石斛堿抑制甲型流感病毒復(fù)制的活性研究
發(fā)布時間：2019-05-27 05:18
【摘要】：研究目的:流感是嚴(yán)重危害人類健康的傳染性疾病。歷史上已出現(xiàn)多次的流感大流行,波及范圍廣,死亡率高。由于流感病毒抗原性轉(zhuǎn)變和抗原性漂移的存在,流感病毒具有極高的變異性,新型流感病毒的出現(xiàn)已對人類的生存造成了嚴(yán)重的影響。流感的防治,尤其是藥物治療,迫在眉睫。迄今為止,在全球臨床應(yīng)用的抗流感藥物主要有神經(jīng)氨酸酶抑制劑和M2離子通道抑制劑兩類,但藥物耐藥性及毒副作用等問題一直困擾著這些藥物的發(fā)展和應(yīng)用。目前用于流感病毒感染預(yù)防和治療的化學(xué)合成藥物十分有限。因此,從天然產(chǎn)物中尋找低毒有效的抗流感病毒中藥顯得尤為重要�！稌r病論》清熱保津法曾收載復(fù)方石斛湯可治溫?zé)嵊泻?風(fēng)熱化火,熱病傷津等,其中石斛具有廣泛的生物學(xué)活性,包括益胃生津,潤肺止咳、滋陰清熱等,常用于口干煩渴、熱病傷津、病后虛熱等多種病癥。石斛堿一種從金釵石斛中提取出來的主要生物堿。本項(xiàng)目通過研究石斛堿抗甲型流感病毒活性及其作用機(jī)制,揭示其作用靶點(diǎn),以期充分挖掘石斛的藥用價值,為開發(fā)具有自主知識產(chǎn)權(quán)的新型抗流感藥物奠定基礎(chǔ),為流感的防治提供新的思路。研究方法:1.MTT法與空斑實(shí)驗(yàn)檢測石斛堿的細(xì)胞毒性及抗甲型流感病毒的活性。2.Western blotting、免疫熒光和qRT-PCR檢測石斛堿對流感病毒復(fù)制的影響。3.血凝抑制實(shí)驗(yàn)、SPR實(shí)驗(yàn)、H5N1假病毒體系、神經(jīng)氨酸酶抑制實(shí)驗(yàn)和time-of-addition實(shí)驗(yàn)研究石斛堿作用機(jī)制。4.Mini-replicon實(shí)驗(yàn)、定點(diǎn)突變技術(shù)、免疫共聚焦和計(jì)算機(jī)模擬分子對接技術(shù)研究石斛堿的作用靶點(diǎn)。實(shí)驗(yàn)結(jié)果:1.石斛堿能夠有效地抑制多種甲型流感病毒的感染,包括A/Aichi/2/68(H3N2),A/FM1/47(H1N1)和 A/Puerto Rico/8/34 H274Y(H1N1),IC50 分別是 5.32±1.68,3.39±0.32,2.16±0.91 μg/ml。2.石斛堿可抑制甲型流感病毒的多輪復(fù)制,且抗病毒作用可達(dá)72h。3.石斛堿對流感病毒的進(jìn)入無抑制作用。4.石斛堿在病毒感染細(xì)胞后的0-4 h能夠明顯抑制流感病毒HA的表達(dá)。5.石斛堿可降低vRNP的活性,能夠與NP蛋白結(jié)合,且能抑制NP的出核,降低NP的核內(nèi)表達(dá)。6.石斛堿可通過R267、V270、F338和E339與NP蛋白結(jié)合,且4個位點(diǎn)的保守性大于99%。實(shí)驗(yàn)結(jié)論:1.石斛堿具有抗甲型流感病毒的活性,且能抑制病毒的多輪復(fù)制。2.石斛堿有效地抑制流感病毒的早期復(fù)制。3.石斛堿抗流感病毒的作用靶點(diǎn)為NP蛋白。4.石斛堿與NP蛋白結(jié)合的位點(diǎn)是NP蛋白的保守功能區(qū)域。5.石斛堿通過與NP蛋白的結(jié)合抑制其出核以及寡聚化,從而阻礙流感病毒的復(fù)制。
[Abstract]:Objective: influenza is an infectious disease that seriously endangers human health. There have been many influenza pandemics in history, with a wide range of spread and high mortality. Because of the antigenic transformation and antigenic drift of influenza virus, influenza virus has a high variability. The emergence of new influenza virus has had a serious impact on human survival. The prevention and treatment of influenza, especially drug treatment, is urgent. Up to now, there are mainly two kinds of anti-influenza drugs used in the world: neurosinase inhibitor and M2 ion channel inhibitor, but drug resistance and toxic and side effects have been perplexing the development and application of these drugs. At present, the chemical synthetic drugs used in the prevention and treatment of influenza virus infection are very limited. Therefore, it is particularly important to find low toxic and effective anti-influenza virus traditional Chinese medicine from natural products. "time disease theory" Qingre Baojin method once included compound Dendrobium decoction to treat warm heat and sweat, wind heat and heat, heat injury, etc. Among them, Dendrobium has a wide range of biological activities, including tonifying stomach, relieving cough, nourishing yin and clearing heat, etc., which are often used in many diseases, such as dry thirst, febrile injury, deficiency and heat after disease and so on. A major alkaloid extracted from Dendrobium nobile. By studying the activity and mechanism of Dendrobium alkaloid against influenza A virus, this project reveals its target in order to fully excavate the medicinal value of Dendrobium candidum and lay a foundation for the development of new anti-influenza drugs with independent intellectual property rights. It provides a new way of thinking for the prevention and treatment of influenza. Methods: 1.MTT assay and plaque test were used to detect the cytotoxicity and anti-influenza A virus activity of Dendrobium. 