3OC12HSL参与人角膜上皮细胞与绿脓杆菌间免疫调控机制的研究

发布时间：2018-08-25 11:23

【摘要】：研究背景:铜绿假单胞菌引起的角膜炎是一种潜在的致盲性细菌感染角膜炎,正常完整的角膜上皮对绿脓杆菌有很强的抵抗力,绿脓杆菌性角膜炎只有在当角膜上皮受损屏障被破坏之后才会发生。多种因素例如创伤,眼表疾病,免疫缺陷和长期佩戴隐形眼镜都与绿脓杆菌性角膜炎的发生发展有关。迁延不愈的绿脓杆菌性角膜炎能够造成角膜正常生理结构的破坏,角膜瘢痕的产生,是最为严重的角膜急性化脓性炎症,如未能及时采取有效措施,可引起严重后果。铜绿假单胞菌影响宿主免疫能力并逃避免疫监视是通过群体感应机制(Quorum sensing,QS系统)对于基因的表达调控而发挥作用。通过检测高丝氨酸内酯(AHL)的分泌可以反映铜绿假单胞菌菌群密度的大小。铜绿假单胞菌主要产生两种AHL,3-氧-十二烷酰-高丝氨酸内酯(3-oxododecanoy1-homoserine lactone,3OC12HSL)和丁酰高丝氨酸内酯(C4-HSL,也称为PAI-2或BHL)。虽然这两种AHLs在细菌内外都可扩散,但是3OC12HSL也可以由MexAB OprM外排系统由菌内排出到菌外环境。如铜绿假单胞菌菌群密度增加,在环境中AHLs的含量也会增加。当达到阈值浓度时,AHLs结合并激活转录因子随后开启重要基因的的表达其表达量增多的同时也会导致细菌毒性的增强。近来,已经初步证实的是,3OC12HSL不仅是重要的是调节细菌毒力分子,也可以通过与真核细胞相互作用并刺激免疫反应。3OC12HSL刺激人角膜上皮细胞,诱导IL-6、IL-8等趋化因子的产生。这种诱导通过丝裂原活化蛋白激酶途径的激活来调节,随后导致转录因子NF-kB的激活。3OC12HSL也激活经典的细胞免疫。有研究显示3OC12HSL能够直接刺激T细胞从而导致IFN的表达增加。这些数据表明由绿脓杆菌分泌的3OC12HSL是多种不同真核细胞的有效激活剂并且可能会极大地影响绿脓杆菌的致病性。3OC12HSL是微生物之间、以及微生物和机体之间进行信号通信的分子基础,是绿脓杆菌毒力不可缺少的分子。当角膜感染绿脓杆菌后,绿脓杆菌会释放3OC12HSL分子,人体免疫系统察觉到该信号分子后巨噬细胞和上皮细胞就会对该信号分子产生反应,进而产生炎症、分泌各种炎症因子等免疫调节反应,这也是绿脓杆菌性角膜炎迁延不愈的重要原因,因此,对其影响免疫调控功能的作用机制进行研究有着积极的意义。本文中我们主要就3OC12HSL参与人角膜上皮细胞与绿脓杆菌间免疫调控机制及其所起的作用加以研究。目的:3OC12HSL是绿脓杆菌性角膜炎发生发展过程中进行信号通信的重要号分子,本研究拟探索3OC12HSL是否参与人角膜上皮细胞与绿脓杆菌间固有免疫的调控,探讨3OC12HSL在感染性角膜炎中可能的作用。方法:本研究通过体外培养永生化人角膜上皮细胞,分别加入不同浓度的3OC12HSL,通过结晶紫染色后显微镜下观察细胞状态和CCK-8检测细胞增殖活性的变化。通过Real-time PCR检测不同刺激条件下角膜上皮细胞中Toll样受体(TLR2,4,5,6)的mRNA表达水平。Western Blotting检测TOLL样受体(TLR2,4)的蛋白表达。为进一步证明3OC12HSL对角膜上皮细胞免疫反应的调控是否通过TLRs以及其下游的NF-κB信号通路,我们通过免疫荧光染色和Western Blotting观察并检测转录因子NF-kB的入核情况。利用Real-time PCR检测细胞内炎症因子IL-6、IL-8、IL-10、TNF-α的mRNA分泌情况。