没食子酸体外抗人四型腺病毒（HAdV-4）作用及机制研究

发布时间：2019-05-24 12:51

【摘要】：目的:探索没食子酸(Gallic acid,GA)对人四型腺病毒(Human adenovirus type four,HAdV-4)的体外抑制作用及其机制。方法:本研究采用了一系列实验方法,通过采用细胞培养,细胞病变效应(CPE,cytopathic effect)观察等实验来确定没食子酸的安全用药范围,采用SPSS软件probit回归求得最大无毒浓度(TC0),半数中毒浓度(TC50);TCID50法测定腺病毒的毒力;MTT细胞毒性实验测得没食子酸对腺病毒的体外抑制作用;实时荧光定量PCR技术来测定没食子酸对腺病毒基因拷贝数的影响;蛋白免疫印迹法(western blot)检测没食子酸对腺病毒蛋白外壳合成的抑制作用。结果:没食子酸对A549细胞的最大无毒浓度为TC0=32.75μM,半数中毒浓度TC50=279.906μM。采用 Reed-Munch 法计算 HAdV-4 在 A549 细胞中的 TCID50 为10-6.5/100μL。MTT法测得在四种加药方式对腺病毒的抑制率分别为:灭活作用(73.19%*)吸附抑制作用(59.68%*)生物合成抑制作用(13.06%*)对细胞的保护作用(3.15%#)((*P0.05),(#P0.05))。实时荧光定量PCR法和western blot法测得其对腺病毒的抑制作用与MTT法基本一致。结论:没食子酸抗人四型腺病毒作用主要在于其体外灭活病毒,从而影响病毒对细胞的吸附作用;其次对人四型腺病毒在生物合成阶段有一定抑制作用,包括可以抑制腺病毒的核酸和蛋白的合成,但其对保护细胞阻止腺病毒入侵无明显作用。
[Abstract]:Aim: to investigate the inhibitory effect of Gallic acid (Gallic acid,GA) on human adenoviruses (Human adenovirus type four,HAdV-4 in vitro and its mechanism. Methods: a series of experimental methods were used to determine the safe range of Gallic acid by cell culture and cytopathic effect (CPE,cytopathic effect). The maximum toxic concentration (TC0) and half toxic concentration (TC50) were obtained by probit regression with SPSS software. The toxicity of adenoviruses was determined by TCID50, the inhibitory effect of Gallic acid on adenoviruses in vitro was measured by MTT cytotoxicity test, and the effect of Gallic acid on the copy number of adenoviruses was determined by real-time fluorescence quantitative PCR. The inhibitory effect of Gallic acid on the synthesis of adenoviral protein coat was detected by (western blot). Results: the maximum non-toxic concentration of Gallic acid to A549 cells was TC0= 32.75 渭 M, and half of the toxic concentration TC50= 279.906 渭 M. The TCID50 of HAdV-4 in A549 cells was calculated by Reed-Munch assay to be 10 鈮，

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