弓形虫对DNA受体天然免疫通路的激活及机制研究

发布时间：2018-06-05 03:26

本文选题：弓形虫 + 天然免疫　；参考：《暨南大学》2014年硕士论文

【摘要】：研究目的：弓形虫能感染哺乳动物所有的有核细胞，全球约30%的人曾感染弓形虫。天然免疫是抵抗弓形虫感染的第一道防线，天然免疫细胞通过模式识别受体对弓形虫的病原体相关分子模式进行识别和活化，分泌细胞因子和趋化因子从而清除弓形虫感染；同时，弓形虫也分泌毒力因子蛋白抑制天然免疫信号通路从而进行免疫逃逸。弓形虫侵入宿主细胞后形成纳虫空泡，研究报道弓形虫的纳虫空泡可在IFN-γ的作用下破裂，在宿主细胞质中可检测到弓形虫的DNA。细胞质中出现的DNA是非常重要的危险信号，外源性的DNA或者自身DNA在胞质内的积累可触发强烈的天然免疫反应，诱导产生一系列的细胞因子，如IFN-β等。cGAS作为新发现的胞质DNA受体，通过激活STING(干扰素基因刺激分子)、TBK1（TANK结合激酶1）、IRF3（干扰素调节因子3）诱导Ⅰ干扰素产生。基于以上研究背景，本实验旨在研究弓形虫纳虫空泡破裂后，其DNA进入宿主细胞质中能否被DNA受体识别激活下游信号通路，诱导I型干扰素的产生以及对该通路的活化是否依赖于STING、cGAS和TBK1等关键基因，阐明弓形虫感染的胞内天然免疫机制，为理解胞内病原体的天然免疫机制提供新内容。研究方法： 1.采用人包皮成纤维细胞（HFF）培养刚地弓形虫ME49-PTG株，抽提弓形虫DNA。HEK293细胞转染弓形虫DNA，用荧光素酶报告基因技术检测IFN-β、ISRE、NF-κB、AP-1基因的启动子表达水平。 2.用稳定表达ISRE荧光素酶报告基因的2FTGH细胞测定I型干扰素的蛋白水平和生物活性。 3.采用非变性聚丙烯酰胺凝胶电泳和免疫印迹方法检测转录因子IRF-3二聚体形成和活化。 4.通过过表达Sting、cGAS分析Sting和cGAS基因在弓形虫DNA活化通路中的功能。 5.弓形虫ME49-PTG株速殖子感染HEK293细胞，用荧光素酶报告基因技术检测IFN-β、ISRE、NF-κB、AP-1基因的启动子表达水平。 6. Western Blot鉴定TBK1基因敲除（TBK1-/-）和野生型TBK1+/+的小鼠胚胎成纤维细胞（MEF）中TBK1蛋白表达，并以弓形虫速殖子感染TBK1-/-和TBK1+/+MEF细胞，q-PCR检测IL-6、IL-18、STING、ISG15、SAG1等细胞因子基因mRNA的转录变化，分析TBK1基因在弓形虫激活天然免疫中的作用。研究结果： 1. HEK293细胞中IFN-β、ISRE启动子的表达随着转染弓形虫DNA量的升高而升高。 2. I型干扰素生物活性实验显示：转染弓形虫DNA后，I型干扰素的生物活性显著性增强，说明弓形虫DNA可以诱导I型干扰素的产生，并具有生物活性。 3.为进一步揭示弓形虫DNA在感染宿主时的作用机制，我们检测了IFN-β上游信号通路中转录因子IRF-3的活化情况，结果证明弓形虫DNA可以使IRF-3发生二聚化，激活了IRF-3转录因子，说明弓形虫DNA能够通过IRF3激活IFN-β信号通路。 4.弓形虫DNA转染HEK293细胞，同时过表达cGAS和STING真核质粒，发现IFN-β启动子的表达量进一步升高，同时I型干扰素生物活性测定显示IFNs活性增强，揭示弓形虫DNA诱导I型干扰素的产生可能通过cGAS-STING信号通路。 5.弓形虫ME49-PTG株速殖子可以促进NF-κB启动子的表达，揭示弓形虫可能活化了NF-κB信号通路，而过表达cGAS-STING对NF-κB无影响，同时弓形虫ME49-PTG株速殖子可以轻微激活IFN-β，但对ISRE和AP-1影响不大。 6. Western Blot结果鉴定了在TBK1+/+MEF细胞中表达TBK1蛋白，而在TBK1基因敲除的MEF细胞（TBK1-/-）中TBK1蛋白不表达。用弓形虫速殖子感染TBK1+/+MEF细胞和TBK1-/-MEF细胞，，q-PCR结果显示，弓形虫内参基因表面抗原蛋白1（SAG1）mRNA转录水平随着时间的增加而升高，说明在小鼠胚胎成纤维细胞中弓形虫的感染和增殖情况；同时，弓形虫速殖子可以抑制IL-6、IL-18、ISG15、STING的mRNA转录。在TBK1-/-细胞感染弓形虫的结果中发现，TBK1基因敲除后，ISG15和IL-18的mRNA水平显著下降；IL-6的mRNA水平有所升高。结论： 1.弓形虫DNA能够激活IRF3，使其二聚化，诱导IFN-β的产生，进而促进ISRE的表达，该激活途径有可能通过cGAS-STING信号通路。 2.弓形虫速殖子可以促进NF-κB启动子表达，活化NF-κB信号通路。 3.弓形虫速殖子可以抑制IL-6、IL-18、ISG15、STING的mRNA转录。TBK1基因在弓形虫速殖子对IL-18、ISG15mRNA转录的抑制作用中起着一定的作用；弓形虫依赖于TBK1基因从而抑制IL-6mRNA的转录。
[Abstract]:The purpose of the study is:
