本文關(guān)鍵詞： 弓形蟲 速殖子 緩殖子 應(yīng)激 轉(zhuǎn)化 差異蛋白 細胞因子　出處：《華中農(nóng)業(yè)大學》2015年碩士論文　論文類型：學位論文

【摘要】：剛地弓形蟲(Toxoplasma gondii)是一種專性細胞內(nèi)寄生的原蟲,可以引起嚴重的人畜共患寄生蟲病。弓形蟲宿主范圍極其廣泛,可以感染家禽、家畜、伴侶動物以及人類在內(nèi)的所有溫血動物。對于免疫功能健全的個體,弓形蟲的感染不表現(xiàn)出明顯的臨床癥狀,蟲體多以半休眠的組織包囊形式在宿主腦部、肌肉及眼部定居,與機體長期共存。但是包囊并不是無威脅的存在,當宿主的免疫力低下時,包囊會再活化,進而由緩殖子轉(zhuǎn)變?yōu)樗僦匙?引起急性感染。因此,弓形蟲速殖子和緩殖子的轉(zhuǎn)化在弓形蟲致病過程中扮演著關(guān)鍵的角色。研究表明,運輸應(yīng)激可以降低宿主的免疫力,從而導致多種疾病的復發(fā),據(jù)此推測,弓形蟲作為一種機會性致病病原體,運輸應(yīng)激可能誘發(fā)弓形蟲慢性感染小鼠腦內(nèi)包囊的再活化。弓形蟲速殖子和緩殖子轉(zhuǎn)化過程中,多種期特異性蛋白、熱休克蛋白以及代謝酶類的表達發(fā)生明顯變化,它們對弓形蟲的生長以及代謝有著重要的調(diào)控作用。因此,本研究針對弓形蟲速殖子和緩殖子轉(zhuǎn)化過程中可能的差異蛋白進行篩選及鑒定。本研究首先建立運輸應(yīng)激誘導弓形蟲緩殖子向速殖子轉(zhuǎn)化的動物模型,驗證了運輸應(yīng)激誘使包囊再活化,即由緩殖子轉(zhuǎn)化成速殖子;隨后模擬運輸應(yīng)激,利用細胞因子抗體芯片檢測小鼠血清中細胞因子的變化,結(jié)果顯示小鼠外周血血清中γ干擾素(IFN-γ),白細胞介素(IL-1α),白細胞介素(IL-4),白細胞介素(IL-10),白細胞介素(IL-12)以及巨噬細胞集落刺激因子(M-CSF)的表達水平在應(yīng)激后顯著下降;最后利用雙向電泳以及質(zhì)譜鑒定技術(shù)篩選并鑒定出了緩殖子向速殖子轉(zhuǎn)化過程中的47個鼠源蛋白和15個弓形蟲蛋白。具體工作包括以下幾個方面:(1)弓形蟲緩殖子向速殖子轉(zhuǎn)化動物模型的建立利用運輸應(yīng)激成功誘導弓形蟲腦包囊的再活化,即由緩殖子轉(zhuǎn)變成速殖子。將弓形蟲PRU株慢性感染小鼠模擬運輸應(yīng)激3天后,利用Real-time PCR分別檢測弓形蟲速殖子期特異性基因SAG1和緩殖子期特異性基因BAG1的m RNA相對表達水平。結(jié)果顯示在運輸應(yīng)激3天后,BAG1的相對表達水平下降,SAG1的相對表達水平顯著上升,表明運輸應(yīng)激誘導包囊的再活化,即從緩殖子向速殖子轉(zhuǎn)化。(2)運輸應(yīng)激影響弓形蟲慢性感染小鼠的免疫水平利用弓形蟲緩殖子向速殖子轉(zhuǎn)化的動物模型,分別收集運輸應(yīng)激前,應(yīng)激1天,應(yīng)激2天,應(yīng)激3天的小鼠外周血血清,利用細胞因子抗體芯片檢測小鼠細胞因子的變化。結(jié)果顯示,IFN-γ,IL-1α,IL-4,IL-10,IL-12以及M-CSF 6種細胞因子水平隨著應(yīng)激時間的延長逐漸下降,在應(yīng)激3天后顯著下降。表明運輸應(yīng)激降低慢性感染小鼠的免疫水平。(3)弓形蟲緩殖子向速殖子轉(zhuǎn)化差異蛋白的篩選及鑒定利用弓形蟲緩殖子向速殖子轉(zhuǎn)化的動物模型,分別收集并純化運輸應(yīng)激前,應(yīng)激1天,應(yīng)激2天,應(yīng)激3天的小鼠腦組織包囊,提取包囊總蛋白,利用雙向電泳技術(shù)篩選緩殖子向速殖子轉(zhuǎn)化過程中的差異蛋白并進行質(zhì)譜鑒定。結(jié)果顯示,47個鼠源蛋白和15個弓形蟲相關(guān)蛋白的表達量在緩殖子向速殖子轉(zhuǎn)化過程中顯著變化;隨后利用Real-time PCR驗證了弓形蟲肌動蛋白(Actin),微線體蛋白13(MIC13),致密顆粒蛋白9(GRA9),致密顆粒蛋白1(GRA1)和烯醇酶1(ENO1)的m RNA水平在運輸應(yīng)激后顯著上升,與雙向電泳結(jié)果一致,表明緩殖子向速殖子轉(zhuǎn)化過程中多種宿主和蟲體的蛋白表達發(fā)生變化。本研究利用小鼠運輸應(yīng)激模型證實了應(yīng)激導致機體免疫力下降;成功篩選并鑒定出了弓形蟲緩殖子向速殖子轉(zhuǎn)化過程中的47個鼠源蛋白和15個弓形蟲相關(guān)蛋白,為闡明弓形蟲速殖子和緩殖子轉(zhuǎn)化機制提供了理論依據(jù)。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is an obligate intracellular parasitic protozoa, can cause serious zoonotic parasitic diseases. Toxoplasma gondii can infect a broad host range, poultry, livestock, all warm blooded animal companion animal and human beings. The immune function of healthy individuals, Toxoplasma infection do not show obvious clinical symptoms, the body in the form of semi dormant tissue cysts in the host brain, eye and muscle settled. But long-term coexistence with the body, cyst is not threatened, when the host's immune system is low, will be activated and the cyst, bradyzoite into tachyzoites that caused by acute infection. Therefore, the transformation of Toxoplasma gondii tachyzoite and bradyzoite of plays a key role in the pathogenesis of toxoplasmosis. The results show that transport stress can reduce host immunity, leading to a variety of diseases We speculated that the recurrence