【摘要】：目的探討Toll樣受體2和4（TLR2和TLR4）在α-干擾素（IFN-α）抗病毒效應(yīng)中的作用以及抗病毒蛋白2',5'-OAS和PKR在體外對抗HBV活性的研究。 方法以人肝胚瘤細(xì)胞HepG2為細(xì)胞模型，通過LPS刺激并聯(lián)合IFN-α處理細(xì)胞，以RT-PCR法檢測細(xì)胞表面TLR2和TLR4的表達(dá)，再以Western blot和RT-PCR檢測HepG2細(xì)胞中IFN-α JAK-STAT途徑分子STAT1、STAT2和IRF9的表達(dá)，，進(jìn)一步分析IFN-α誘導(dǎo)的MxA、2',5'-OAS、PKR等抗病毒蛋白的表達(dá)。 以穩(wěn)定轉(zhuǎn)染HBV全基因組，并能分泌完整HBV病毒顆粒子的肝胚瘤細(xì)胞株HepG2.2.15細(xì)胞為細(xì)胞模型，將未處理的HepG2.2.15細(xì)胞作為空白組；將轉(zhuǎn)染質(zhì)粒pEGFP-2',5'-OAS的HepG2.2.15細(xì)胞作為2',5'-OAS組；將轉(zhuǎn)染質(zhì)粒pEGFP-PKR的HepG2.2.15細(xì)胞作為PKR組；將同時轉(zhuǎn)染質(zhì)粒pEGFP-2',5'-OAS和pEGFP-PKR的HepG2.2.15細(xì)胞的作為共轉(zhuǎn)染組。Western blot檢測細(xì)胞內(nèi)PKR和2',5'-OAS蛋白的表達(dá)，電化學(xué)發(fā)光方法檢測HepG2.2.15細(xì)胞分泌的HBsAg和HBeAg量；熒光定量PCR法檢測上清液中HBV DNA的量。 結(jié)果研究表明，HepG2細(xì)胞在LPS的刺激下，細(xì)胞表面TLR2、TLR4mRNA的表達(dá)水平升高（P＜0.05），而STAT1、STAT2、IRF9等相關(guān)信號轉(zhuǎn)導(dǎo)分子的表達(dá)卻無顯著變化；與IFN-α單獨(dú)處理相比，LPS和IFN-α聯(lián)合處理HepG2細(xì)胞，可使細(xì)胞內(nèi)JAK-STAT途徑信號分子STAT1、STAT2、IRF9及抗病毒蛋白2',5'-OAS、PKR表達(dá)增加（P＜0.05），而抗病毒蛋白MxA的表達(dá)水平無顯著變化（P＞0.05）。 HepG2.2.15細(xì)胞經(jīng)質(zhì)粒轉(zhuǎn)染能夠表達(dá)2',5'-OAS、PKR蛋白，且2',5'-OAS組和PKR組細(xì)胞上清液中HBsAg和HBeAg的量低于空白對照組（P＜0.05）。共轉(zhuǎn)染組上清液中HBsAg和HBeAg的量明顯低于空白對照組（P＜0.01）；不過，各組上清液中HBV DNA的表達(dá)無顯著性差異。 結(jié)論（1）HepG2細(xì)胞表面TLR2、TLR4被LPS刺激表達(dá)后，可影響細(xì)胞內(nèi)IFN-αJAK-STAT信號轉(zhuǎn)導(dǎo)途徑，提高抗病毒蛋白PKR和2',5'-OAS的表達(dá)，從而增強(qiáng)了IFN-α的抗病毒效應(yīng)。（2）在體外，抗病毒蛋白PKR和2',5'-OAS具有獨(dú)立的抗HBV活性，兩者在抗病毒效應(yīng)上具有協(xié)同作用。
[Abstract]:Objective to investigate the role of Toll like receptors 2 and 4 (TLR2 and TLR4) in the antiviral effect of interferon 偽 (IFN- 偽) and the antiviral activity of antiviral proteins 2, 5 and PKR against HBV in vitro. Methods Human hepatic embryoma cell line HepG2 was used as the cell model. The cells were stimulated by LPS and treated with IFN- 偽. The expression of TLR2 and TLR4 on the cell surface was detected by RT-PCR method. The IFN- 偽 JAK-STAT pathway molecule STAT1, was detected by Western blot and RT-PCR in HepG2 cells. The expression of STAT2 and IRF9, and the expression of MxA,2',5'-OAS,PKR and other antiviral proteins induced by IFN- 偽 were further analyzed. The hepatoembryonic tumor cell line HepG2.2.15 cell line which stably transfected the whole genome of HBV and secreted intact HBV virus particles was used as the cell model. The untreated HepG2.2.15 cells were taken as the blank group. The HepG2.2.15 cells transfected with plasmid pEGFP-2',5'-OAS and HepG2.2.15 cells transfected with plasmid pEGFP-PKR were used as group 2 and PKR respectively. HepG2.2.15 cells transfected with plasmids pEGFP-2',5'-OAS and pEGFP-PKR were used as cotransfected. Western blot to detect the expression of PKR and 2O5-OS-OAS protein. The amount of HBsAg and HBeAg secreted by HepG2.2.15 cells was detected by electrochemiluminescence (ECL). Fluorescence quantitative PCR method was used to determine the content of HBV DNA in supernatant. The results showed that the expression of TLR2,TLR4mRNA on the surface of HepG2 cells increased under the stimulation of LPS (P < 0. 05), but the expression of STAT1,STAT2,IRF9 and other related signal transduction molecules did not change significantly. Compared with IFN- 偽 alone, LPS combined with IFN- 偽 could increase the expression of JAK-STAT signaling molecule STAT1,STAT2,IRF9 and antiviral protein 2OAS-OAS-PKR in HepG2 cells (P < 0. 05). There was no significant change in the expression of antiviral protein MxA (P > 0. 05). HepG2.2.15 cells were transfected with plasmids and the expression levels of HBsAg and HBeAg in the supernatants of 2OAS-OAS-OAS and PKR groups were lower than those of the control group (P < 0. 05), and the expression of HBsAg and HBeAg in the supernatants of the two groups were significantly lower than those in the control group (P < 0. 05). The amount of HBsAg and HBeAg in the supernatant of the cotransfection group was significantly lower than that in the control group (P < 0. 01), but there was no significant difference in the expression of HBV DNA in the supernatant of each group. Conclusion (1) the expression of TLR2,TLR4 on the surface of HepG2 cells stimulated by LPS could affect the signal transduction pathway of IFN- 偽 JAK-STAT, and increase the expression of PKR and 2OS-OAS. Therefore, the antiviral effect of IFN- 偽 was enhanced. (2) in vitro, the antiviral protein PKR and the 2ACE-5OS-OAS had independent antiviral activities, and they had synergistic antiviral effects.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R512.62

【共引文獻(xiàn)】

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