【作者】 张清；

【导师】 孙坚；

【作者基本信息】 南昌大学 ， 内科学， 2012， 硕士

【摘要】 目的：探讨黄芩及黄芩提取成分对甲型流感病毒核蛋白基因（NP基因）表达的影响，和对核蛋白（NP）表达的影响，为寻找新的抗流感病毒中药提供实验依据。方法：本实验首先采用细胞病变法（CPE）确定各种药物的实验浓度；然后将真核重组质粒pcDNA3.1（+）/NP通过脂质体介导瞬时转染到HeLa细胞中，将成功转染的HeLa细胞作为靶细胞，转染后48h，取细胞培养上清液，采用胶体金免疫层析试纸法测NP蛋白的表达；取转染后的细胞提取RNA，合成cDNA，采用荧光定量RT-PCR法测NP基因的起始核酸量。本实验设HeLa细胞组、空质粒转染组（转染pcDNA3.1（+））、脂质体组、重组质粒转染组（转染pcDNA3.1（+）/NP）、黄芩水煎剂组、黄芩总黄酮组、黄芩苷组和黄芩素组，共八组。结果：采用细胞病变法得出各种药物对HeLa细胞的无毒界限分别为：黄芩水煎剂：3906.25μg·ml^-1，黄芩总黄酮：0.9765625μg·ml^-1，黄芩苷：62.5μg·ml^-1，黄芩素：31.25μg·ml^-1；药物的实验浓度分别为：黄芩水煎剂：1953.125μg·ml^-1，黄芩总黄酮：0.48828125μg·ml^-1，黄芩苷：31.25μg·ml^-1，黄芩素：15.625μg·ml^-1。转染48h后，取细胞培养上清液，采用胶体金免疫层析试纸法测得NP蛋白表达：重组质粒转染组、黄芩总黄酮组、黄芩苷组、黄芩素组为阳性，细胞对照组、脂质体组、空质粒转染组和黄芩水煎剂组为阴性；从转染后的细胞中提取RNA，合成cDNA，并进行荧光定量RT-PCR测试，得到一条标准曲线：Y=-3.455X+48.119；根据标准曲线方程计算出各组的NP基因起始核酸量分别为：细胞对照组：（0.6613±0.1020）×10⁵copies·μl^-1，空质粒转染组：（0.2520±0.0946）×10⁵copies·μl^-1，脂质体组：（0.4750±0.1943）×10⁵copies·μl^-1，重组质粒转染组为（81.4500±29.6597）×10⁵copies·μl^-1，黄芩水煎剂组：（29.6250±7.6596）×10⁵copies·μl^-1，黄芩总黄酮组：（61.0250±25.3261）×10⁵copies·μl^-1，黄芩苷组：（27.7250±12.8484）×10⁵copies·μl^-1，黄芩素组：（31.8250±8.2152）×10⁵copies·μl^-1。采用t检验对数据进行统计学分析，重组质粒转染组与细胞对照组比较，t=5.448，P=0.012＜0.05，具有统计学意义；与空质粒转染组比较，t=5.475，P=0.012＜0.05，具有统计学意义；与脂质体组比较，t=5.460，P=0.012＜0.05，具有统计学意义；与黄芩水煎剂组比较，t=3.384，P=0.015＜0.05，具有统计学意义；与黄芩总黄酮组比较，t=1.047，P=0.335＞0.05，不具有统计学意义；与黄芩苷组比较，t=3.324，P=0.016＜0.05，具有统计学意义；与黄芩素组比较，t=3.225，P=0.018＜0.05，具有统计学意义。结论：黄芩水煎剂能够抑制甲型流感病毒NP基因和NP蛋白的表达。黄芩总黄酮对甲型流感病毒NP基因和NP蛋白的表达无影响。黄芩苷和黄芩素能够下调甲型流感病毒NP基因起始量。更多还原

【Abstract】 OBJECTIVE:To investigate the effect of Scutellaria baicalensis Georgei and its extracts against influenza A virus NP gene, and the effect of the expression of NP to provide experimental basis for the search of new antiviral drugs which can anti-influenza.METHODS:First, used cytopathic effect (CPE) method to determine the concentration of drugs subsequent experiments; then eukaryotic recombinant plasmid pcDNA3.1(+)/NP by fat-plasmid transfection method was transfected into HeLa cells.The successfully transfected HeLa cells as target cells,48hours after transfection, the supernatant was used to test the expression of NP measured by colloidal gold immunoassay chromatography;at the same time, RNA was extracted from cells after transient transfection, afterwards, synthetised cDNA, the starting amount of NP gene was tested by means of fluorescent quantitative RT-PCR. In our research, installed eight groups as follows:HeLa cell group, empty plasmid group (transfected pcDNA3.1(+)), liposome group, and eukaryotic recombinant plasmid group (transfected pcDNA3.1(+)/NP), skullcap water decoction group, total flavonoids group, baicalin group, baicalein group.RESULTS:Used cytopathic effect method to get the toxic boundaries of various drugs on HeLa cells, the skullcap water decoction:3906.25μg·ml-1, total flavonoids:0.976562525μg·ml-1, baicalin:62.525μg·ml-1, baicalein:31.2525μg·ml-1,respectively; so, the experimental concentrations of each drug was:the skullcap water decoction:1953.125μg·ml-1, total flavonoids:0.48828125μg·ml-1, baicalin:31.25μg·ml-1, baicalein:15.625μg·ml-1.48hours after transfection, the supernatant was used testing the expression of NP by colloidal gold immunoassay chromatography:the eukaryotic recombinant plasmid group、total flavonoids group、baicalin group and baicalein group was positive, to the contrary,the HeLa cell group、liposome group、empty plasmid group and skullcap water decoction group was negative. At the same time, we used the cDNA that synthesized from the total RNA which extracted from HeLa cells to measure the the starting amount of NP gene by means of fluorescent quantitative RT-PCR. We got a standard curve:Y=-3.455X+48.119. According to the formula, we calculated the starting amount of NP gene of each group:HeLa cell group:(0.6613±0.1020) X105copies·μl-1, empty plasmid group:(0.2520±0.0946) X105copies·μl-1, liposome group:(0.4750±0.1943)×105opies·μl-1, eukaryotic recombinant plasmid group:(81.4500±29.6597) X105copies·μl-1, skullcap water decoction group:(29.6250±7.6596) X105copies·μl-1, total flavonoids group:(61.0250±25.3261)×105copies·μl-1, baicalin group:(27.7250±12.8484) X105copies·μl-1, baicalein group:(31.8250±8.2152)×105copies·μl-1, respectively. Student t test was used for statistical analysis, in comparison with eukaryotic recombinant plasmid group, the HeLa cell group with statistically significant,t=5.448,P=0.012<0.05; statistically significant of empty plasmid group,t=5.475,P=0.012<0.05; liposome group also with statistically significant,t=5.460,P=0.012<0.05; statistically significant of skullcap water decoction group,t=3.384, P=0.015<0.05; without statistically significant of eukaryotic recombinant plasmid group,t=1.047, P=.335>0.05; statistically significant of baicalin group,t=3.324,P=0.016<0.05; baicalein group with statistically significant,t=3.225,P=0.018<0.05.CONCLUTIONS:The skullcap water decoction could inhibit the expression of influenza A virus NP and decrease the starting amount of influenza A virus NP gene; total flavonoids have no effect on the expression of influenza A virus NP and NP gene; baicalin and baicalein could decrease the starting amount of influenza A virus NP gene.更多还原