绿原酸体外抑制肠道病毒71型的效应机制

发布时间：2019-03-26 17:50

【摘要】：目的：探讨绿原酸对肠道病毒71型（EV71）的抑制作用及分子机制。方法：（1）斑块形成试验检测不同浓度绿原酸对EV71感染的抑制作用；（2）噻唑蓝（MTT）法检测不同浓度绿原酸和利巴韦林对RD细胞的细胞毒作用；病毒斑块减少试验分析绿原酸对EV71吸附作用的影响；（3）时间过程试验检测绿原酸抑制效用与病毒生命周期的相关性；（4）绿原酸处理RD细胞后，采用RT-PCR和Western-Blotting法检测不同时间点EV71/VP1、2A、3C和3D mRNA及蛋白酶的表达。结果：（1）绿原酸在5、10和20μg/mL剂量时对EV71具有良好的抑制率，分别为83.4±3.0%、88.7±1.9%和94.7±1.8%，其半数抑制浓度（IC50）=45.7μg/mL。利巴韦林在20、40和80μg/mL剂量时对EV71具有良好的抑制率，，分别为84.5±1.1%、95.6±2.2%和85.4±4.6%，其IC50=195.2μg/mL；（2）绿原酸低于50μg/mL剂量处理时，细胞活率与空白对照组相比较并没有显著下降，其半数细胞毒性浓度（CC_50）=143.5μg/mL。而利巴韦林低于150μg/mL时对RD细胞的存活率亦无明显影响，其CC_50=1187.2μg/mL；（3）20μg/mL绿原酸与病毒同时加入或病毒吸附1h后加入，两种方法不同时间点细胞裂解液上清病毒滴度的降低差异无统计意义（P0.05）；（4）绿原酸在EV71感染RD细胞后0-8h加入，能显著抑制EV71复制；而在EV71感染后10-24h加入绿原酸，仅有部分或轻微抑制EV71复制效应；（5）EV71感染RD细胞后4h和8h绿原酸可阻碍EV71/2A mRNA表达，但未影响EV71/VP1、3C和3DmRNA表达；（6）绿原酸处理EV71感染RD细胞后4h和8h，未能检测出EV71/2A蛋白酶，但EV71/VP1、3C和3D蛋白表达未受影响。结论：（1）绿原酸具有良好的抑制EV71病毒复制效用；（2）绿原酸对EV71感染的抑制效应与阻止病毒吸附无关；（3）绿原酸主要通过干扰EV71复制早期2AmRNA表达和蛋白翻译而发挥抗病毒效应。
[Abstract]:Objective: to investigate the inhibitory effect and molecular mechanism of chlorogenic acid on enterovirus 71 (EV71). Methods: (1) plaque formation test was used to detect the inhibitory effect of different concentrations of chlorogenic acid on EV71 infection, (2) the cytotoxic effects of different concentrations of chlorogenic acid and ribavirin on RD cells were detected by thiazolyl blue (MTT) assay. The effect of chlorogenic acid on the adsorption of EV71 was analyzed by viral plaque reduction test. (3) the correlation between inhibition effect of chlorogenic acid and virus life cycle was detected by time process test. (4) after RD cells were treated with chlorogenic acid, the expression of EV71/VP1,2A,3C, 3D mRNA and protease were detected by RT-PCR and Western-Blotting at different time points. Results: (1) the inhibition rate of chlorogenic acid on EV71 was 83.4 卤3.0%, 88.7 卤1.9% and 94.7 卤1.8% at 5, 10 and 20 渭 g / mL, respectively. The half inhibitory concentration (IC50) of chlorogenic acid was 45.7 渭 g / mL.. Ribavirin had a good inhibitory effect on EV71 (84.5 卤1.1%, 95.6 卤2.2% and 85.4 卤4.6%) at the doses of 20,40 and 80 渭 g / mL, respectively. The IC50= of ribavirin was 195.2 渭 g / mL;. (2) when the concentration of chlorogenic acid was lower than 50 渭 g / mL, the cell viability did not decrease significantly compared with the control group, and its CC_50 was 143.5 渭 g / mL.. However, when ribavirin was lower than 150 渭 g / mL, there was no significant effect on the survival rate of RD cells, and its CC_50= 1187.2 渭 g / mL;. (3) when 20 渭 g / mL chlorogenic acid and virus were added at the same time or 1 hour after virus adsorption, there was no significant difference in virus titers between the two methods at different time points (P0.05). (4) the addition of chlorogenic acid at 0-8 h after EV71 infected RD cells could significantly inhibit the replication of EV71, while the addition of chlorogenic acid at 24 h after EV71 infection only partially or slightly inhibited the EV71 replication effect. (5) chlorogenic acid inhibited the expression of EV71/2A mRNA at 4h and 8h after EV71 infection in RD cells, but did not affect the expression of EV71/VP1,3C and 3DmRNA. (6) the expression of EV71/2A protease was not detected in RD cells treated with chlorogenic acid for 4 h and 8 h after EV71 infection, but the expression of EV71/VP1,3C and 3D protein was not affected. Conclusion: (1) chlorogenic acid has good inhibitory effect on EV71 virus replication, (2) the inhibitory effect of chlorogenic acid on EV71 infection has nothing to do with preventing virus adsorption. (3) chlorogenic acid plays an antiviral role by interfering with 2AmRNA expression and protein translation in the early stage of EV71 replication.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

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