NS2在呼吸道合胞病毒感染A549细胞活化TLR7中的作用研究

发布时间：2018-04-17 20:12

本文选题：A549细胞 + NS2　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的探讨呼吸道合胞病毒(Respiratory syncytial virus,RSV)非结构蛋白NS2在RSV感染人Ⅱ型肺泡上皮细胞(A549)过程中转录水平的变化情况,研究NS2在RSV感染A549细胞活化TLR7中的作用机理,信号转导等,为防治呼吸道合胞病毒感染提供新的思路。方法以RSV感染体外培养的A549细胞,设立正常对照组、RSV感染组、RSV NS2小干扰RNA(NS2 si RNA)沉默组及TLR7激动剂(Resiquimod,R848)组。各组分别于病毒感染后的4h、12h、24h、48h收集细胞和培养上清液。(1)实时荧光定量PCR法(real-time PCR)检测各组不同时间点RSV NS2,TLR7 m RNA表达量变化;(2)免疫印迹法(Western-blot)检测各组不同时间点IFN-βToll样受体相关区域连结蛋白(TIR domain-containing adapter inducing interferon IFN-β,TRIF),肿瘤坏死因子相关因子6(TNF receptor associated factor 6,TRAF6)及磷酸化NF-κB/P65抑制蛋白(Phosphorylated inhibitor of nuclear factor kappa B kinase,p-IκB-α)蛋白水平的表达变化;(3)酶联免疫吸附法(ELISA)检测各组细胞培养上清液中INF-α,INF-β含量的变化。结果(1)Real-time PCR结果显示:RSV感染后TLR7 m RNA表达上升,在感染的48h达到了正常对照组的8倍,差异有统计学意义(P0.01);在R848+RSV组,TLR7 m RNA表达量显著升高,在感染的48h达到了正常对照组的17.3倍,与RSV感染组相比,TLR7 m RNA表达量升高,感染24h表达量为RSV感染组的1.2倍,具有统计学意义(P0.01);在NS2 si RNA+RSV组,TLR7 m RNA表达量较正常对照组显著上升,但较RSV感染组下降,感染的48h表达量为RSV感染组的70%,差异从12h开始具有统计学意义(P0.05)。RSV感染后NS2 m RNA表达上升,在R848+RSV组,NS2 m RNA表达量具有感染时间依赖性,与RSV感染组相比,NS2 m RNA表达量升高,感染24h时表达量为RSV感染组的1.1倍,差异无统计学意义(P0.01)。在NS2 si RNA+RSV组,NS2 m RNA表达量较RSV感染组下降,且从感染12h开始显著下降,感染的24h仅为RSV感染组的38%,48h为39%,差异具有统计学意义(12h,P0.05;24h、48h,P0.01)。结果表明RSV感染能够上调TLR7 m RNA的表达,在RSV感染A549细胞活化TLR7过程中,NS2发挥一定的促进作用。(2)Western-Blot结果显示:正常对照组中,TRIF、TRAF6和p-IκB-α蛋白的表达量较低,经RSV感染之后,随感染时间增加,表达量增加,升高有显著性差异(P0.01);在R848+RSV组,TRIF、TRAF6和p-IκB-α蛋白表达量较RSV感染组增加,差异有统计学意义(P0.05);在NS2 si RNA+RSV组,TRIF、TRAF6和p-IκB-α蛋白表达量较正常对照组上调,但相对于RSV感染组,表达下降,差异有统计学意义(P0.05)。结果表明RSV NS2可以活化TRIF,TRAF6及p-IκB-α蛋白的表达。(3)ELISA法结果显示:与正常组细胞培养上清相比,RSV感染组IFN-α,IFN-β含量升高,从4h开始升高有统计学意义(P0.01),随着感染时间的延长而增加;在R848+RSV组,IFN-α,IFN-β含量亦随感染时间的延长逐渐增加,感染4h升高有统计学意义(P0.05);在NS2 si RNA沉默组,IFN-α,IFN-β含量随感染时间量逐渐增加,与RSV感染组相比,有所升高,且从感染4h开始,差异有显著性(P0.01),说明在病毒感染过程中,NS2抑制了IFN-α,IFN-β的表达。结论NS2在RSV感染A549细胞活化TLR7过程中起到一定的促进作用,活化TRAF6、TRIF、p-IκB-α表达,导致下游I型干扰素IFN-α,IFN-β表达量下降。
[Abstract]:Objective to investigate the respiratory syncytial virus (Respiratory syncytial, virus, RSV) NS2 non structural protein in RSV infected human type II alveolar epithelial cells (A549) changes in gene expression in the process of the research of NS2 in RSV infection mechanism of activation in TLR7 A549 cells, signal transduction, for the prevention and treatment of respiratory syncytial virus infection and provide a new the idea of RSV infection. Methods A549 cells cultured in vitro, a normal control group, RSV infection group, RSV NS2 (NS2 Si RNA small interfering RNA silencing) group and TLR7 agonists (Resiquimod, R848) group. Rats in each group were infected after 4h, 12h, 24h, 48h cells were collected and cultured the supernatant. (1) by real-time quantitative PCR (real-time PCR) were detected at different time points RSV NS2, TLR7 m RNA expression; (2) Western blot (Western-blot) were detected at different time points of IFN- beta Toll like receptor related protein (TIR domain-c link area Ontaining adapter inducing interferon IFN- beta, TRIF), tumor necrosis factor related factor 6 (TNF receptor associated factor 6, TRAF6) and phosphorylated NF- kappa B/P65 inhibitory protein (Phosphorylated inhibitor of nuclear factor kappa B kinase, p-I kappa B- alpha) protein expression level changes; (3) ELISA (ELISA) was detected by cell culture supernatant of INF- alpha, INF- beta content changes. Results (1) Real-time