【摘要】：背景： 流感可引起季節(jié)性流行和大流行，嚴(yán)重危害人民健康。目前抗流感病毒西藥因毒副作用較大或耐藥性的增加，限制了其應(yīng)用。祖國(guó)傳統(tǒng)中醫(yī)藥在預(yù)防與治療流感的臨床和基礎(chǔ)理論方面的研究，受到國(guó)內(nèi)外的廣泛關(guān)注，尋找新的抗流感中藥及探索其作用機(jī)制，有助于將中藥推向世界和造福人類。 目的： 探討連翹提取物(連翹苷、連翹醇提液、連翹水煎劑)對(duì)甲型流感病毒核蛋白基因體外干預(yù)的影響，為連翹抗流感病毒提供科學(xué)的理論依據(jù)。方法： 1.實(shí)驗(yàn)分組： HeLa細(xì)胞組（HeLa細(xì)胞)、空質(zhì)粒組（空質(zhì)粒和HeLa細(xì)胞）、脂質(zhì)體組（HeLa細(xì)胞和脂質(zhì)體）、重組質(zhì)粒組（重組質(zhì)�；蜣D(zhuǎn)染HeLa細(xì)胞）、連翹苷組（連翹苷加入重組質(zhì)粒組中）、連翹水煎劑組（連翹水煎劑加入重組質(zhì)粒組中）、連翹醇提液組（連翹醇提液加入重組質(zhì)粒組中）七組。 2.細(xì)胞病變法確定連翹提取物最大無(wú)毒界限及實(shí)驗(yàn)用濃度。 3.脂質(zhì)體瞬時(shí)轉(zhuǎn)染法轉(zhuǎn)染HeLa細(xì)胞，并以其作為實(shí)驗(yàn)用靶細(xì)胞。 4.膠體金免疫層析試紙法檢測(cè)各實(shí)驗(yàn)組細(xì)胞培養(yǎng)上清和細(xì)胞內(nèi)核蛋白的表達(dá)。 5.提取膠體金檢測(cè)陰性組及重組質(zhì)粒組細(xì)胞總RNA，合成cDNA。 6.熒光定量逆轉(zhuǎn)錄PCR檢測(cè)重組質(zhì)粒組及膠體金檢測(cè)陰性組的核蛋白基因拷貝數(shù)，用統(tǒng)計(jì)學(xué)軟件進(jìn)行分析。 結(jié)果： 1.重組質(zhì)粒組細(xì)胞培養(yǎng)上清及細(xì)胞內(nèi)膠體金法檢測(cè)均為陽(yáng)性；連翹醇提液組、連翹水煎劑組兩組細(xì)胞培養(yǎng)上清為弱陽(yáng)性，醇提液組更弱些；連翹苷組、連翹醇提液組、連翹水煎劑組三組細(xì)胞內(nèi)為弱陽(yáng)性，連翹苷組最弱，連翹醇提液組次之；細(xì)胞對(duì)照組、脂質(zhì)體組、空質(zhì)粒組和連翹苷組四組細(xì)胞培養(yǎng)上清及細(xì)胞對(duì)照組、脂質(zhì)體組、空質(zhì)粒組三組細(xì)胞內(nèi)均為陰性。 2.連翹苷組胞內(nèi)核蛋白基因表達(dá)量為(2.07±0.2)×105copies·μl-1，重組質(zhì)粒組胞內(nèi)核蛋白基因表達(dá)量為(61.2±13)×105copies·μl-1，連翹苷組胞內(nèi)核蛋白基因表達(dá)量低于重組質(zhì)粒組胞內(nèi)，差異有統(tǒng)計(jì)學(xué)意義(t=9.836，，p0.05)。 結(jié)論： 1.連翹苷可以下調(diào)瞬時(shí)轉(zhuǎn)染HeLa細(xì)胞的甲型流感病毒核蛋白基因量，并能較強(qiáng)的抑制其核蛋白的表達(dá)。 2.連翹水煎劑及連翹醇提液也能抑制瞬時(shí)轉(zhuǎn)染HeLa細(xì)胞的甲型流感病毒核蛋白的表達(dá)，但均較連翹苷弱，連翹水煎劑最弱。
[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 鹵0.2) 脳 105copies 渭 l -1 in the Forsythione group and (61.2 鹵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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R285;R511.7

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