新孢子虫激活宿主EGFR信号转导调控细胞凋亡的分子机制
发布时间：2019-03-17 13:54
【摘要】：犬新孢子虫(Neospora caninum)属于顶复门,是一种专门寄生于哺乳宿主细胞内的致病性原虫。它能感染大多数哺乳类动物,其中对牛的危害最为严重。肉牛或奶牛感染N.caninum主要引起流产,给全世界养牛业带来巨大的经济损失。然而,N.caninum的细胞感染机制还没有被完全阐明,尤其N.caninum对宿主信号转导和细胞生存或死亡的调控机制报道甚少。EGFR(epidermal growth factor receptor)通路是参与细胞存活调控的重要信号通路。已经有报道弓形虫能够抑制感染细胞的凋亡;弓形虫感染激活EGFR-AKT信号通路,抑制自噬蛋白对虫体的靶标和杀伤作用。但在新孢子虫,是否其感染能够调控EGFR信号报道较少,能否通过EGFR信号转导控制宿主细胞的凋亡仍未知。因此,在本文中我们将针对N.caninum对宿主EGFR信号转导通路及细胞凋亡调控的分子机制作详细的研究,论文包括以下几个部分:1.通过对新孢子虫刺激后宿主EGFR通路信号蛋白和凋亡相关蛋白的表达检测,结果表明N.caninum感染能迅速激活宿主EGFR、EGFR-AKT和EGFR-MAPK信号通路,进而调控凋亡相关蛋白的表达;2.通过对细胞凋亡相关指标的检测,评估新孢子虫感染对宿主细胞活力和细胞凋亡的影响。结果发现,N.caninum感染早期能够抑制细胞凋亡,且其对凋亡的抑制主要通过EGFR-AKT通路的活化介导;3.对新孢子虫激活EGFR信号转导和抑制细胞凋亡的分子机制进行了探讨,结果发现N.caninum通过EGF-MIC和宿主细胞表面EGF受体的互作,激活宿主EGFR信号转导,进而抑制细胞凋亡;4.使用特异性抑制剂、siRNA阻断和干预宿主EGFR通路后,对新孢子虫细胞感染的影响进行了检测,结果表明抑制EGFR信号通路的活化或沉默EGFR、AKT后,新孢子虫的细胞感染被显著抑制;总之,本研究对新孢子虫调控细胞凋亡的分子机制进行了探讨,结果发现:N.caninum感染能够抑制宿主细胞凋亡,并且其对凋亡的抑制是通过含EGF样结构域的微线蛋白(EGF-MIC)来发挥作用的,N.caninum MIC-EGF通过与宿主EGFR的互作,介导EGFR-AKT信号通路的活化,并进一步调控凋亡相关蛋白的表达,结果发挥抑制细胞凋亡的效应。这些新的信息将有助于我们更深入地认识EGFR信号转导在虫体感染过程中的功能及新孢子虫的细胞感染和生存机制;为新孢子虫参与宿主功能的调控提供了理论依据,对今后寻找和设计用于化学干预新孢子虫病的有效药物具有重要意义。
[Abstract]:Neosporidium canis (Neospora caninum) is a kind of pathogenic protozoa that parasitize the host cells of lactation. It can infect most mammals, among which the most serious harm to cattle. Beef cattle or cows infected with N.caninum mainly cause abortion and bring huge economic losses to cattle industry all over the world. However, the mechanism of N.caninum cell infection has not been fully clarified. In particular, there are few reports on the regulation of host signal transduction and cell survival or death by N.caninum. EGFR (epidermal growth factor receptor) pathway is an important signal pathway involved in the regulation of cell survival. It has been reported that Toxoplasma gondii can inhibit the apoptosis of infected cells and that Toxoplasma gondii infection activates EGFR-AKT signaling pathway and inhibits the target and killing effect of autophagy protein on infected cells. However, in Neosporidium, whether the infection can regulate the EGFR signal is less reported, whether the EGFR signal transduction can control the apoptosis of host cells is still unknown. Therefore, in this paper, we will make a detailed study of the molecular mechanism of N.caninum on the host EGFR signal transduction pathway and apoptosis regulation. The thesis includes the following parts: 1. After stimulation with neosporidium, the expression of EGFR signaling protein and apoptosis-related protein in host was detected. The results showed that N.caninum infection could activate the EGFR,EGFR-AKT and EGFR-MAPK signaling pathway of host rapidly, and then regulate the expression of apoptosis-related protein. 2. The effect of Neosporidium infection on cell viability and apoptosis was evaluated by detecting apoptosis-related indexes. The results showed that N.caninum infection could inhibit apoptosis in early stage, and its inhibition was mainly mediated by activation of EGFR-AKT pathway. The molecular mechanism of neosporidium activating EGFR signal transduction and inhibiting cell apoptosis was discussed. The results showed that N.caninum could activate host EGFR signal transduction through interaction between EGF-MIC and host cell surface EGF receptor, and then inhibit cell apoptosis; 4. The effects of siRNA on Neosporidium cell infection were detected after blocking and interfering the host EGFR pathway with specific inhibitors. The results showed that after inhibiting the activation of EGFR signaling pathway or silencing EGFR,AKT, the cell infection of Neosporidium was significantly inhibited. In conclusion, this study discussed the molecular mechanism of neosporidium regulating cell apoptosis. The results showed that N.caninum infection could inhibit the apoptosis of host cells. The inhibition of apoptosis is mediated by the EGF-like domain EGF-MIC, which mediates the activation of the EGFR-AKT signaling pathway by interacting with the host EGFR. And further regulate the expression of apoptosis-related proteins, resulting in the inhibition of cell apoptosis effect. These new information will help us to further understand the function of EGFR signal transduction in the process of parasite infection and the cell infection and survival mechanism of Neosporidium. It provides a theoretical basis for neosporidium to participate in the regulation of host function, and it is of great significance to find and design effective drugs for chemical intervention of neosporidiosis in the future.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.7
