【摘要】：目的：手足口病是一種常見的傳染疾病,以嬰幼兒發(fā)病多見,腸道病毒71型(Enterovirus71, EV71),屬于小核糖核酸病毒家族中的腸道病毒屬成員,是引起手足口病的常見病因,到目前為止,臨床上還沒有十分有效的治療方案或者疫苗研制成功。在中國近幾年的數(shù)次手足口病爆發(fā)的過程中,中草藥顯示出了良好的臨床治療效果,灰樹花多糖被發(fā)現(xiàn)具有多種抗癌,抗炎,抗病毒等藥用價值,然而,在灰樹花多糖抗EV71病毒的研究方面,尚未見報道。因此,我們探討了灰樹花多糖的抗EV71病毒活性,同時對多糖的結(jié)構(gòu)進行了分析,以期為進一步了解這一生物活性物質(zhì)的構(gòu)效關(guān)系提供一定的基礎(chǔ)。方法：實驗中采用傅里葉紅外光譜儀和核磁共振儀(包括核磁共振氫譜儀和核磁共振碳譜儀)被用于研究純化后的灰樹花多糖的結(jié)構(gòu),排除測定藥物對細胞增值的影響后,我們初步用結(jié)晶紫染色法檢測藥物的抗EV71病毒作用,采用蛋白印跡法和實時定量PCR進一步說明了灰樹花多糖的抗病毒作用及其作用的初步機制,通過時間進程實驗我們也進一步研究了灰樹花多糖抗病毒的時間點。結(jié)果：通過體外實驗發(fā)現(xiàn)灰樹花多糖可以通過抑制病毒前體蛋白合成和RNA合成來阻礙EV71的復(fù)制,在病毒感染的早期,灰樹花多糖可以有效地抑制EV71的作用,通過對VP1蛋白水平檢測及caspase-3蛋白的檢測,證明灰樹花多糖通過抑制EV71誘導(dǎo)的caspase-3的剪切活性,從而起到抑制宿主細胞凋亡的作用。EV71感染可以引起細胞內(nèi)IκBα的下調(diào),灰樹花多糖可以通過抑制這種由EV71感染誘導(dǎo)的宿主細胞IκBα蛋白下調(diào)的表達達到抗病毒的作用。結(jié)論：實驗中所用灰樹花多糖含有α—吡喃型糖殘基和β—葡萄糖殘基糖苷鍵,具有良好的抑制EV71病毒復(fù)制的作用。
[Abstract]:Objective: hand, foot and mouth disease is a common infectious disease. Enterovirus 71 (Enterovirus71, EV71), a member of small RNA virus family, is a common cause of hand, foot and mouth disease. So far, there is no very effective treatment or vaccine development. In the course of several outbreaks of hand, foot and mouth disease in China in recent years, Chinese herbal medicine has shown a good clinical therapeutic effect. Polysaccharides from Grifola frondosa have been found to have many medicinal values such as anticancer, anti-inflammatory, anti-virus, etc. However, The study of Grifola frondosa polysaccharides against EV71 virus has not been reported. Therefore, we discussed the antiviral activity of Grifola frondosa polysaccharides and analyzed the structure of polysaccharides in order to provide a basis for further understanding the structure-activity relationship of this bioactive substance. Methods: Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) were used to study the structure of purified Grifola frondosa polysaccharides. The antiviral effect of the drug against EV71 was detected by crystal violet staining. The antiviral effect of Grifola frondosa polysaccharides and its mechanism were further explained by Western blotting and real-time quantitative PCR. We also studied the antiviral time point of Grifola frondosa polysaccharide by time process experiment. Results: it was found in vitro that Grifola frondosa polysaccharides could inhibit the replication of EV71 by inhibiting the synthesis of virus precursor protein and RNA. In the early stage of virus infection, Grifola frondosa polysaccharides could effectively inhibit the effect of EV71. By detecting the level of VP1 protein and caspase-3 protein, it was proved that Grifola frondosa polysaccharide inhibited the apoptosis of host cells by inhibiting the shearing activity of caspase-3 induced by EV71. EV71 infection could induce the down-regulation of I 魏 B 偽 in cells. Grifola frondosa polysaccharides can inhibit the down-regulation of I 魏 B 偽 protein in host cells induced by EV71 infection. Conclusion: the polysaccharides of Grifola frondosa contained 偽 -pyrrolidene glycosyl residues and 尾 -glucose residues glycosides, which could inhibit the replication of EV71 virus.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R725.1

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