连翘提取物对甲型流感病毒核蛋白表达影响的体外实验研究

发布时间：2018-11-27 09:07

【摘要】：背景：流感可引起季节性流行和大流行，严重危害人民健康。目前抗流感病毒西药因毒副作用较大或耐药性的增加，限制了其应用。祖国传统中医药在预防与治疗流感的临床和基础理论方面的研究，受到国内外的广泛关注，寻找新的抗流感中药及探索其作用机制，有助于将中药推向世界和造福人类。目的：探讨连翘提取物(连翘苷、连翘醇提液、连翘水煎剂)对甲型流感病毒核蛋白基因体外干预的影响，为连翘抗流感病毒提供科学的理论依据。方法： 1.实验分组： HeLa细胞组（HeLa细胞)、空质粒组（空质粒和HeLa细胞）、脂质体组（HeLa细胞和脂质体）、重组质粒组（重组质粒基因转染HeLa细胞）、连翘苷组（连翘苷加入重组质粒组中）、连翘水煎剂组（连翘水煎剂加入重组质粒组中）、连翘醇提液组（连翘醇提液加入重组质粒组中）七组。 2.细胞病变法确定连翘提取物最大无毒界限及实验用浓度。 3.脂质体瞬时转染法转染HeLa细胞，并以其作为实验用靶细胞。 4.胶体金免疫层析试纸法检测各实验组细胞培养上清和细胞内核蛋白的表达。 5.提取胶体金检测阴性组及重组质粒组细胞总RNA，合成cDNA。 6.荧光定量逆转录PCR检测重组质粒组及胶体金检测阴性组的核蛋白基因拷贝数，用统计学软件进行分析。结果： 1.重组质粒组细胞培养上清及细胞内胶体金法检测均为阳性；连翘醇提液组、连翘水煎剂组两组细胞培养上清为弱阳性，醇提液组更弱些；连翘苷组、连翘醇提液组、连翘水煎剂组三组细胞内为弱阳性，连翘苷组最弱，连翘醇提液组次之；细胞对照组、脂质体组、空质粒组和连翘苷组四组细胞培养上清及细胞对照组、脂质体组、空质粒组三组细胞内均为阴性。 2.连翘苷组胞内核蛋白基因表达量为(2.07±0.2)×105copies·μl-1，重组质粒组胞内核蛋白基因表达量为(61.2±13)×105copies·μl-1，连翘苷组胞内核蛋白基因表达量低于重组质粒组胞内，差异有统计学意义(t=9.836，，p0.05)。结论： 1.连翘苷可以下调瞬时转染HeLa细胞的甲型流感病毒核蛋白基因量，并能较强的抑制其核蛋白的表达。 2.连翘水煎剂及连翘醇提液也能抑制瞬时转染HeLa细胞的甲型流感病毒核蛋白的表达，但均较连翘苷弱，连翘水煎剂最弱。
[Abstract]:Background: influenza can cause seasonal epidemics and pandemics, seriously endangering people's health. At present, the use of anti-influenza virus western drugs is limited by the increase of side effects or drug resistance. The research on the clinical and basic theory of traditional Chinese medicine in the prevention and treatment of influenza has received extensive attention both at home and abroad. Finding new anti-influenza Chinese medicine and exploring its mechanism of action will help to bring Chinese medicine to the world and benefit mankind. Objective: to study the effect of Forsythia suspensa extract (Forsythia suspensa alcohol extract, Forsythia suspensa decoction) on the in vitro intervention of nucleoprotein gene of influenza A virus, and to provide scientific theoretical basis for anti-influenza virus of Forsythia suspensa. Methods: 1. The experimental groups were as follows: HeLa cell group (HeLa cell), empty plasmid group (empty plasmid and HeLa cell), liposome group (HeLa cell and liposome), recombinant plasmid group (recombinant plasmid gene transfected HeLa cell). Forsythrin group (Forsythia suspensin added to recombinant plasmid group), Forsythia suspensa decoction group (Forsythia suspensa decoction added to recombinant plasmid group), Forsythia suspensa alcohol extract group (forsythia suspensa alcohol extract added to recombinant plasmid group) group. 2. The maximum nontoxic limit and experimental concentration of Forsythia suspensa extract were determined by cytopathic method. 3. HeLa cells were transfected by liposome transient transfection and used as experimental target cells. 4. The expression of nuclear protein in culture supernatants and cells of each experimental group was detected by colloidal gold immunochromatographic assay. 5. Total RNA, Synthesis of cDNA. in cells of negative and Recombinant plasmid groups by extraction of Colloidal Gold 6. The copy number of nucleoprotein gene in recombinant plasmid group and colloidal gold negative group was detected by fluorescence quantitative reverse transcriptase (PCR) and analyzed by statistical software. Results: 1. The supernatant of cell culture and colloidal gold assay were positive in the recombinant plasmid group, and weak positive in the alcohol extract group and Forsythia suspensa decoction group, but weaker in the alcohol extract group. Forsythin group, Forsythia suspensa alcohol extract group, Forsythia suspensa decoction group three groups were weakly positive in cell, Forsythin group was the weakest, Forsythia suspensa alcohol extract group was the second. The cell culture supernatant and cell control group, liposome group, empty plasmid group and suspensin group were negative. 2. The expression of nuclear protein gene was (2.07 卤0.2) 脳 105copies 渭 l -1 in the Forsythione group and (61.2 卤13) 脳 105copies 渭 l -1 in the recombinant plasmid group. The expression of nuclear protein gene in Forsythione group was significantly lower than that in the recombinant plasmid group (t = 9.836, p0.05). Conclusion: 1. Phillyrin could down-regulate the nucleoprotein gene of influenza A virus and inhibit the expression of nucleoprotein of influenza A virus in HeLa cells. 2. Forsythia suspensa decoction and Forsythia suspensa alcohol extract could also inhibit the expression of influenza A virus nucleoprotein in HeLa cells, but both of them were weaker than forsythia glucoside, and Forsythia suspensa decoction was the weakest.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R285;R511.7

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