本文選題：豬鏈球菌 + 腦微血管內(nèi)皮細(xì)胞�。� 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：豬鏈球菌二型(Streptococcus suis serotype 2)是一種重要的人獸共患病病原菌,對人或者動物均具有高致病率并導(dǎo)致高死亡率。腦膜炎是SS2感染導(dǎo)致的重要臨床癥狀,但是SS2感染致腦膜炎特有的分子機制仍然有待研究。本研究主要探究EGFR在SS2菌株SC19致腦膜炎的過程中,介導(dǎo)神經(jīng)炎癥反應(yīng)以及破壞細(xì)胞骨架的作用。主要結(jié)果如下:1.SS2菌株SC19介導(dǎo)嚴(yán)重的神經(jīng)炎癥反應(yīng)SS2菌株SC19感染h BMEC,隨著互作時間的延長,SC19黏附h BMEC的菌量顯著上升。SC19感染CD1小鼠,發(fā)現(xiàn)出現(xiàn)腦膜增厚、出血,腦組織出現(xiàn)炎性細(xì)胞浸潤以及噬神經(jīng)元的病理變化。CD1小鼠在感染SC19后,多種細(xì)胞因子和趨化因子發(fā)生顯著上調(diào)。同時,SC19感染h BMEC后炎性因子的轉(zhuǎn)錄也均有顯著上調(diào)。2.SC19刺激h BMEC導(dǎo)致胞內(nèi)蛋白酪氨酸磷酸化隨后采用SC19感染h BMEC,檢測細(xì)胞內(nèi)蛋白分子的酪氨酸磷酸化,發(fā)現(xiàn)在180,100,和60k Da的位置均有蛋白發(fā)生明顯的酪氨酸磷酸化。并證實180k Da大小的蛋白為EGFR。3.SC19感染介導(dǎo)配體依賴性的EGFR激活與二聚體化SC19感染h BMEC后,檢測Erb B家族(EGFR/Erb B1、Erb B2、Erb B3和Erb B4)蛋白轉(zhuǎn)錄水平和翻譯水平,發(fā)現(xiàn)EGFR、Erb B2和Erb B4的轉(zhuǎn)錄水平不變,而Erb B3的轉(zhuǎn)錄下調(diào),翻譯水平與轉(zhuǎn)錄水平結(jié)果一致。同時,通過免疫共沉淀和sh RNA技術(shù)分析發(fā)現(xiàn),EGFR和Erb B3在SC19感染后發(fā)生酪氨酸磷酸化,并且介導(dǎo)EGFR和Erb B3形成異源二聚體。進(jìn)一步比較活菌和熱滅活的SC19感染h BMEC后EGFR的激活情況,發(fā)現(xiàn)活菌刺激激活EGFR,而在熱滅活菌株刺激下EGFR不發(fā)生酪氨酸磷酸化。同時,SC19活菌感染能夠上調(diào)EGFR的配體分子AREG、EREG和HB-EGF,而熱滅活SC19刺激并不能上調(diào)上述配體的轉(zhuǎn)錄。并且,采用MMPs抑制劑抑制EGFR配體的剪切釋放后發(fā)現(xiàn),EGFR的激活隨著抑制劑濃度的增高而減弱,表明SC19感染h BMEC介導(dǎo)配體依賴性EGFR的激活。4.體內(nèi)體外實驗表明抑制EGFR的激活不影響細(xì)菌的黏附與定殖為探究EGFR激活在SC19介導(dǎo)腦膜炎過程中的作用,我們隨后采用EGFR的抑制劑AG1478進(jìn)行預(yù)處理,并發(fā)現(xiàn)AG1478處理并不能減少SC19對h BMEC的黏附。同樣,在SC19感染CD1小鼠時,比較AG1478給藥組和不給藥組的小鼠血液、腦組織和肺組織的細(xì)菌定殖量,并未發(fā)現(xiàn)表現(xiàn)出顯著差別。5.EGFR的激活參與SC19感染導(dǎo)致的神經(jīng)炎癥反應(yīng)隨后,我們分析了AG1478處理對于SC19感染誘導(dǎo)神經(jīng)炎癥的影響。體外,我們發(fā)現(xiàn)AG1478處理能顯著降低IL-6、IL-8、MCP-1、MIP-2以及GRO-α的轉(zhuǎn)錄。同樣,體內(nèi)在抑制EGFR的激活后,循環(huán)血液中炎性因子的表達(dá)沒有明顯改變,但在腦組織中細(xì)胞因子的表達(dá)量顯著降低。6.EGFR通過MAPK-ERK1/2和NF-κB通路引起神經(jīng)炎癥反應(yīng)使用NF-κB和MAPK-ERK1/2通路抑制劑處理h BMEC后發(fā)現(xiàn),SC19感染誘導(dǎo)的IL-6和MCP-1轉(zhuǎn)錄顯著被抑制。同時我們發(fā)現(xiàn)p65和ERK1/2在SC19感染后發(fā)生磷酸化,且感染前使用AG1478處理能顯著降低p65和ERK1/2的磷酸化。此外,免疫熒光發(fā)現(xiàn)p65在h BMEC感染后明顯入核,證實SC19感染后的確激活NF-κB通路,同時NF-κB通路的激活與MAPK-ERK1/2的激活無關(guān)。上述結(jié)果表明,SC19感染h BMEC介導(dǎo)EGFR激活后,通過MAPK-ERK1/2和NF-κB通路引起神經(jīng)炎癥反應(yīng)。7.EGFR競爭結(jié)合ACTN4繼而影響細(xì)胞骨架的穩(wěn)定隨后,通過免疫共沉淀實驗發(fā)現(xiàn),EGFR發(fā)生酪氨酸磷酸化的同時,其與ACTN4的結(jié)合有所增加。此外,ACTN4隨感染也能發(fā)生酪氨酸磷酸化,并且在感染前,ACTN4主要與actin結(jié)合來維持細(xì)胞骨架的穩(wěn)定;而在感染后,ACTN4被EGFR競爭結(jié)合,導(dǎo)致與actin的結(jié)合明顯減少,繼而影響了細(xì)胞骨架的穩(wěn)定。綜上所述,SS2菌株SC19感染h BMEC過程中,通過上調(diào)EGFR的相關(guān)配體分子AREG、EREG和HB-EGF,來介導(dǎo)EGFR的激活及EGFR/Erb B3異源二聚體的形成。EGFR的激活通過MAPK-ERK1/2和NF-κB通路引起h BMEC或腦組織中細(xì)胞因子和趨化因子的產(chǎn)生,導(dǎo)致神經(jīng)炎癥反應(yīng)。此外,SC19刺激EGFR激活后還能競爭性的結(jié)合ACTN4,繼而破壞由ACTN4維持的細(xì)胞骨架的穩(wěn)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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.611

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