为进一步验证角膜上皮细胞炎症因子的分泌情况,分别采用特异性TLR2、4、5、6单克隆抗体功能性阻断TLR2、4、5、6和TLR2、4、5、6专一激动剂预激活TLR2、4、5、6后再给与100uM浓度的3OC12HSL刺激,通过ELISA检测细胞上清液中的IL-8的分泌,并进行统计学分析。利用结晶紫定量检测不同浓度地塞米松对绿脓杆菌生物被膜的影响。实验中数据统计分析采用GraphPad Prism 7.0 软件。结果:通过结晶紫染色后显微镜下观察细胞状态和CCK-8检测细胞增殖活性的变化,发现随着刺激时间延长(OH、4H、8H、12H、24H),角膜上皮细胞的增殖活性逐渐减弱,12H时达到最低水平;随着给药浓度增加(0、25、50、100、200umol/l),角膜上皮细胞增殖活性呈现逐渐减弱的趋势,在100umol/1浓度3OC12HSL刺激时其活性基本到达最低水平,继续增加给药浓度其活性变化不显著。通过RT-PCR我们发现相同浓度(100 umol/1)的3OC12HSL刺激在不同时间(OH、4H、8H、12H、24H)刺激后TLR2和TLR4的mRNA表达水平呈现出时间依赖性,随着处理时间的延长,其mRNA含量逐渐增加,而TLR5和TLR6无明显差异;在不同浓度(0、25、50、100、200umol/l)的 3OC12HSL 刺激相同时间(12H)后发现 TLR2 和 TLR4 的mRNA含量呈现浓度依赖性,逐渐增加并在100umol/l时其含量最高,继续增大浓度至200 umol/l发现其含量略有下降,而TLR5和TLR6亦无明显差异。通过Western Blotting验证了 TLR2和TLR4的蛋白表达和其mRNA的表达趋势基本一致,证明了Tol 1样受体尤其是TLR2和TLR4在3OC12HSL调控人角膜上皮细胞免疫反应中的重要作用。通过免疫荧光染色和Western Blotting我们发现3OC12HSL能促进角膜上皮细胞内NF-κB的入核,并且在处理1H后入核最明显,处理2H甚至4H后NF-κB基本恢复至未处理水平。此外,通过RT-PCR我们发现100umol/l的3OC12HSL处理12H后的人角膜上皮细胞IL-6、IL-8、IL-10、TNF-α的mmRNA含量增加;并且通过ELISA验证了 IL-8的分泌和3OC12HSL的处理浓度和时间相关,其相关性和TLR2和TLR4的mRNA的表达趋势基本保持一致;为了探究IL-8的表达和TOLL样受体的关系,本研究进一步采用特异性TLR2、4、5、6单克隆抗体功能性阻断TLR2、4、5、6和TLR2、4、5、6专一激动剂预激活TLR2、4、5、6后,通过ELISA检测发现TLR2特异抑制剂处理后IL-8的表达明显下降,其余激动剂以及阻断剂无明显效应。最后,通过定量检测绿脓杆菌生物被膜含量,发现地塞米松预处理能够抑制绿脓杆菌生物被膜的形成,而3OC12HSL预处理未观察到对绿脓杆菌生物被膜的显著影响。结论:3OC12HSL能够介导免疫相关炎症因子的表达增加;3OC12HSL对角膜上皮细胞固有免疫的调控存在特异性TLRs激活机制,尤其是TLR2和TLR4的激活;特异性TLRs激活机制和核转录因子NF-κB通路的激活相关,3OC12HSL能够促进NF-κB的快速入核,NF-κB可能是3OC12HSL参与调控免疫环节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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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