表皮生长因子受体在猪链球菌2型致脑膜炎过程中的作用及机制研究

发布时间：2018-05-27 17:12

本文选题：猪链球菌 + 脑微血管内皮细胞　；参考：《华中农业大学》2017年硕士论文

【摘要】：猪链球菌二型(Streptococcus suis serotype 2)是一种重要的人兽共患病病原菌,对人或者动物均具有高致病率并导致高死亡率。脑膜炎是SS2感染导致的重要临床症状,但是SS2感染致脑膜炎特有的分子机制仍然有待研究。本研究主要探究EGFR在SS2菌株SC19致脑膜炎的过程中,介导神经炎症反应以及破坏细胞骨架的作用。主要结果如下:1.SS2菌株SC19介导严重的神经炎症反应SS2菌株SC19感染h BMEC,随着互作时间的延长,SC19黏附h BMEC的菌量显著上升。SC19感染CD1小鼠,发现出现脑膜增厚、出血,脑组织出现炎性细胞浸润以及噬神经元的病理变化。CD1小鼠在感染SC19后,多种细胞因子和趋化因子发生显著上调。同时,SC19感染h BMEC后炎性因子的转录也均有显著上调。2.SC19刺激h BMEC导致胞内蛋白酪氨酸磷酸化随后采用SC19感染h BMEC,检测细胞内蛋白分子的酪氨酸磷酸化,发现在180,100,和60k Da的位置均有蛋白发生明显的酪氨酸磷酸化。并证实180k Da大小的蛋白为EGFR。3.SC19感染介导配体依赖性的EGFR激活与二聚体化SC19感染h BMEC后,检测Erb B家族(EGFR/Erb B1、Erb B2、Erb B3和Erb B4)蛋白转录水平和翻译水平,发现EGFR、Erb B2和Erb B4的转录水平不变,而Erb B3的转录下调,翻译水平与转录水平结果一致。同时,通过免疫共沉淀和sh RNA技术分析发现,EGFR和Erb B3在SC19感染后发生酪氨酸磷酸化,并且介导EGFR和Erb B3形成异源二聚体。进一步比较活菌和热灭活的SC19感染h BMEC后EGFR的激活情况,发现活菌刺激激活EGFR,而在热灭活菌株刺激下EGFR不发生酪氨酸磷酸化。同时,SC19活菌感染能够上调EGFR的配体分子AREG、EREG和HB-EGF,而热灭活SC19刺激并不能上调上述配体的转录。并且,采用MMPs抑制剂抑制EGFR配体的剪切释放后发现,EGFR的激活随着抑制剂浓度的增高而减弱,表明SC19感染h BMEC介导配体依赖性EGFR的激活。4.体内体外实验表明抑制EGFR的激活不影响细菌的黏附与定殖为探究EGFR激活在SC19介导脑膜炎过程中的作用,我们随后采用EGFR的抑制剂AG1478进行预处理,并发现AG1478处理并不能减少SC19对h BMEC的黏附。同样,在SC19感染CD1小鼠时,比较AG1478给药组和不给药组的小鼠血液、脑组织和肺组织的细菌定殖量,并未发现表现出显著差别。5.EGFR的激活参与SC19感染导致的神经炎症反应随后,我们分析了AG1478处理对于SC19感染诱导神经炎症的影响。体外,我们发现AG1478处理能显著降低IL-6、IL-8、MCP-1、MIP-2以及GRO-α的转录。同样,体内在抑制EGFR的激活后,循环血液中炎性因子的表达没有明显改变,但在脑组织中细胞因子的表达量显著降低。6.EGFR通过MAPK-ERK1/2和NF-κB通路引起神经炎症反应使用NF-κB和MAPK-ERK1/2通路抑制剂处理h BMEC后发现,SC19感染诱导的IL-6和MCP-1转录显著被抑制。同时我们发现p65和ERK1/2在SC19感染后发生磷酸化,且感染前使用AG1478处理能显著降低p65和ERK1/2的磷酸化。此外,免疫荧光发现p65在h BMEC感染后明显入核,证实SC19感染后的确激活NF-κB通路,同时NF-κB通路的激活与MAPK-ERK1/2的激活无关。上述结果表明,SC19感染h BMEC介导EGFR激活后,通过MAPK-ERK1/2和NF-κB通路引起神经炎症反应。7.EGFR竞争结合ACTN4继而影响细胞骨架的稳定随后,通过免疫共沉淀实验发现,EGFR发生酪氨酸磷酸化的同时,其与ACTN4的结合有所增加。此外,ACTN4随感染也能发生酪氨酸磷酸化,并且在感染前,ACTN4主要与actin结合来维持细胞骨架的稳定;而在感染后,ACTN4被EGFR竞争结合,导致与actin的结合明显减少,继而影响了细胞骨架的稳定。综上所述,SS2菌株SC19感染h BMEC过程中,通过上调EGFR的相关配体分子AREG、EREG和HB-EGF,来介导EGFR的激活及EGFR/Erb B3异源二聚体的形成。EGFR的激活通过MAPK-ERK1/2和NF-κB通路引起h BMEC或脑组织中细胞因子和趋化因子的产生,导致神经炎症反应。此外,SC19刺激EGFR激活后还能竞争性的结合ACTN4,继而破坏由ACTN4维持的细胞骨架的稳定。
[Abstract]:Streptococcus suis type two (Streptococcus suis serotype 2) is an important pathogeny of zoonosis, which has high pathogenic rate and high mortality for both human and animal. Meningitis is an important clinical symptom caused by SS2 infection, but the specific molecular mechanism of meningitis caused by SS2 infection remains to be studied. This study mainly explores EGFR in SS 2 strain SC19 induced meningitis, mediating neuroinflammatory reaction and destroying cytoskeleton. The main results are as follows: 1.SS2 strain SC19 mediates severe neuroinflammatory response, SS2 strain SC19 infection h BMEC. With the prolongation of the interaction time, SC19 adhered to h BMEC significantly increased.SC19 infection of CD1 mice, and found meningeal thickening, Hemorrhage, inflammatory cell infiltration in brain tissue and pathological changes of phagocytic neurons in.CD1 mice, after infection of SC19, various cytokines and chemokines were significantly up-regulated. At the same time, the transcription of inflammatory factors after SC19 infection of H BMEC also significantly increased.2.SC19 stimulation of H BMEC, resulting in intracellular protein tyrosine phosphorylation and then SC19 infection H. BMEC, detection of tyrosine phosphorylation of protein molecules in cells, found that protein tyrosine phosphorylation was observed in 180100 and 60K Da sites, and confirmed that 180K Da size protein was EGFR.3.SC19 infection mediated ligand dependent EGFR activation and two polycrystalline SC19 infected h BMEC. The transcriptional level and translation level of Erb B4 protein showed that the transcriptional level of EGFR, Erb B2 and Erb B4 was unchanged, and the transcriptional level of Erb B3 was down, and the translation level