叶黄素抗单核细胞增生性李斯特菌感染的研究

发布时间：2018-01-15 08:04

本文关键词：叶黄素抗单核细胞增生性李斯特菌感染的研究　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：单核细胞增生性李斯特菌(Listeria monocytogenes,L.monocytogenes)是一种革兰氏阳性机会致病菌,主要威胁孕妇、儿童和老年人等免疫机能较弱的群体,可引起涉及多种器官的全身性感染,最严重可引发细菌性脑膜炎。李斯特菌感染相对较为罕见,但其患者有极高的死亡率。临床一般选取高剂量的青霉素G和阿莫西林联合使用庆大霉素的抗生素疗法对单增李斯特菌感染进行治疗。虽然单增李斯特菌对多种抗生素敏感,但近年来耐药型单增李斯特菌不断出现,因此抗生素替代药物的研发值得引起我们的重视。单增李斯特菌感染发病的过程需要多种毒力因子的参与,溶血素(Listeriolysin O,LLO)就是其中最重要的一员。LLO可与吞噬泡膜上的胆固醇结合,形成寡聚体并在膜上成孔,协助细菌逃避吞噬溶酶体的裂解。到达细胞溶质的细菌迅速增殖,这一过程对细菌感染至关重要。另外,LLO缺失株几乎丧失了致病能力,提示LLO是单增李斯特菌感染药物研发的重要潜在靶标。叶黄素是一种广泛存在于蔬菜和水果之中的天然植物色素,属于类胡萝卜素类化合物,它对于年龄相关的黄斑变性,以及非霍奇金淋巴瘤的治疗具有一定的积极作用。本研究发现天然植物色素叶黄素可以有效地抑制LLO寡聚,从而抑制其溶血活性。细菌生长曲线以及MIC结果提示,叶黄素对单增李斯特菌几乎无抑菌活性(MIC≥1792μM)。我们使用巨噬细胞RAW 264.7体外建立了细菌感染模型,细胞毒性实验表明叶黄素对李斯特菌介导的巨噬细胞损伤具有一定的缓解作用。细胞内生长曲线结果显示,叶黄素并未抑制细菌的内化,而是抑制了细菌的逃逸及增殖。我们使用了免疫荧光法,对感染不同阶段细胞内的单增李斯特菌和肌动蛋白染色,并通过激光扫描共聚焦显微镜直接观察,结果显示叶黄素有效阻止了细菌从吞噬泡的逃逸,从而阻断了其感染的最关键步骤。最后,我们应用Balb/c小鼠建立了体内细菌感染模型,探究了叶黄素对单增李斯特菌感染动物的实验治疗学作用。结果显示,皮下注射叶黄素可缓解非致死剂量细菌感染小鼠肝脏和脾脏的损伤,并显著降低器官内菌落的定殖数。在致死剂量细菌感染实验中,叶黄素治疗延迟了小鼠死亡时间,并提高了其生存率。综上所述,本研究结果表明天然化合物叶黄素可通过直接作用于LLO而抑制其介导的成孔活性,从而于体外和体内降低单增李斯特菌致病力,提示叶黄素可能是一种潜在的抗感染药物,具有深入研究的价值。
[Abstract]:Monocytogenes bacteria Lester (Listeria monocytogenes, L.monocytogenes) is a gram positive opportunistic bacteria, the main threat to pregnant women, children and the elderly immune function of weak groups, can cause systemic infection involving multiple organs, the most serious cause of bacterial meningitis. Lester infection is relatively rare, but the patients have a high mortality rate. Clinical antibiotic therapy usually takes high doses of penicillin G and amoxicillin combined with gentamicin on Listeria infection treatment. Although Lester Lester Listeria bacteria sensitive to many antibiotics, but in recent years, resistance to Listeria bacteria Lester appear constantly, so the alternative antibiotics drug development deserves our attention Lester. Listeria pathogenesis infection requires several virulence factors involved, hemolysin (Listeriolysin O LLO) is the The most important part of the.LLO with the phagosome membrane cholesterol binding, formation of oligomers and the membrane pore, assist the bacteria evade phagolysosome cracking. To reach the cell solute bacteria rapid proliferation, the process of infection. In addition, the LLO mutant almost lost its pathogenicity, tips LLO is an important potential target for drug development of Listeria Lester infection. Lutein is a widely present in vegetables and fruits of the natural plant pigment belongs to the carotenoid compounds, it is for age related macular degeneration, has a positive effect as well as the treatment of non Hodgkin lymphoma. This study found that natural plant pigment lutein can effectively inhibit LLO oligomers, thereby inhibiting its hemolytic activity. Bacterial growth curves and MIC results suggest that lutein almost no antimicrobial activity against Listeria bacteria (MIC = Lester 1792 mu M). We use RAW 264.7 macrophages in vitro established models of bacterial infection, cell toxicity experiment showed that lutein can relieve the damage of macrophage mediated. List Rand cell growth curve showed that lutein did not inhibit bacterial internalization, but inhibited the proliferation and escape of bacteria. We use immunofluorescence method in different stages of infection, intracellular Listeria bacteria Lester and actin staining, and by laser scanning confocal microscope to directly observe and results showed that lutein can effectively prevent bacteria escape from phagocytic vesicles, thereby blocking the key steps of the infection. Finally, we used Balb/c mouse model was established for bacterial infection in vivo, inquiry the lutein on Listeria bacteria infection in animal experimental treatment of Lester effect. The results showed that subcutaneous injection of lutein can alleviate non lethal agent The amount of bacterial infection in liver and spleen injury in mice, and significantly reduce the number of colonization organs within the colony. The lethal dose of bacterial infection in the experiment, the lutein treatment delay the death time of mice, and improve their survival rate. In conclusion, the results of this study show that pore activity of natural compounds leaves Huang Suke through direct interaction with LLO and inhibition the mediated in vitro and in vivo, thus to reduce Listeria Lester virulence, suggesting that lutein may be a potential anti infection drugs, has the value of further study.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378

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