本文選題：弓形蟲 + 天然免疫��； 參考：《暨南大學》2014年碩士論文

【摘要】：研究目的： 弓形蟲能感染哺乳動物所有的有核細胞，全球約30%的人曾感染弓形蟲。天然免疫是抵抗弓形蟲感染的第一道防線，天然免疫細胞通過模式識別受體對弓形蟲的病原體相關分子模式進行識別和活化，分泌細胞因子和趨化因子從而清除弓形蟲感染；同時，弓形蟲也分泌毒力因子蛋白抑制天然免疫信號通路從而進行免疫逃逸。弓形蟲侵入宿主細胞后形成納蟲空泡，研究報道弓形蟲的納蟲空泡可在IFN-γ的作用下破裂，在宿主細胞質(zhì)中可檢測到弓形蟲的DNA。細胞質(zhì)中出現(xiàn)的DNA是非常重要的危險信號，外源性的DNA或者自身DNA在胞質(zhì)內(nèi)的積累可觸發(fā)強烈的天然免疫反應，誘導產(chǎn)生一系列的細胞因子，如IFN-β等。cGAS作為新發(fā)現(xiàn)的胞質(zhì)DNA受體，通過激活STING(干擾素基因刺激分子)、TBK1（TANK結合激酶1）、IRF3（干擾素調(diào)節(jié)因子3）誘導Ⅰ干擾素產(chǎn)生。基于以上研究背景，本實驗旨在研究弓形蟲納蟲空泡破裂后，其DNA進入宿主細胞質(zhì)中能否被DNA受體識別激活下游信號通路，誘導I型干擾素的產(chǎn)生以及對該通路的活化是否依賴于STING、cGAS和TBK1等關鍵基因，闡明弓形蟲感染的胞內(nèi)天然免疫機制，為理解胞內(nèi)病原體的天然免疫機制提供新內(nèi)容。 研究方法： 1.采用人包皮成纖維細胞（HFF）培養(yǎng)剛地弓形蟲ME49-PTG株，抽提弓形蟲DNA。HEK293細胞轉(zhuǎn)染弓形蟲DNA，用熒光素酶報告基因技術檢測IFN-β、ISRE、NF-κB、AP-1基因的啟動子表達水平。 2.用穩(wěn)定表達ISRE熒光素酶報告基因的2FTGH細胞測定I型干擾素的蛋白水平和生物活性。 3.采用非變性聚丙烯酰胺凝膠電泳和免疫印跡方法檢測轉(zhuǎn)錄因子IRF-3二聚體形成和活化。 4.通過過表達Sting、cGAS分析Sting和cGAS基因在弓形蟲DNA活化通路中的功能。 5.弓形蟲ME49-PTG株速殖子感染HEK293細胞，用熒光素酶報告基因技術檢測IFN-β、ISRE、NF-κB、AP-1基因的啟動子表達水平。 6. Western Blot鑒定TBK1基因敲除（TBK1-/-）和野生型TBK1+/+的小鼠胚胎成纖維細胞（MEF）中TBK1蛋白表達，并以弓形蟲速殖子感染TBK1-/-和TBK1+/+MEF細胞，q-PCR檢測IL-6、IL-18、STING、ISG15、SAG1等細胞因子基因mRNA的轉(zhuǎn)錄變化，分析TBK1基因在弓形蟲激活天然免疫中的作用。 研究結果： 1. HEK293細胞中IFN-β、ISRE啟動子的表達隨著轉(zhuǎn)染弓形蟲DNA量的升高而升高。 2. I型干擾素生物活性實驗顯示：轉(zhuǎn)染弓形蟲DNA后，I型干擾素的生物活性顯著性增強，說明弓形蟲DNA可以誘導I型干擾素的產(chǎn)生，并具有生物活性。 3.為進一步揭示弓形蟲DNA在感染宿主時的作用機制，我們檢測了IFN-β上游信號通路中轉(zhuǎn)錄因子IRF-3的活化情況，結果證明弓形蟲DNA可以使IRF-3發(fā)生二聚化，激活了IRF-3轉(zhuǎn)錄因子，說明弓形蟲DNA能夠通過IRF3激活IFN-β信號通路。 4.弓形蟲DNA轉(zhuǎn)染HEK293細胞，同時過表達cGAS和STING真核質(zhì)粒，發(fā)現(xiàn)IFN-β啟動子的表達量進一步升高，同時I型干擾素生物活性測定顯示IFNs活性增強，揭示弓形蟲DNA誘導I型干擾素的產(chǎn)生可能通過cGAS-STING信號通路。 5.弓形蟲ME49-PTG株速殖子可以促進NF-κB啟動子的表達，揭示弓形蟲可能活化了NF-κB信號通路，而過表達cGAS-STING對NF-κB無影響，同時弓形蟲ME49-PTG株速殖子可以輕微激活IFN-β，但對ISRE和AP-1影響不大。 6. Western Blot結果鑒定了在TBK1+/+MEF細胞中表達TBK1蛋白，而在TBK1基因敲除的MEF細胞（TBK1-/-）中TBK1蛋白不表達。用弓形蟲速殖子感染TBK1+/+MEF細胞和TBK1-/-MEF細胞，，q-PCR結果顯示，弓形蟲內(nèi)參基因表面抗原蛋白1（SAG1）mRNA轉(zhuǎn)錄水平隨著時間的增加而升高，說明在小鼠胚胎成纖維細胞中弓形蟲的感染和增殖情況；同時，弓形蟲速殖子可以抑制IL-6、IL-18、ISG15、STING的mRNA轉(zhuǎn)錄。在TBK1-/-細胞感染弓形蟲的結果中發(fā)現(xiàn)，TBK1基因敲除后，ISG15和IL-18的mRNA水平顯著下降；IL-6的mRNA水平有所升高。 結論： 1.弓形蟲DNA能夠激活IRF3，使其二聚化，誘導IFN-β的產(chǎn)生，進而促進ISRE的表達，該激活途徑有可能通過cGAS-STING信號通路。 2.弓形蟲速殖子可以促進NF-κB啟動子表達，活化NF-κB信號通路。 3.弓形蟲速殖子可以抑制IL-6、IL-18、ISG15、STING的mRNA轉(zhuǎn)錄。TBK1基因在弓形蟲速殖子對IL-18、ISG15mRNA轉(zhuǎn)錄的抑制作用中起著一定的作用；弓形蟲依賴于TBK1基因從而抑制IL-6mRNA的轉(zhuǎn)錄。
[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.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R392

【參考文獻】

相關期刊論文 前1條

1 馬海濤;;全球創(chuàng)新型城市建設的模式提煉[J];科學;2013年04期

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