【摘要】：研究背景:銅綠假單胞菌引起的角膜炎是一種潛在的致盲性細菌感染角膜炎,正常完整的角膜上皮對綠膿桿菌有很強的抵抗力,綠膿桿菌性角膜炎只有在當角膜上皮受損屏障被破壞之后才會發(fā)生。多種因素例如創(chuàng)傷,眼表疾病,免疫缺陷和長期佩戴隱形眼鏡都與綠膿桿菌性角膜炎的發(fā)生發(fā)展有關(guān)。遷延不愈的綠膿桿菌性角膜炎能夠造成角膜正常生理結(jié)構(gòu)的破壞,角膜瘢痕的產(chǎn)生,是最為嚴重的角膜急性化膿性炎癥,如未能及時采取有效措施,可引起嚴重后果。銅綠假單胞菌影響宿主免疫能力并逃避免疫監(jiān)視是通過群體感應機制(Quorum sensing,QS系統(tǒng))對于基因的表達調(diào)控而發(fā)揮作用。通過檢測高絲氨酸內(nèi)酯(AHL)的分泌可以反映銅綠假單胞菌菌群密度的大小。銅綠假單胞菌主要產(chǎn)生兩種AHL,3-氧-十二烷酰-高絲氨酸內(nèi)酯(3-oxododecanoy1-homoserine lactone,3OC12HSL)和丁酰高絲氨酸內(nèi)酯(C4-HSL,也稱為PAI-2或BHL)。雖然這兩種AHLs在細菌內(nèi)外都可擴散,但是3OC12HSL也可以由MexAB OprM外排系統(tǒng)由菌內(nèi)排出到菌外環(huán)境。如銅綠假單胞菌菌群密度增加,在環(huán)境中AHLs的含量也會增加。當達到閾值濃度時,AHLs結(jié)合并激活轉(zhuǎn)錄因子隨后開啟重要基因的的表達其表達量增多的同時也會導致細菌毒性的增強。近來,已經(jīng)初步證實的是,3OC12HSL不僅是重要的是調(diào)節(jié)細菌毒力分子,也可以通過與真核細胞相互作用并刺激免疫反應。3OC12HSL刺激人角膜上皮細胞,誘導IL-6、IL-8等趨化因子的產(chǎn)生。這種誘導通過絲裂原活化蛋白激酶途徑的激活來調(diào)節(jié),隨后導致轉(zhuǎn)錄因子NF-kB的激活。3OC12HSL也激活經(jīng)典的細胞免疫。有研究顯示3OC12HSL能夠直接刺激T細胞從而導致IFN的表達增加。這些數(shù)據(jù)表明由綠膿桿菌分泌的3OC12HSL是多種不同真核細胞的有效激活劑并且可能會極大地影響綠膿桿菌的致病性。3OC12HSL是微生物之間、以及微生物和機體之間進行信號通信的分子基礎(chǔ),是綠膿桿菌毒力不可缺少的分子。當角膜感染綠膿桿菌后,綠膿桿菌會釋放3OC12HSL分子,人體免疫系統(tǒng)察覺到該信號分子后巨噬細胞和上皮細胞就會對該信號分子產(chǎn)生反應,進而產(chǎn)生炎癥、分泌各種炎癥因子等免疫調(diào)節(jié)反應,這也是綠膿桿菌性角膜炎遷延不愈的重要原因,因此,對其影響免疫調(diào)控功能的作用機制進行研究有著積極的意義。本文中我們主要就3OC12HSL參與人角膜上皮細胞與綠膿桿菌間免疫調(diào)控機制及其所起的作用加以研究。目的:3OC12HSL是綠膿桿菌性角膜炎發(fā)生發(fā)展過程中進行信號通信的重要 號分子,本研究擬探索3OC12HSL是否參與人角膜上皮細胞與綠膿桿菌間固有免疫的調(diào)控,探討3OC12HSL在感染性角膜炎中可能的作用。方法:本研究通過體外培養(yǎng)永生化人角膜上皮細胞,分別加入不同濃度的3OC12HSL,通過結(jié)晶紫染色后顯微鏡下觀察細胞狀態(tài)和CCK-8檢測細胞增殖活性的變化。通過Real-time PCR檢測不同刺激條件下角膜上皮細胞中Toll樣受體(TLR2,4,5,6)的mRNA表達水平。Western Blotting檢測TOLL樣受體(TLR2,4)的蛋白表達。為進一步證明3OC12HSL對角膜上皮細胞免疫反應的調(diào)控是否通過TLRs以及其下游的NF-κB信號通路,我們通過免疫熒光染色和Western Blotting觀察并檢測轉(zhuǎn)錄因子NF-kB的入核情況。利用Real-time PCR檢測細胞內(nèi)炎癥因子IL-6、IL-8、IL-10、TNF-α的mRNA分泌情況。為進一步驗證角膜上皮細胞炎癥因子的分泌情況,分別采用特異性TLR2、4、5、6單克隆抗體功能性阻斷TLR2、4、5、6和TLR2、4、5、6專一激動劑預激活TLR2、4、5、6后再給與100uM濃度的3OC12HSL刺激,通過ELISA檢測細胞上清液中的IL-8的分泌,并進行統(tǒng)計學分析。利用結(jié)晶紫定量檢測不同濃度地塞米松對綠膿桿菌生物被膜的影響。實驗中數(shù)據(jù)統(tǒng)計分析采用GraphPad Prism 7.0 軟件。結(jié)果:通過結(jié)晶紫染色后顯微鏡下觀察細胞狀態(tài)和CCK-8檢測細胞增殖活性的變化,發(fā)現(xiàn)隨著刺激時間延長(OH、4H、8H、12H、24H),角膜上皮細胞的增殖活性逐漸減弱,12H時達到最低水平;隨著給藥濃度增加(0、25、50、100、200umol/l),角膜上皮細胞增殖活性呈現(xiàn)逐漸減弱的趨勢,在100umol/1濃度3OC12HSL刺激時其活性基本到達最低水平,繼續(xù)增加給藥濃度其活性變化不顯著。通過RT-PCR我們發(fā)現(xiàn)相同濃度(100 umol/1)的3OC12HSL刺激在不同時間(OH、4H、8H、12H、24H)刺激后TLR2和TLR4的mRNA表達水平呈現(xiàn)出時間依賴性,隨著處理時間的延長,其mRNA含量逐漸增加,而TLR5和TLR6無明顯差異;在不同濃度(0、25、50、100、200umol/l)的 3OC12HSL 刺激相同時間(12H)后發(fā)現(xiàn) TLR2 和 TLR4 的mRNA含量呈現(xiàn)濃度依賴性,逐漸增加并在100umol/l時其含量最高,繼續(xù)增大濃度至200 umol/l發(fā)現(xiàn)其含量略有下降,而TLR5和TLR6亦無明顯差異。通過Western Blotting驗證了 TLR2和TLR4的蛋白表達和其mRNA的表達趨勢基本一致,證明了Tol 1樣受體尤其是TLR2和TLR4在3OC12HSL調(diào)控人角膜上皮細胞免疫反應中的重要作用。