Toxoplasma can infect all mammalian nucleated cells. About 30% of the world have been infected with Toxoplasma gondii. Natural immunity is the first line of defense against Toxoplasma infection. Natural immune cells recognize and activate toxoplasmosis related molecular patterns through pattern recognition receptors, secrete cytokine and chemokines to clear the bow. At the same time, Toxoplasma also secretes a toxic factor protein that inhibits the natural immune signaling pathway to escape. Toxoplasma invades the host cell to form a nematode vacuole. It is reported that the vacuoles of the Toxoplasma gondii can be broken under the action of IFN- gamma, and can be detected in the cytoplasm of the DNA. cytoplasm of Toxoplasma gondii in the cytoplasm of the host. DNA is a very important risk signal. The accumulation of exogenous DNA or its own DNA in the cytoplasm triggers a strong natural immune response and induces a series of cytokines, such as IFN- beta and.CGAS as a newly discovered cytoplasmic DNA receptor, by activating the STING (interferon gene stimulator), TBK1 (TANK binding kinase 1), IRF3 (interferon modulation). Based on the above background, the purpose of this experiment is to investigate whether the DNA enters the host cytoplasm and can be identified by the DNA receptor to activate the downstream signal pathway, and to induce the production of type I interferon and whether the activation of the pathway is dependent on the key genes of STING, cGAS and TBK1. The innate immunity mechanism of Toxoplasma gondii infection provides new contents for understanding the natural immune mechanism of intracellular pathogens.
Research methods:
1. the ME49-PTG strain of Toxoplasma gondii was cultured by human foreskin fibroblast (HFF), and the DNA.HEK293 cells of Toxoplasma gondii were transfected into Toxoplasma DNA, and the promoter expression level of IFN- beta, ISRE, NF- kappa B, and AP-1 gene was detected by luciferase reporter gene technique.
2. the protein level and biological activity of type I interferon were determined by 2FTGH cells expressing ISRE luciferase reporter gene.
