【摘要】：目的:探索沒食子酸(Gallic acid,GA)對(duì)人四型腺病毒(Human adenovirus type four,HAdV-4)的體外抑制作用及其機(jī)制。方法:本研究采用了一系列實(shí)驗(yàn)方法,通過采用細(xì)胞培養(yǎng),細(xì)胞病變效應(yīng)(CPE,cytopathic effect)觀察等實(shí)驗(yàn)來確定沒食子酸的安全用藥范圍,采用SPSS軟件probit回歸求得最大無毒濃度(TC0),半數(shù)中毒濃度(TC50);TCID50法測(cè)定腺病毒的毒力;MTT細(xì)胞毒性實(shí)驗(yàn)測(cè)得沒食子酸對(duì)腺病毒的體外抑制作用;實(shí)時(shí)熒光定量PCR技術(shù)來測(cè)定沒食子酸對(duì)腺病毒基因拷貝數(shù)的影響;蛋白免疫印跡法(western blot)檢測(cè)沒食子酸對(duì)腺病毒蛋白外殼合成的抑制作用。結(jié)果:沒食子酸對(duì)A549細(xì)胞的最大無毒濃度為TC0=32.75μM,半數(shù)中毒濃度TC50=279.906μM。采用 Reed-Munch 法計(jì)算 HAdV-4 在 A549 細(xì)胞中的 TCID50 為10-6.5/100μL。MTT法測(cè)得在四種加藥方式對(duì)腺病毒的抑制率分別為:滅活作用(73.19%*)吸附抑制作用(59.68%*)生物合成抑制作用(13.06%*)對(duì)細(xì)胞的保護(hù)作用(3.15%#)((*P0.05),(#P0.05))。實(shí)時(shí)熒光定量PCR法和western blot法測(cè)得其對(duì)腺病毒的抑制作用與MTT法基本一致。結(jié)論:沒食子酸抗人四型腺病毒作用主要在于其體外滅活病毒,從而影響病毒對(duì)細(xì)胞的吸附作用;其次對(duì)人四型腺病毒在生物合成階段有一定抑制作用,包括可以抑制腺病毒的核酸和蛋白的合成,但其對(duì)保護(hù)細(xì)胞阻止腺病毒入侵無明顯作用。
[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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