通過免疫熒光染色和Western Blotting我們發(fā)現(xiàn)3OC12HSL能促進角膜上皮細胞內(nèi)NF-κB的入核,并且在處理1H后入核最明顯,處理2H甚至4H后NF-κB基本恢復至未處理水平。此外,通過RT-PCR我們發(fā)現(xiàn)100umol/l的3OC12HSL處理12H后的人角膜上皮細胞IL-6、IL-8、IL-10、TNF-α的mmRNA含量增加;并且通過ELISA驗證了 IL-8的分泌和3OC12HSL的處理濃度和時間相關(guān),其相關(guān)性和TLR2和TLR4的mRNA的表達趨勢基本保持一致;為了探究IL-8的表達和TOLL樣受體的關(guān)系,本研究進一步采用特異性TLR2、4、5、6單克隆抗體功能性阻斷TLR2、4、5、6和TLR2、4、5、6專一激動劑預激活TLR2、4、5、6后,通過ELISA檢測發(fā)現(xiàn)TLR2特異抑制劑處理后IL-8的表達明顯下降,其余激動劑以及阻斷劑無明顯效應。最后,通過定量檢測綠膿桿菌生物被膜含量,發(fā)現(xiàn)地塞米松預處理能夠抑制綠膿桿菌生物被膜的形成,而3OC12HSL預處理未觀察到對綠膿桿菌生物被膜的顯著影響。結(jié)論:3OC12HSL能夠介導免疫相關(guān)炎癥因子的表達增加;3OC12HSL對角膜上皮細胞固有免疫的調(diào)控存在特異性TLRs激活機制,尤其是TLR2和TLR4的激活;特異性TLRs激活機制和核轉(zhuǎn)錄因子NF-κB通路的激活相關(guān),3OC12HSL能夠促進NF-κB的快速入核,NF-κB可能是3OC12HSL參與調(diào)控免疫環(huán)節(jié)中早期的重要通路之一。
[Abstract]:BACKGROUND: Pseudomonas aeruginosa-induced keratitis is a potentially blinding bacterial keratitis. Normal intact corneal epithelium is strongly resistant to Pseudomonas aeruginosa. Pseudomonas aeruginosa keratitis occurs only when the damaged corneal epithelial barrier is destroyed. Protracted Pseudomonas aeruginosa keratitis is the most serious acute suppurative inflammation of the cornea. If effective measures are not taken in time, it can cause serious consequences. Pseudomonas aeruginosa affects host immunity and escapes epidemic surveillance through quorum sensing (QS system) regulation of gene expression. Detection of hyperserine lactone (AHL) secretion can reflect the density of Pseudomonas aeruginosa. Pseudomonas aeruginosa mainly produces two kinds of AHL, 3-oxygen-10. Dialkyl-homoserine lactone (3-oxododecanoy 1-homoserine lactone, 3OC12HSL) and butyryl-homoserine lactone (C4-HSL, also known as PAI-2 or BHL). Although both AHLs can diffuse inside and outside the bacteria, 3OC12HSL can also be excreted from the bacterial environment by the MexAB OprM efflux system. For example, Pseudomonas aeruginosa bacterial population density increases in the bacterial community, in the bacterial environment. When the threshold concentration is reached, the expression of the transcription factors that bind to and activate the transcription factors and then turn on the important genes will also increase the bacterial toxicity. 3OC12HSL stimulates human corneal epithelial cells and induces the production of chemokines such as IL-6 and IL-8. This induction is regulated by activation of mitogen-activated protein kinase pathway, which then leads to activation of transcription factor NF-kB. 3OC12HSL also activates classical cellular immunity. Studies have shown that 3OC12HSL can directly induce the production of chemokines such as IL-6 and IL-8. These data suggest that the 3OC12HSL secreted by Pseudomonas aeruginosa is an effective activator of many different eukaryotic cells and may greatly affect the pathogenicity of Pseudomonas aeruginosa. 