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  本文選題：A549細胞 + NS2�。� 參考：《安徽醫(yī)科大學》2017年碩士論文

【摘要】：目的探討呼吸道合胞病毒(Respiratory syncytial virus,RSV)非結構蛋白NS2在RSV感染人Ⅱ型肺泡上皮細胞(A549)過程中轉錄水平的變化情況,研究NS2在RSV感染A549細胞活化TLR7中的作用機理,信號轉導等,為防治呼吸道合胞病毒感染提供新的思路。方法以RSV感染體外培養(yǎng)的A549細胞,設立正常對照組、RSV感染組、RSV NS2小干擾RNA(NS2 si RNA)沉默組及TLR7激動劑(Resiquimod,R848)組。各組分別于病毒感染后的4h、12h、24h、48h收集細胞和培養(yǎng)上清液。(1)實時熒光定量PCR法(real-time PCR)檢測各組不同時間點RSV NS2,TLR7 m RNA表達量變化;(2)免疫印跡法(Western-blot)檢測各組不同時間點IFN-βToll樣受體相關區(qū)域連結蛋白(TIR domain-containing adapter inducing interferon IFN-β,TRIF),腫瘤壞死因子相關因子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)蛋白水平的表達變化;(3)酶聯(lián)免疫吸附法(ELISA)檢測各組細胞培養(yǎng)上清液中INF-α,INF-β含量的變化。結果(1)Real-time PCR結果顯示:RSV感染后TLR7 m RNA表達上升,在感染的48h達到了正常對照組的8倍,差異有統(tǒng)計學意義(P0.01);在R848+RSV組,TLR7 m RNA表達量顯著升高,在感染的48h達到了正常對照組的17.3倍,與RSV感染組相比,TLR7 m RNA表達量升高,感染24h表達量為RSV感染組的1.2倍,具有統(tǒng)計學意義(P0.01);在NS2 si RNA+RSV組,TLR7 m RNA表達量較正常對照組顯著上升,但較RSV感染組下降,感染的48h表達量為RSV感染組的70%,差異從12h開始具有統(tǒng)計學意義(P0.05)。RSV感染后NS2 m RNA表達上升,在R848+RSV組,NS2 m RNA表達量具有感染時間依賴性,與RSV感染組相比,NS2 m RNA表達量升高,感染24h時表達量為RSV感染組的1.1倍,差異無統(tǒng)計學意義(P0.01)。在NS2 si RNA+RSV組,NS2 m RNA表達量較RSV感染組下降,且從感染12h開始顯著下降,感染的24h僅為RSV感染組的38%,48h為39%,差異具有統(tǒng)計學意義(12h,P0.05;24h、48h,P0.01)。結果表明RSV感染能夠上調(diào)TLR7 m RNA的表達,在RSV感染A549細胞活化TLR7過程中,NS2發(fā)揮一定的促進作用。(2)Western-Blot結果顯示:正常對照組中,TRIF、TRAF6和p-IκB-α蛋白的表達量較低,經(jīng)RSV感染之后,隨感染時間增加,表達量增加,升高有顯著性差異(P0.01);在R848+RSV組,TRIF、TRAF6和p-IκB-α蛋白表達量較RSV感染組增加,差異有統(tǒng)計學意義(P0.05);在NS2 si RNA+RSV組,TRIF、TRAF6和p-IκB-α蛋白表達量較正常對照組上調(diào),但相對于RSV感染組,表達下降,差異有統(tǒng)計學意義(P0.05)。結果表明RSV NS2可以活化TRIF,TRAF6及p-IκB-α蛋白的表達。(3)ELISA法結果顯示:與正常組細胞培養(yǎng)上清相比,RSV感染組IFN-α,IFN-β含量升高,從4h開始升高有統(tǒng)計學意義(P0.01),隨著感染時間的延長而增加;在R848+RSV組,IFN-α,IFN-β含量亦隨感染時間的延長逐漸增加,感染4h升高有統(tǒng)計學意義(P0.05);在NS2 si RNA沉默組,IFN-α,IFN-β含量隨感染時間量逐漸增加,與RSV感染組相比,有所升高,且從感染4h開始,差異有顯著性(P0.01),說明在病毒感染過程中,NS2抑制了IFN-α,IFN-β的表達。結論NS2在RSV感染A549細胞活化TLR7過程中起到一定的促進作用,活化TRAF6、TRIF、p-IκB-α表達,導致下游I型干擾素IFN-α,IFN-β表達量下降。
[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.

【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R373

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本文編號：1765097