3. non denaturing polyacrylamide gel electrophoresis and Western blotting were used to detect the formation and activation of transcription factor IRF-3 two polymer.
4. the function of Sting and cGAS genes in DNA activation pathway of Toxoplasma gondii was analyzed by over expression of Sting and cGAS.
5. Toxoplasma gondii ME49-PTG strain tachychite was infected with HEK293 cells. Luciferase reporter gene technology was used to detect the expression level of IFN- beta, ISRE, NF- kappa B and AP-1 gene promoter.
The expression of TBK1 protein in mouse embryonic fibroblasts (MEF) of TBK1 gene knockout (TBK1-/-) and wild type TBK1+/+ was identified by 6. Western Blot, and TBK1-/- and TBK1+/+MEF cells were infected with Toxoplasma gondii tachytachype. Q-PCR IL-6 was detected by q-PCR. The role of the epidemic.
The results of the study:
1. the expression of IFN- beta and ISRE promoter in HEK293 cells increased with the increase of DNA content in transfected Toxoplasma gondii.
The biological activity test of type 2. I interferon showed that after transfection of Toxoplasma DNA, the biological activity of type I interferon was significantly enhanced, indicating that Toxoplasma DNA could induce the production of I type interferon and have biological activity.
3. in order to further reveal the mechanism of Toxoplasma DNA in the infection of the host, we detected the activation of the transcription factor IRF-3 in the IFN- beta upstream signal pathway. The results show that the Toxoplasma DNA can cause the dimerization of IRF-3 and activates the IRF-3 transcription factor, indicating that the Toxoplasma DNA can activate the IFN- beta signaling pathway through IRF3.
4. Toxoplasma DNA transfected into HEK293 cells and expressed the true nuclear particles of cGAS and STING. It was found that the expression of IFN- beta promoter was further increased. Meanwhile, the bioactivity of I type interferon showed that the activity of IFNs was enhanced. The production of I type interferon induced by Toxoplasma DNA may pass through the cGAS-STING signaling pathway.
5. Toxoplasma ME49-PTG tachyonus could promote the expression of NF- kappa B promoter, suggesting that Toxoplasma may activate NF- kappa B signaling pathway, while overexpression cGAS-STING has no effect on NF- kappa B, while ME49-PTG strain tachyonus of Toxoplasma gondii can activate IFN- beta slightly, but it has little effect on ISRE and AP-1.
6. Western Blot results showed that TBK1 protein was expressed in TBK1+/+MEF cells, and TBK1 protein was not expressed in MEF cells (TBK1-/-) of TBK1 gene knockout. TBK1+/+MEF and TBK1-/-MEF cells were infected with Toxoplasma tachygonite. The q-PCR results showed that the transcription level of surface antigen protein 1 (SAG1) of Toxoplasma gondii increased with time. The increase, indicating the infection and proliferation of Toxoplasma gondii in mouse embryonic fibroblasts, and at the same time, the tachyonus of Toxoplasma can inhibit the mRNA transcription of IL-6, IL-18, ISG15 and STING. In the results of TBK1-/- cells infected with Toxoplasma gondii, the mRNA level of ISG15 and IL-18 decreased significantly after the TBK1 gene knockout, and IL-6 mRNA levels increased.
Conclusion:
1. the DNA of Toxoplasma gondii can activate IRF3, make it dimer, induce the production of IFN- beta, and then promote the expression of ISRE, which may be mediated by cGAS-STING signaling pathway.
2. Toxoplasma tachygoni can promote the expression of NF- kappa B promoter and activate the NF- kappa B signaling pathway.
3. Toxoplasma tachytachis can inhibit the mRNA transcriptional.TBK1 gene of IL-6, IL-18, ISG15 and STING, which plays a role in the inhibition of the transcription of IL-18 and ISG15mRNA by Toxoplasma tachygonite, and Toxoplasma relies on the TBK1 gene to inhibit the transcription of IL-6mRNA.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392

【参考文献】