本文選題：弓形蟲　切入點(diǎn)：感染　出處：《安徽醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的 弓形蟲是一種在世界范圍廣泛傳播的機(jī)會(huì)致病寄生蟲。世界上約有1／3的人血清試驗(yàn)呈陽性,但是大多數(shù)人表現(xiàn)為沒有顯著臨床癥狀的隱性感染。對(duì)孕產(chǎn)婦則為顯性感染,主要表現(xiàn)有流產(chǎn),死胎,先天畸形等,其致病機(jī)制尚不清楚。本研究旨在闡述弓形蟲感染后宿主固有免疫變化與弓形蟲致病間的關(guān)系,選取弓形蟲感染后NLRP3炎癥小體為研究對(duì)象,探討弓形蟲感染后NLRP3炎癥小體活性及其激活機(jī)制,為弓形蟲病治療提供新的策略。 方法 1,弓形蟲感染與NLRP3激活的相關(guān)性研究：弓形蟲速殖子以1：2(速殖子：細(xì)胞)的比例感染THP-1細(xì)胞,對(duì)照組加入與速殖子等體積的PBS緩沖液。半定量PCR分析THP-1細(xì)胞中NLRP3和caspase-1mRNA的變化。Western blot分析THP-1細(xì)胞中NLRP3和caspase-1蛋白的變化。ELISA分析THP-1細(xì)胞上清中白介素1p(IL-1p)和IL-18的變化。將昆明鼠隨機(jī)分為實(shí)驗(yàn)組和對(duì)照組。實(shí)驗(yàn)組腹腔注射2×107弓形蟲速殖子,對(duì)照組注射等體積的PBS緩沖液。半定量PCR分析小鼠脾臟中NLRP3和caspase-1mRNA的變化。ELISA分析小鼠外周血血清中IL-1p和IL-18的變化。 2,弓形蟲感染激活NLRP3炎癥小體可能機(jī)制：弓形蟲速殖子以1：2(速殖子：細(xì)胞)的比例感染對(duì)照組和實(shí)驗(yàn)組THP-1細(xì)胞,空白組不感染弓形蟲速殖子,實(shí)驗(yàn)組分別加入K+70Mm/孔、N-乙�；�-L-半胱氨酸(NAC)25Mm/孔和CA-074Me10μm/孔。ELISA分析THP-1細(xì)胞上清中IL-1β和IL-18的變化。 結(jié)果 1,弓形蟲感染與NLRP3激活的相關(guān)性研究：弓形蟲感染THP-1細(xì)胞后NLRP3、 caspase-1mRNA和蛋白表達(dá)水平增高,IL-1β、IL-18含量升高。弓形蟲感染小鼠的脾臟中NLRP3、caspase-1mRNA同樣增高,其外周血血清中IL-1α、IL-18含量升高。, 2,弓形蟲感染激活NLRP3炎癥小體的可能機(jī)制：加入K+后實(shí)驗(yàn)組IL-1p和IL-18含量與空白組比較升高,但與對(duì)照組比較無顯著變化,說明其活化可能與K+外排無關(guān)。加入NAC后實(shí)驗(yàn)組IL-1p和IL-18含量與空白組比較顯著降低,與對(duì)照組比較也顯著降低,說明其活化可能與活性氧(reactive oxygen species,ROS)有關(guān)。加入CA-074Me后實(shí)驗(yàn)組IL-1p和IL-18含量與空白組比較升高,但與對(duì)照組比較無顯著變化,說明其活化可能與組織蛋白酶無關(guān)。 結(jié)論 弓形蟲感染NLRP3炎癥小體活性增加,繼而活化caspase,剪接IL-1p和IL-18前體,IL-1β和IL-18表達(dá)水平的升高。這種由弓形蟲感染而激活NLRP3炎癥小體的可能機(jī)制與ROS有關(guān)。
[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 偽 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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R531.8

【共引文獻(xiàn)】

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本文編號(hào)：1678252