【摘要】：新孢子蟲病是由新孢子蟲寄生在宿主機體內引起的原蟲病，呈世界性分布。新孢子蟲可感染犬、牛、羊、馬、鹿和狐貍等多種動物，并引起腦膜腦炎、多發(fā)性肌炎、神經根神經炎以及孕畜的流產、死胎、木乃伊胎和新生兒的運動障礙等。對畜牧業(yè)和養(yǎng)殖業(yè)造成重大的經濟損失。 EGFR信號通路是生命活動中重要的信號通路，調節(jié)細胞的增殖、凋亡、遷移、存活等一系列復雜的過程。研究發(fā)現弓形蟲感染早期可激活宿主細胞EGFR信號通路，阻止自噬蛋白對弓形蟲的殺傷。但新孢子蟲侵入對宿主細胞EGFR信號通路的作用尚未見報道。本研究通過新孢子蟲速殖子體外培養(yǎng)方法建立、速殖子侵入對宿主細胞EGFR信號通路的激活和抑制EGFR信號通路分子對速殖子侵入的影響的系統(tǒng)研究，闡明新孢子蟲侵入對宿主細胞EGFR信號通路的調控和該通路在蟲體侵入過程中的作用，對揭示新孢子蟲與宿主互作機制、致病機制等具有重要學術價值和科學意義。 主要研究內容如下： 新孢子蟲速殖子體外培養(yǎng)分別用293T、HCT-8、Hela和Vero細胞培養(yǎng)新孢子蟲速殖子，觀察并計數速殖子數量和繪制生長曲線。吖啶噔染色并用激光共聚焦顯微鏡觀察細胞內速殖子。結果表明，新孢子蟲速殖子可以在293T、HCT-8、Vero和Hela細胞中培養(yǎng)。在293T、HCT-8和Vero細胞中生長較快，感染的第3天開始增多，分別在第5、7和6天達到高峰。而在Hela細胞中速殖子生長較慢，蟲體數量一直處于較低水平。在293T、HCT-8和Vero細胞中納蟲泡較大且速殖子數量較多，而在Hela細胞中納蟲泡較小且速殖子數量較少。為新孢子蟲速殖子體外培養(yǎng)提供新的細胞系—293T和HCT-8。 新孢子蟲侵入對宿主細胞EGFR信號通路的激活在蟲體感染293T細胞后，不同時間（10min、20min、30min、60min）收集細胞，提取細胞總RNA和總蛋白，，分別用Real time PCR和Western Blot方法檢測EGFR及其下游PI3K/Akt信號通路、細胞凋亡和細胞骨架調節(jié)通路分子的變化。結果表明，蟲體侵入能明顯上調EGFR、PI3K、Akt、Bad、Bcl-2、MUC1、Caveolin和E-cadherin基因的表達，下調Bax和P53基因的表達，但不影響Paxillin和β-catenin基因的表達。新孢子蟲侵入可磷酸化EGFR(Tyr1173)、Ak（tSer473）和Bad（Ser112）。用EGFR抑制劑AG1478可減少新孢子蟲對宿主細胞EGFR和Akt的磷酸化。且PI3K抑制劑LY294002可減少新孢子蟲對Akt和Bad的磷酸化。綜上表明，新孢子蟲速殖子侵入可激活宿主細胞EGFR信號通路。 抑制EGFR信號通路分子對新孢子蟲侵入宿主細胞的影響分別用EGFR信號通路分子抑制劑（EGFR抑制劑AG1478、PI3K抑制劑LY294002和Akt抑制劑IV）處理293T細胞，速殖子感染后觀察蟲體感染細胞數量和細胞內蟲體數量。結果表明，在24hAG1478和IV處理組感染細胞數量和細胞內蟲體數量明顯減少。而LY294002處理組與對照組相比無明顯變化。表明宿主細胞EGFR信號通路的活化與新孢子蟲速殖子的侵入有關。
[Abstract]:Neosporidiosis is a parasite caused by neosporidium in host. Neosporidium can infect dogs, cattle, sheep, horses, deer and foxes, and cause meningitis, polymyositis, nerve root neuritis and abortion, stillbirth, mummies and movement disorders of newborns. EGFR signaling pathway is an important signal pathway in life activities, regulating cell proliferation, apoptosis, migration, survival and a series of complex processes. It was found that the early infection of Toxoplasma gondii could activate the EGFR signaling pathway of host cells and prevent autophagy from killing Toxoplasma gondii. However, the effect of neosporidium invasion on the EGFR signaling pathway of host cells has not been reported. In this study, a systematic study on the effects of tachyzoite invasion on the activation of EGFR signaling pathway in host cells and the inhibition of EGFR signaling pathway molecules on tachyzoite invasion was carried out by means of in vitro culture method of Neosporidium tachyzoites. It is of great academic value and scientific significance to clarify the regulation of neosporidium invasion on host cell EGFR signaling pathway and its role in the process of parasite invasion. It is of great academic value and scientific significance to reveal the mechanism of interaction between neosporidium and host and the pathogenesis of neosporidium. The main contents of this study were as follows: in vitro culture of Neosporidium tachyzoites was carried