PCR showed that RSV TLR7 m RNA expression increased after infection, infection in 48h reached 8 times the normal control group, the difference was statistically significant (P0.01); in the R848+RSV group, the expression of TLR7 m RNA the amount increased significantly in infected 48h reached 17.3 times compared with the normal control group, RSV infection group, TLR7 m expression of RNA increased, the expression level was 1.2 times the RSV infection group, 24h infection, with statistical significance (P0.01); NS2 Si RNA+RSV TLR7 m RNA group. As compared with the normal control group increased significantly, but compared with RSV infection group decreased, the expression of 48h infection RSV infection group 70%, the difference was statistically significant (P0.05) from 12h NS2 m after.RSV infection, the expression of RNA increased in the R848+RSV group, NS2 m expression of RNA infection with time dependence, compared NS2 m and RSV infection group, RNA expression increased, 24h expression of infection was 1.1 times of the RSV infected group, the difference was not statistically significant (P0.01). In NS2 Si RNA+RSV group, m RNA expression of NS2 was decreased and RSV infection group, 12h infection from 24h infection began to decline significantly, only RSV the infection group was 38%, 48h was 39%, the difference was statistically significant (12h, P0.05; 24h, 48h, P0.01). The results showed that RSV infection can increase the expression of TLR7 m RNA, in RSV infected A549 cell activation in the TLR7 process, NS2 plays a certain role in promoting. (2) Western-Blot showed: TRIF normal control in the group, TRAF The expression of p-I 6 and kappa B- alpha protein is lower by RSV after infection, with the infection time increased, the increase in expression increased significantly (P0.01); TRIF in the R848+RSV group, the expression of TRAF6 and p-I kappa B- alpha protein increased compared with RSV infection group, the difference was statistically significant (P0.05) TRIF NS2 Si; in RNA+RSV group, the expression of TRAF6 and p-I kappa B- alpha protein compared with normal control group increased, but compared with RSV infection group, the expression decreased, the difference was statistically significant (P0.05). The results show that RSV NS2 can activate TRIF, TRAF6 and the expression of p-I kappa B- alpha protein (3) by ELISA. The results showed that: compared with normal group cell culture supernatant, RSV infection group, IFN- alpha, IFN- beta content increased, starting from 4H was significantly higher (P0.01), which increases with the duration of infection; in group R848+RSV, IFN- alpha, IFN- beta content with infection time gradually increased, with statistical significance 4H (P0.05) infection; In the NS2 Si RNA silencing group, IFN- alpha, IFN- beta content gradually increased with the infection time, compared with RSV infection group and 4H infection increased, from the beginning, there was a significant difference (P0.01), that in the course of viral infection, NS2 inhibited the expression of IFN- alpha, IFN- beta NS2 in conclusion. RSV infected A549 cell activation in the process of TLR7 play a role in promoting the activation of TRAF6, TRIF, expression of p-I kappa B- alpha, resulting in downstream of type I interferon IFN- alpha, IFN- beta expression was decreased.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373

【参考文献】