弓形虫感染与NLRP3炎症小体相关性的初步研究

发布时间：2018-03-28 22:05

本文选题：弓形虫　切入点：感染　出处：《安徽医科大学》2013年硕士论文

【摘要】：目的弓形虫是一种在世界范围广泛传播的机会致病寄生虫。世界上约有1／3的人血清试验呈阳性,但是大多数人表现为没有显著临床症状的隐性感染。对孕产妇则为显性感染,主要表现有流产,死胎,先天畸形等,其致病机制尚不清楚。本研究旨在阐述弓形虫感染后宿主固有免疫变化与弓形虫致病间的关系,选取弓形虫感染后NLRP3炎症小体为研究对象,探讨弓形虫感染后NLRP3炎症小体活性及其激活机制,为弓形虫病治疗提供新的策略。方法 1,弓形虫感染与NLRP3激活的相关性研究：弓形虫速殖子以1：2(速殖子：细胞)的比例感染THP-1细胞,对照组加入与速殖子等体积的PBS缓冲液。半定量PCR分析THP-1细胞中NLRP3和caspase-1mRNA的变化。Western blot分析THP-1细胞中NLRP3和caspase-1蛋白的变化。ELISA分析THP-1细胞上清中白介素1p(IL-1p)和IL-18的变化。将昆明鼠随机分为实验组和对照组。实验组腹腔注射2×107弓形虫速殖子,对照组注射等体积的PBS缓冲液。半定量PCR分析小鼠脾脏中NLRP3和caspase-1mRNA的变化。ELISA分析小鼠外周血血清中IL-1p和IL-18的变化。 2,弓形虫感染激活NLRP3炎症小体可能机制：弓形虫速殖子以1：2(速殖子：细胞)的比例感染对照组和实验组THP-1细胞,空白组不感染弓形虫速殖子,实验组分别加入K+70Mm/孔、N-乙酰基-L-半胱氨酸(NAC)25Mm/孔和CA-074Me10μm/孔。ELISA分析THP-1细胞上清中IL-1β和IL-18的变化。结果 1,弓形虫感染与NLRP3激活的相关性研究：弓形虫感染THP-1细胞后NLRP3、 caspase-1mRNA和蛋白表达水平增高,IL-1β、IL-18含量升高。弓形虫感染小鼠的脾脏中NLRP3、caspase-1mRNA同样增高,其外周血血清中IL-1α、IL-18含量升高。, 2,弓形虫感染激活NLRP3炎症小体的可能机制：加入K+后实验组IL-1p和IL-18含量与空白组比较升高,但与对照组比较无显著变化,说明其活化可能与K+外排无关。加入NAC后实验组IL-1p和IL-18含量与空白组比较显著降低,与对照组比较也显著降低,说明其活化可能与活性氧(reactive oxygen species,ROS)有关。加入CA-074Me后实验组IL-1p和IL-18含量与空白组比较升高,但与对照组比较无显著变化,说明其活化可能与组织蛋白酶无关。结论弓形虫感染NLRP3炎症小体活性增加,继而活化caspase,剪接IL-1p和IL-18前体,IL-1β和IL-18表达水平的升高。这种由弓形虫感染而激活NLRP3炎症小体的可能机制与ROS有关。
[Abstract]:Purpose. Toxoplasma gondii is a opportunistic parasite that is widely spread around the world. About a third of the world's human serum tests are positive, but most people show recessive infections without significant clinical symptoms. The main manifestations are abortion, stillbirth, congenital malformation and so on. The aim of this study is to elucidate the relationship between host innate immune changes and pathogenesis of Toxoplasma gondii after infection of Toxoplasma gondii (Toxoplasma gondii). The activity and activation mechanism of NLRP3 inflammatory bodies after Toxoplasma gondii infection were studied in order to provide a new strategy for the treatment of Toxoplasma gondii. Method. 1. Correlation between Toxoplasma gondii infection and NLRP3 activation: Toxoplasma gondii tachyzoites were infected with THP-1 cells at a ratio of 1: 2 (tachyzoites: cells). In control group, PBS buffer of the same volume as tachyzoites was added. The changes of NLRP3 and caspase-1mRNA in THP-1 cells were analyzed by semi-quantitative PCR. The changes of NLRP3 and caspase-1 proteins in THP-1 cells were analyzed by Western blot. The changes of IL-1pIL-1pand IL-18 in the supernatant of THP-1 cells were detected by Elisa. Kunming mice were randomly divided into experimental group and control group, the experimental group was injected with 2 脳 107 Toxoplasma gondii tachyzoites, The changes of NLRP3 and caspase-1mRNA in spleen of mice were analyzed by semi-quantitative PCR. The changes of IL-1p and IL-18 in peripheral blood serum of mice were analyzed by Elisa. 2. The possible mechanism of Toxoplasma gondii infection and activation of NLRP3 inflammatory bodies: Toxoplasma gondii tachyzoites were infected with Toxoplasma gondii Toxoplasma tachyzoites at the ratio of 1: 2 (tachyzoites: cells) in control group and experimental group, while those in blank group were not infected with Toxoplasma gondi@@. In the experimental group, the changes of IL-1 尾 and IL-18 in the supernatant of THP-1 cells were analyzed by Elisa and 25 mm / well of NACU and CA-074Me10 渭 m / pore respectively. Results. 1. The correlation between Toxoplasma gondii infection and NLRP3 activation: the expression levels of NLRP3, caspase-1mRNA and protein increased after Toxoplasma gondii infection in THP-1 cells. The contents of IL-1 尾 and IL-18 in spleen of Toxoplasma gondii infected mice were also increased, and the contents of IL-1 伪 and IL-18 in peripheral blood serum of Toxoplasma gondii infected mice were also increased. 2. The possible mechanism of Toxoplasma gondii infection in activating NLRP3 inflammatory bodies: the contents of IL-1p and IL-18 in the experimental group were higher than those in the blank group after K addition, but there was no significant change compared with the control group. After adding NAC, the contents of IL-1p and IL-18 in the experimental group were significantly lower than those in the blank group, and those in the control group were also significantly lower than those in the control group. These results suggested that the activation of Ros might be related to reactive oxygen speciesros.The contents of IL-1p and IL-18 in the experimental group were higher than those in the control group after the addition of CA-074Me, but there was no significant change compared with the control group, indicating that the activation of Ros might not be related to cathepsin. Conclusion. Toxoplasma gondii infection increased the activity of NLRP3 inflammatory bodies, and activated caspase, and spliced the expression levels of IL-1p and IL-18 precursor IL-1 尾 and IL-18. This mechanism of NLRP3 inflammatory bodies activated by Toxoplasma gondii infection may be related to ROS.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R531.8

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