本文编号：2442363
[Abstract]:Neosporidium canis (Neospora caninum) is a kind of pathogenic protozoa that parasitize the host cells of lactation. It can infect most mammals, among which the most serious harm to cattle. Beef cattle or cows infected with N.caninum mainly cause abortion and bring huge economic losses to cattle industry all over the world. However, the mechanism of N.caninum cell infection has not been fully clarified. In particular, there are few reports on the regulation of host signal transduction and cell survival or death by N.caninum. EGFR (epidermal growth factor receptor) pathway is an important signal pathway involved in the regulation of cell survival. It has been reported that Toxoplasma gondii can inhibit the apoptosis of infected cells and that Toxoplasma gondii infection activates EGFR-AKT signaling pathway and inhibits the target and killing effect of autophagy protein on infected cells. However, in Neosporidium, whether the infection can regulate the EGFR signal is less reported, whether the EGFR signal transduction can control the apoptosis of host cells is still unknown. Therefore, in this paper, we will make a detailed study of the molecular mechanism of N.caninum on the host EGFR signal transduction pathway and apoptosis regulation. The thesis includes the following parts: 1. After stimulation with neosporidium, the expression of EGFR signaling protein and apoptosis-related protein in host was detected. The results showed that N.caninum infection could activate the EGFR,EGFR-AKT and EGFR-MAPK signaling pathway of host rapidly, and then regulate the expression of apoptosis-related protein. 2. The effect of Neosporidium infection on cell viability and apoptosis was evaluated by detecting apoptosis-related indexes. The results showed that N.caninum infection could inhibit apoptosis in early stage, and its inhibition was mainly mediated by activation of EGFR-AKT pathway. The molecular mechanism of neosporidium activating EGFR signal transduction and inhibiting cell apoptosis was discussed. The results showed that N.caninum could activate host EGFR signal transduction through interaction between EGF-MIC and host cell surface EGF receptor, and then inhibit cell apoptosis; 4. The effects of siRNA on Neosporidium cell infection were detected after blocking and interfering the host EGFR pathway with specific inhibitors. The results showed that after inhibiting the activation of EGFR signaling pathway or silencing EGFR,AKT, the cell infection of Neosporidium was significantly inhibited. In conclusion, this study discussed the molecular mechanism of neosporidium regulating cell apoptosis. The results showed that N.caninum infection could inhibit the apoptosis of host cells. The inhibition of apoptosis is mediated by the EGF-like domain EGF-MIC, which mediates the activation of the EGFR-AKT signaling pathway by interacting with the host EGFR. And further regulate the expression of apoptosis-related proteins, resulting in the inhibition of cell apoptosis effect. These new information will help us to further understand the function of EGFR signal transduction in the process of parasite infection and the cell infection and survival mechanism of Neosporidium. It provides a theoretical basis for neosporidium to participate in the regulation of host function, and it is of great significance to find and design effective drugs for chemical intervention of neosporidiosis in the future.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.7
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1 靳小霞;新孢子虫激活宿主EGFR信号转导调控细胞凋亡的分子机制[D];吉林大学;2017年
，本文编号：2442363
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