA immunofluorescence to verify the ability to inhibit the replication of these two compounds. Moreover, the two compounds tested by fluorescent quantitative PCR CRNA and mRNA can inhibit virus replication. In addition, the mammalian two hybrid experiments proved that the BR interaction between two viral proteins NP and PB2 interference, this interaction may affect viral RNP complex functions. Summarize the above findings, BR and UA A with good anti influenza virus activity, may be the future of new drug candidates in the treatment of influenza.
As for enterovirus 71 (EV71), in recent years, due to the outbreak of many epidemic, the effects of the geographical range more widely, infected population is gradually increased, so that it has become a major public health problem for the Asia Pacific region and the wider range. Severe EV71 infection will lead to neurological diseases, and shock with a high mortality rate. But so far the cure mechanism of EV71 is still not clear, there is no specific drug can be very effective in prevention and treatment. It also increased the necessity and urgency of developing new drugs. In this study, we selected the EV71 strains isolated in our laboratory, using cell screening using RD cells as model, two hundred kinds of medicinal plants in the extraction of pure compounds in the library, in vitro screening Matrine (Mat), ganoderic acid L13A and ganoderic acid L13B has obvious anti EV71 virus activity. In addition, test cell toxicity In the experimental concentration range, the three kinds of medicinal compounds also have a role in promoting the survival and growth of host cells. Further research, we use the drug model experiment preliminary judgment function during the period of three kinds of medicinal compounds, and the use of bioinformatics and molecular docking simulation experiment, the structure and the change of energy confirms three compounds can be directly with the virus particle interaction, by blocking virus uncoating played the role of evident virus effect.
In summary, our study confirms that natural compounds in vitro screened with good antiviral activity against HPAI H5N1 and EV71, extremely low cytotoxicity, antiviral and reveal their molecular mechanisms; providing new drug candidates for the prevention and treatment of two kinds of epidemic infectious diseases, the theoretical and experimental basis, at the same time clinical treatment for infected patients of two viruses provide more theoretical support.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R373

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