3OC12HSL is the molecular basis for signaling between microorganisms, as well as between microorganisms and organisms. When the cornea is infected with Pseudomonas aeruginosa, Pseudomonas aeruginosa releases the 3OC12HSL molecule. When the human immune system detects the signal molecule, macrophages and epithelial cells will react to the signal molecule, and then produce inflammation, secrete various inflammatory factors and other immune regulatory responses. This is also Pseudomonas aeruginosa sex angle. In this paper, we mainly study the mechanism of 3OC12HSL involved in the immune regulation between human corneal epithelial cells and Pseudomonas aeruginosa and its role. Objective: 3OC12HSL is the pathogenesis of Pseudomonas aeruginosa keratitis. The purpose of this study was to explore whether 3OC12HSL was involved in the regulation of innate immunity between human corneal epithelial cells and Pseudomonas aeruginosa and the possible role of 3OC12HSL in infectious keratitis. The expression of Toll-like receptor (TLR2,4,5,6) mRNA in corneal epithelial cells was detected by Real-time PCR. The expression of TOLL-like receptor (TLR2,4) protein was detected by Western Blotting. Whether SL regulates the immune response of corneal epithelial cells through TLRs and its downstream NF-kappa B signaling pathway was observed and detected by immunofluorescence staining and Western Blotting. Real-time PCR was used to detect the secretion of inflammatory cytokines IL-6, IL-8, IL-10 and TNF-alpha. To investigate the secretion of inflammatory cytokines in corneal epithelial cells, TLR2, 4, 5, 6 monoclonal antibodies were used to block TLR2, 4, 5, 6 and TLR2, 4, 5, 6 specific agonists to pre-activate TLR2, 4, 5, 6 and then stimulate them with 100uM 3OC12HSL. The secretion of IL-8 in supernatant was detected by ELISA and analyzed statistically. The effect of dexamethasone on the biofilm of Pseudomonas aeruginosa was quantitatively detected with violet. GraphPad Prism 7.0 software was used for statistical analysis of the experimental data. Results: Cell status and the proliferation activity of CCK-8 cells were observed under microscope after crystal violet staining, and it was found that with the prolongation of stimulation time (OH, 4H, 8H, 12H, 24H), corneal epithelium was observed. The proliferative activity of corneal epithelial cells decreased gradually and reached the lowest level at 12H. The proliferative activity of corneal epithelial cells decreased gradually with