【摘要】：目的：探討綠原酸對腸道病毒71型（EV71）的抑制作用及分子機制。 方法：（1）斑塊形成試驗檢測不同濃度綠原酸對EV71感染的抑制作用；（2）噻唑藍（MTT）法檢測不同濃度綠原酸和利巴韋林對RD細胞的細胞毒作用；病毒斑塊減少試驗分析綠原酸對EV71吸附作用的影響；（3）時間過程試驗檢測綠原酸抑制效用與病毒生命周期的相關性；（4）綠原酸處理RD細胞后，采用RT-PCR和Western-Blotting法檢測不同時間點EV71/VP1、2A、3C和3D mRNA及蛋白酶的表達。 結果：（1）綠原酸在5、10和20μg/mL劑量時對EV71具有良好的抑制率，分別為83.4±3.0%、88.7±1.9%和94.7±1.8%，其半數(shù)抑制濃度（IC50）=45.7μg/mL。利巴韋林在20、40和80μg/mL劑量時對EV71具有良好的抑制率，，分別為84.5±1.1%、95.6±2.2%和85.4±4.6%，其IC50=195.2μg/mL；（2）綠原酸低于50μg/mL劑量處理時，細胞活率與空白對照組相比較并沒有顯著下降，其半數(shù)細胞毒性濃度（CC_50）=143.5μg/mL。而利巴韋林低于150μg/mL時對RD細胞的存活率亦無明顯影響，其CC_50=1187.2μg/mL；（3）20μg/mL綠原酸與病毒同時加入或病毒吸附1h后加入，兩種方法不同時間點細胞裂解液上清病毒滴度的降低差異無統(tǒng)計意義（P0.05）；（4）綠原酸在EV71感染RD細胞后0-8h加入，能顯著抑制EV71復制；而在EV71感染后10-24h加入綠原酸，僅有部分或輕微抑制EV71復制效應；（5）EV71感染RD細胞后4h和8h綠原酸可阻礙EV71/2A mRNA表達，但未影響EV71/VP1、3C和3DmRNA表達；（6）綠原酸處理EV71感染RD細胞后4h和8h，未能檢測出EV71/2A蛋白酶，但EV71/VP1、3C和3D蛋白表達未受影響。 結論：（1）綠原酸具有良好的抑制EV71病毒復制效用；（2）綠原酸對EV71感染的抑制效應與阻止病毒吸附無關；（3）綠原酸主要通過干擾EV71復制早期2AmRNA表達和蛋白翻譯而發(fā)揮抗病毒效應。
[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 鹵3.0%, 88.7 鹵1.9% and 94.7 鹵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 鹵1.1%, 95.6 鹵2.2% and 85.4 鹵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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R392

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