Toll样受体2和4增强IFN-α抗病毒效应机制的初步研究
发布时间：2018-11-10 18:15
【摘要】：目的探讨Toll样受体2和4（TLR2和TLR4）在α-干扰素（IFN-α）抗病毒效应中的作用以及抗病毒蛋白2',5'-OAS和PKR在体外对抗HBV活性的研究。 方法以人肝胚瘤细胞HepG2为细胞模型，通过LPS刺激并联合IFN-α处理细胞，以RT-PCR法检测细胞表面TLR2和TLR4的表达，再以Western blot和RT-PCR检测HepG2细胞中IFN-α JAK-STAT途径分子STAT1、STAT2和IRF9的表达，，进一步分析IFN-α诱导的MxA、2',5'-OAS、PKR等抗病毒蛋白的表达。 以稳定转染HBV全基因组，并能分泌完整HBV病毒颗粒子的肝胚瘤细胞株HepG2.2.15细胞为细胞模型，将未处理的HepG2.2.15细胞作为空白组；将转染质粒pEGFP-2',5'-OAS的HepG2.2.15细胞作为2',5'-OAS组；将转染质粒pEGFP-PKR的HepG2.2.15细胞作为PKR组；将同时转染质粒pEGFP-2',5'-OAS和pEGFP-PKR的HepG2.2.15细胞的作为共转染组。Western blot检测细胞内PKR和2',5'-OAS蛋白的表达，电化学发光方法检测HepG2.2.15细胞分泌的HBsAg和HBeAg量；荧光定量PCR法检测上清液中HBV DNA的量。 结果研究表明，HepG2细胞在LPS的刺激下，细胞表面TLR2、TLR4mRNA的表达水平升高（P＜0.05），而STAT1、STAT2、IRF9等相关信号转导分子的表达却无显著变化；与IFN-α单独处理相比，LPS和IFN-α联合处理HepG2细胞，可使细胞内JAK-STAT途径信号分子STAT1、STAT2、IRF9及抗病毒蛋白2',5'-OAS、PKR表达增加（P＜0.05），而抗病毒蛋白MxA的表达水平无显著变化（P＞0.05）。 HepG2.2.15细胞经质粒转染能够表达2',5'-OAS、PKR蛋白，且2',5'-OAS组和PKR组细胞上清液中HBsAg和HBeAg的量低于空白对照组（P＜0.05）。共转染组上清液中HBsAg和HBeAg的量明显低于空白对照组（P＜0.01）；不过，各组上清液中HBV DNA的表达无显著性差异。 结论（1）HepG2细胞表面TLR2、TLR4被LPS刺激表达后，可影响细胞内IFN-αJAK-STAT信号转导途径，提高抗病毒蛋白PKR和2',5'-OAS的表达，从而增强了IFN-α的抗病毒效应。（2）在体外，抗病毒蛋白PKR和2',5'-OAS具有独立的抗HBV活性，两者在抗病毒效应上具有协同作用。
[Abstract]:Objective to investigate the role of Toll like receptors 2 and 4 (TLR2 and TLR4) in the antiviral effect of interferon 伪 (IFN- 伪) 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- 伪. The expression of TLR2 and TLR4 on the cell surface was detected by RT-PCR method. The IFN- 伪 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- 伪 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- 伪 alone, LPS combined with IFN- 伪 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- 伪 JAK-STAT, and increase the expression of PKR and 2OS-OAS. Therefore, the antiviral effect of IFN- 伪 was enhanced. (2) in vitro, the antiviral protein PKR and the 2ACE-5OS-OAS had independent antiviral activities, and they had synergistic antiviral effects.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62
[Abstract]:Objective to investigate the role of Toll like receptors 2 and 4 (TLR2 and TLR4) in the antiviral effect of interferon 伪 (IFN- 伪) 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- 伪. The expression of TLR2 and TLR4 on the cell surface was detected by RT-PCR method. The IFN- 伪 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- 伪 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- 伪 alone, LPS combined with IFN- 伪 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- 伪 JAK-STAT, and increase the expression of PKR and 2OS-OAS. Therefore, the antiviral effect of IFN- 伪 was enhanced. (2) in vitro, the antiviral protein PKR and the 2ACE-5OS-OAS had independent antiviral activities, and they had synergistic antiviral effects.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62
【共引文献】
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