out with 293T HCT-8 Hela and Vero cells respectively. The number of tachyzoites was observed and counted and the growth curve was plotted. Acridine dyed and observed by laser confocal microscope. The results showed that the Tachyzoites of Neosporidium could be cultured in 293T HCT-8 Vero and Hela cells. The growth rate of HCT-8 and Vero cells increased rapidly on the 3rd day of infection and reached the peak on the 7th and 6th day, respectively. However, in Hela cells, the growth of tachyzoites was slow and the number of larvae was at a low level. In 293TnHCT-8 and Vero cells, the vesicles were larger and the number of tachyzoites was larger, but in Hela cells the vesicles were smaller and the number of tachyzoites was less. A new cell line-293T and HCT-8 were provided for the culture of Tachyzoites of Neosporidium in vitro. Activation of EGFR signaling pathway in host cells induced by neosporidium. After infection with 293T cells, cells were collected at different times (10 min, 20 min, 30 min or 60 min), total RNA and total protein were extracted, and EGFR and its downstream PI3K/Akt signaling pathway were detected by Real time PCR and Western Blot, respectively. Apoptosis and cytoskeleton regulatory pathway molecule changes. The results showed that the expression of Caveolin and E-cadherin genes was up-regulated by EGFR PI3KPI3KHN AktBcl-2UC1C1Caveolin and E-cadherin genes, and the expression of Bax and p53 genes was down-regulated, but the expression of Paxillin and 尾 -catenin genes was not affected. Neosporidium invades phosphorylated EGFR (Tyr1173), Ak (tSer473) and Bad (Ser112). EGFR inhibitor AG1478 can reduce the phosphorylation of EGFR and Akt by neosporidium. PI3K inhibitor LY294002 could reduce the phosphorylation of Akt and Bad by neosporidium. It is concluded that the invasion of Tachyzoites by Neosporidium activates the EGFR signaling pathway of host cells. Inhibitory effects of EGFR signaling pathway molecules on invasion of neosporidium into host cells 293T cells were treated with EGFR signaling pathway inhibitors (EGFR inhibitor AG1478 PI3K inhibitor LY294002 and Akt inhibitor IV), respectively. The number of infected cells and the number of intracellular worms were observed after tachyzoite infection. The results showed that the number of infected cells and the number of intracellular parasites decreased significantly in 24hAG1478 and IV groups. There was no significant change in LY294002 treatment group compared with control group. These results suggest that the activation of EGFR signaling pathway in host cells is related to the invasion of Tachyzoites of Neosporidium.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.7

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1 金春梅;于龍政;張守發(fā);;新孢子蟲致密顆粒蛋白截短型NcGRA7t的體外表達及免疫活性檢測[J];畜牧與獸醫(yī);2012年12期

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