was in accordance with the transcriptional level. The activation of EGFR after SC19 h BMEC infection by live and thermal inactivated SC19 was further compared. It was found that the active bacteria stimulated EGFR, and EGFR did not produce tyrosine phosphorylation under the stimulation of the thermal inactivated strain. At the same time, SC19 live bacteria infection could increase the ligand molecules AREG, EREG and HB-EGF of EGFR, and the thermal inactivated SC19 stimulus could not increase the above. The transcription of ligands. And, after the use of MMPs inhibitors to inhibit the shear release of EGFR ligands, the activation of EGFR decreased with the increase of inhibitor concentration, indicating that SC19 infection h BMEC mediated ligand dependent EGFR activation.4. in vitro and in vitro experiments showed that inhibition of EGFR activation did not affect bacterial adhesion and colonization to explore EGFR activation in SC19. In mediating the role of meningitis, we then pretreated with EGFR inhibitor AG1478, and found that AG1478 treatment did not reduce the adhesion of SC19 to h BMEC. Similarly, when SC19 infected CD1 mice, the bacterial colonization of the blood, brain tissue and lung tissue of the AG1478 and non drug groups was not found to show significant performance in the SC19 infected CD1 mice. After the activation of differential.5.EGFR was involved in the neuroinflammatory response induced by SC19 infection, we analyzed the effect of AG1478 treatment on the induced neuroinflammation induced by SC19 infection. In vitro, we found that AG1478 treatment could significantly reduce the transcription of IL-6, IL-8, MCP-1, MIP-2, and GRO- alpha. Similarly, inflammatory causes in circulating blood after inhibition of EGFR activation in the body The expression of the cytokine was not significantly altered, but the expression of cytokine in the brain significantly decreased.6.EGFR through the MAPK-ERK1/2 and NF- kappa B pathway to induce the use of NF- kappa B and the MAPK-ERK1/2 pathway inhibitor to treat h BMEC, and the IL-6 and MCP-1 transcripts induced by SC19 infection were inhibited. 19 after infection, the phosphorylation of p65 and ERK1/2 could be significantly reduced before infection. In addition, the immunofluorescence showed that p65 was obviously nucleated after H BMEC infection, and confirmed that NF- kappa B pathway was activated after SC19 infection, and the activation of NF- kappa B pathway was not related to the activation of MAPK-ERK1/2. After activation of GFR, MAPK-ERK1/2 and NF- kappa B pathways cause neuroinflammatory response to.7.EGFR competition binding to ACTN4 and subsequent to the stability of cytoskeleton. By immunoprecipitation experiments, it is found that EGFR occurs tyrosine phosphorylation and its binding to ACTN4 increases. In addition, ACTN4 can also occur tyrosine phosphorylation with infection, and Before infection, ACTN4 is mainly combined with actin to maintain the stability of the cytoskeleton, and after infection, ACTN4 is combined with EGFR, resulting in a significant reduction in the combination of actin and the stability of the cytoskeleton. To sum up, the SS2 strain SC19 is involved in H BMEC process by up regulation of EGFR related ligand molecule AREG, EREG and enrichment. Activation and the activation of the EGFR/Erb B3 heterogenous two polymer.EGFR activation through MAPK-ERK1/2 and NF- kappa B pathway causes the production of cytokines and chemokines in H BMEC or brain tissue, leading to neuroinflammatory reactions. In addition, SC19 stimulates EGFR to activate a competitive binding ACTN4 and then destroys the stability of the cytoskeleton maintained by ACTN4.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.611

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