新孢子虫侵入对宿主细胞EGFR信号通路的作用
发布时间：2018-08-18 14:46
【摘要】：新孢子虫病是由新孢子虫寄生在宿主机体内引起的原虫病，呈世界性分布。新孢子虫可感染犬、牛、羊、马、鹿和狐狸等多种动物，并引起脑膜脑炎、多发性肌炎、神经根神经炎以及孕畜的流产、死胎、木乃伊胎和新生儿的运动障碍等。对畜牧业和养殖业造成重大的经济损失。 EGFR信号通路是生命活动中重要的信号通路，调节细胞的增殖、凋亡、迁移、存活等一系列复杂的过程。研究发现弓形虫感染早期可激活宿主细胞EGFR信号通路，阻止自噬蛋白对弓形虫的杀伤。但新孢子虫侵入对宿主细胞EGFR信号通路的作用尚未见报道。本研究通过新孢子虫速殖子体外培养方法建立、速殖子侵入对宿主细胞EGFR信号通路的激活和抑制EGFR信号通路分子对速殖子侵入的影响的系统研究，阐明新孢子虫侵入对宿主细胞EGFR信号通路的调控和该通路在虫体侵入过程中的作用，对揭示新孢子虫与宿主互作机制、致病机制等具有重要学术价值和科学意义。 主要研究内容如下： 新孢子虫速殖子体外培养分别用293T、HCT-8、Hela和Vero细胞培养新孢子虫速殖子，观察并计数速殖子数量和绘制生长曲线。吖啶噔染色并用激光共聚焦显微镜观察细胞内速殖子。结果表明，新孢子虫速殖子可以在293T、HCT-8、Vero和Hela细胞中培养。在293T、HCT-8和Vero细胞中生长较快，感染的第3天开始增多，分别在第5、7和6天达到高峰。而在Hela细胞中速殖子生长较慢，虫体数量一直处于较低水平。在293T、HCT-8和Vero细胞中纳虫泡较大且速殖子数量较多，而在Hela细胞中纳虫泡较小且速殖子数量较少。为新孢子虫速殖子体外培养提供新的细胞系—293T和HCT-8。 新孢子虫侵入对宿主细胞EGFR信号通路的激活在虫体感染293T细胞后，不同时间（10min、20min、30min、60min）收集细胞，提取细胞总RNA和总蛋白，，分别用Real time PCR和Western Blot方法检测EGFR及其下游PI3K/Akt信号通路、细胞凋亡和细胞骨架调节通路分子的变化。结果表明，虫体侵入能明显上调EGFR、PI3K、Akt、Bad、Bcl-2、MUC1、Caveolin和E-cadherin基因的表达，下调Bax和P53基因的表达，但不影响Paxillin和β-catenin基因的表达。新孢子虫侵入可磷酸化EGFR(Tyr1173)、Ak（tSer473）和Bad（Ser112）。用EGFR抑制剂AG1478可减少新孢子虫对宿主细胞EGFR和Akt的磷酸化。且PI3K抑制剂LY294002可减少新孢子虫对Akt和Bad的磷酸化。综上表明，新孢子虫速殖子侵入可激活宿主细胞EGFR信号通路。 抑制EGFR信号通路分子对新孢子虫侵入宿主细胞的影响分别用EGFR信号通路分子抑制剂（EGFR抑制剂AG1478、PI3K抑制剂LY294002和Akt抑制剂IV）处理293T细胞，速殖子感染后观察虫体感染细胞数量和细胞内虫体数量。结果表明，在24hAG1478和IV处理组感染细胞数量和细胞内虫体数量明显减少。而LY294002处理组与对照组相比无明显变化。表明宿主细胞EGFR信号通路的活化与新孢子虫速殖子的侵入有关。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7
本文编号：2189804
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7
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相关期刊论文 前1条
1 金春梅;于龙政;张守发;;新孢子虫致密颗粒蛋白截短型NcGRA7t的体外表达及免疫活性检测[J];畜牧与兽医;2012年12期
本文编号：2189804
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