of Toxoplasma gondii as an opportunistic pathogen, transport stress may re activation induced by chronic Toxoplasma infected mouse brain cyst. The transformation process of Toxoplasma tachyzoites and in a variety of stage specific protein, heat shock protein and metabolic enzymes expression changed significantly, with regulation an important role of Toxoplasma growth and metabolism. Therefore, screening and identification of possible differences in protein transformation process of this research and the tachyzoites of Toxoplasma gondii tachyzoites in animal models. In this study we developed transport stress induced bow gondii bradyzoite into tachyzoites, verify the transport stress inducing cyst re activation from tachyzoites into tachyzoites; then simulated transport stress, detecting the changes of cytokines in the serum of mice using cytokine antibody array, results showed that the mice of Zhou Xuexue In the interferon gamma (IFN- gamma), interleukin (IL-1 alpha), interleukin (IL-4), interleukin (IL-10), interleukin (IL-12) and macrophage colony-stimulating factor (M-CSF) expression level decreased significantly after stress; finally by two-dimensional electrophoresis and the screening and identification of mass spectrometry and identified 47 mouse protein tachyzoites into tachyzoites of Toxoplasma gondii and 15 protein. The specific work includes the following aspects: (1) Toxoplasma gondii tachyzoites to establish animal model of tachyzoites reactivation induced cerebral Toxoplasma cysts the use of transport stress, from tachyzoites into tachyzoites. The chronic infection of Toxoplasma gondii PRU strain mice simulated transport stress after 3 days, we detected m RNA of Toxoplasma gondii tachyzoite stage specific gene SAG1 and tachyzoites stage specific gene BAG1 the expression level by using Real-time PCR. The results showed In the transport stress after 3 days, the relative expression level of BAG1 decreased significantly increased the relative expression level of SAG1, showed that the reactivation of transport stress induced cysts, which transformed from tachyzoites to tachyzoites. (2) the transport of animal model of chronic infection of Toxoplasma gondii in mice with immune level arch insect bradyzoite into tachyzoites stress effects were collected before 1 days of transport stress, stress, stress for 2 days, the serum of mice 3 days of stress, the changes of cytokines were detected by cytokine antibody array. The results showed that IFN- gamma, IL-1 alpha, IL-4, IL-10, IL-12 and M-CSF 6 kinds of cytokines decreased gradually with the prolonging of stress time, stress in 3 days decreased significantly. That transport stress reduce chronic infection of mice immune level. (3) insect bow shape using screening and identification of Toxoplasma gondii tachyzoites into proteins to tachyzoite to bradyzoite tachyzoites 杞寲鐨勫姩鐗╂ā鍨，

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