2. Western blotting, immunofluorescence and qRT-PCR were used to detect the effect of Dendrobium on influenza virus replication. Hemagglutination inhibition test, SPR test, H5N1 pseudovirus system, neuraminase inhibition test and time-of-addition experiment were used to study the mechanism of Dendrobium alkaloid action. 4.Mini-replicon test, site-directed mutation technique, Immunoconfocal and computer simulated molecular docking techniques were used to study the target of Dendrobium alkaloid. The experimental results are as follows: 1. Dendrobium can effectively inhibit the infection of a variety of influenza A viruses, including A/Aichi/2/68 (H3N2), A/FM1/47 (H1N1) and A/Puerto Rico/8/34 H274Y (H1N1). IC50 was 5.32 鹵1.68, 3.39 鹵0.32, 2.16 鹵0.91 渭 g 鈮，
本文編號：2485923
[Abstract]:Objective: influenza is an infectious disease that seriously endangers human health. There have been many influenza pandemics in history, with a wide range of spread and high mortality. Because of the antigenic transformation and antigenic drift of influenza virus, influenza virus has a high variability. The emergence of new influenza virus has had a serious impact on human survival. The prevention and treatment of influenza, especially drug treatment, is urgent. Up to now, there are mainly two kinds of anti-influenza drugs used in the world: neurosinase inhibitor and M2 ion channel inhibitor, but drug resistance and toxic and side effects have been perplexing the development and application of these drugs. At present, the chemical synthetic drugs used in the prevention and treatment of influenza virus infection are very limited. Therefore, it is particularly important to find low toxic and effective anti-influenza virus traditional Chinese medicine from natural products. "time disease theory" Qingre Baojin method once included compound Dendrobium decoction to treat warm heat and sweat, wind heat and heat, heat injury, etc. Among them, Dendrobium has a wide range of biological activities, including tonifying stomach, relieving cough, nourishing yin and clearing heat, etc., which are often used in many diseases, such as dry thirst, febrile injury, deficiency and heat after disease and so on. A major alkaloid extracted from Dendrobium nobile. By studying the activity and mechanism of Dendrobium alkaloid against influenza A virus, this project reveals its target in order to fully excavate the medicinal value of Dendrobium candidum and lay a foundation for the development of new anti-influenza drugs with independent intellectual property rights. It provides a new way of thinking for the prevention and treatment of influenza. Methods: 1.MTT assay and plaque test were used to detect the cytotoxicity and anti-influenza A virus activity of Dendrobium. 2. Western blotting, immunofluorescence and qRT-PCR were used to detect the effect of Dendrobium on influenza virus replication. Hemagglutination inhibition test, SPR test, H5N1 pseudovirus system, neuraminase inhibition test and time-of-addition experiment were used to study the mechanism of Dendrobium alkaloid action. 4.Mini-replicon test, site-directed mutation technique, Immunoconfocal and computer simulated molecular docking techniques were used to study the target of Dendrobium alkaloid. The experimental results are as follows: 1. Dendrobium can effectively inhibit the infection of a variety of influenza A viruses, including A/Aichi/2/68 (H3N2), A/FM1/47 (H1N1) and A/Puerto Rico/8/34 H274Y (H1N1). IC50 was 5.32 鹵1.68, 3.39 鹵0.32, 2.16 鹵0.91 渭 g 鈮，
本文編號：2485923
本文鏈接：http://sikaile.net/falvlunwen/zhishichanquanfa/2485923.html
最近更新
教材專著