the increase of dosage (0,25,50,100,200 umol/l). The activity reached the lowest level at the stimulation of 100umol/1 concentration of 3OC12HSL, and there was no significant change with the increase of dosage. They found that the mRNA expression levels of TLR2 and TLR4 were time-dependent after stimulation with the same concentration (100 umol/1) of 3OC12HSL at different time (OH, 4H, 8H, 12H, 24H). The mRNA content of TLR2 and TLR4 increased gradually with the treatment time, but there was no significant difference between them at different concentrations (0, 25, 50, 100, 200 umol/l). TLR2 and TLR4 mRNA content increased gradually and reached the highest level at 100 umol/l, and decreased slightly at 200 umol/l, while there was no significant difference between TLR5 and TLR6. Western Blotting confirmed that the expression of TLR2 and TLR4 protein and its mRNA expression trend were basically the same. By immunofluorescence staining and Western Blotting, we found that 3OC12HSL could promote the entry of NF-kappa B into the nucleus of corneal epithelial cells, especially TLR2 and TLR4. In addition, we found that the levels of IL-6, IL-8, IL-10 and TNF-alpha in human corneal epithelial cells increased after 12H treatment with 100umol/l 3OC12HSL by RT-PCR, and the correlation between the secretion of IL-8 and the concentration and time of 3OC12HSL treatment was confirmed by ELISA, and the expression trend of TLR2 and TLR4 mRNA was basically consistent. To investigate the relationship between the expression of IL-8 and TOLL-like receptor, we further used specific TLR2, 4, 5, 6 monoclonal antibodies to block TLR2, 4, 5, 6 and TLR2, 4, 5, 6 specific agonists to pre-activate TLR2, 4, 5, 6, and found that the expression of IL-8 was significantly decreased after TLR2 specific inhibitors were treated by ELISA. Finally, through quantitative detection of the biofilm content of Pseudomonas aeruginosa, it was found that dexamethasone pretreatment could inhibit the biofilm formation of Pseudomonas aeruginosa, while 3OC12HSL pretreatment had no significant effect on the biofilm of Pseudomonas aeruginosa. Specific TLRs activation mechanism, especially TLR2 and TLR4 activation, exists in the regulation of innate immunity of epithelial cells. Specific TLRs activation mechanism is related to the activation of nuclear transcription factor NF-kappa B pathway. 3OC12HSL can promote the rapid entry of NF-kappa B into the nucleus. NF-kappa B may be one of the important pathways in the early regulation of immune response by 3OC12HSL.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392

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