弓形虫TgCtwh3和RH株调控宿主巨噬细胞凋亡的研究

发布时间：2019-07-05 05:46

【摘要】：背景弓形虫是一种专性寄生于有核细胞内的顶复门原虫，具有机会致病性。猫科动物为其终宿主，中间宿主为包括人在内的几乎所有温血动物。已知北美和欧洲的弓形虫基因型主要为I、II和III型；我国的优势基因型为Chinese1型。大量研究证实，弓形虫通过抑制宿主细胞的凋亡在宿主体内建立广泛而有效的感染，维持自身的生存与繁殖。目前，，针对弓形虫调控宿主细胞凋亡机制的研究主要集中于死亡受体和线粒体两个经典凋亡途径，而关于内质网应激途径的相关研究较少。此外，研究的虫株多为I型的RH强毒株，而针对我国流行的优势基因型Chinese1型（TgCtwh3，强毒株）调控宿主细胞凋亡的研究较为罕见。目的观察感染弓形虫两种分离株（TgCtwh3和RH）的巨噬细胞（Raw264.7）凋亡，观察各种凋亡相关因子的转录表达，旨在探讨我国流行的弓形虫TgCtwh3株与I型的RH株调控宿主细胞凋亡机制的差异，以期为我国弓形虫病的防治提供理论与实验依据。方法1）将研究对象分为3组：正常对照组、TgCtwh3感染组和RH感染组，分别给予凋亡诱导剂星状孢子素（staurosporine）处理，通过流式细胞术检测3组巨噬细胞的凋亡；2）应用qRT-PCR检测3组细胞的促凋亡家族成员（Bax，Bad）、抗凋亡家族成员（Bcl-2，Bcl-xl），内质网应激标志物CHOP的转录水平；3）应用Western blotting检测3组巨噬细胞的促凋亡蛋白（Bax，Bad）、抗凋亡蛋白（Bcl-2，Bcl-xl）、内质网应激标志物CHOP以及凋亡关键酶Caspase9的蛋白表达水平。结果1）感染组巨噬细胞早期凋亡率较正常对照均明显下降。2）qRT-PCR结果显示，感染组Bcl-2，Bcl-xl及CHOP的转录水平均明显升高，Bax和Bad的转录水平无明显变化。此外，TgCtwh3组的Bcl-xl转录水平明显高于RH组，而两组的Bcl-2转录水平无明显差异。3）Western Blot结果显示，感染组巨噬细胞的Bcl-2，Bcl-xl及CHOP蛋白表达水平均显著升高，Bax和Bad的蛋白表达水平无明显变化。其中，TgCtwh3组的Bcl-xl蛋白表达水平明显高于RH感染组，而两组的Bcl-2的蛋白表达水平无明显差异。结论在弓形虫感染早期阶段，TgCtwh3和RH分离株通过上调Bcl-2，Bcl-xl的表达均显著抑制了宿主细胞凋亡；TgCtwh3株抑制凋亡的作用较RH强，虽同为强毒株，TgCtwh3株毒力稍弱。促凋亡相关基因除CHOP外，Bax、Bad、Caspase9均无明显变化。感染早期抑制凋亡作用强于促凋亡作用。
[Abstract]:Background Toxoplasma gondii (Toxoplasma gondii) is a kind of parasite parasitic in nucleated cells, which has the opportunity to be pathogenic. Cats are their final hosts, and the intermediate hosts are almost all warm-blooded animals, including humans. It is known that the main genotypic of Toxoplasma gondii in North America and Europe is I, type II and type III, and the dominant genotype in China is Chinese1. A large number of studies have confirmed that Toxoplasma gondii can maintain its own survival and reproduction by inhibiting the apoptosis of host cells and establishing extensive and effective infection in host body. At present, the mechanism of Toxoplasma gondii regulating host cell apoptosis mainly focuses on two classical apoptotic pathways: death receptor and mitochondria, but there are few studies on endoplasmic reticulum stress pathway. In addition, most of the insect strains studied were type I RH virulent strains, but the dominant genotype Chinese1 (TgCtwh3, virulent strain) in China was rare to regulate host cell apoptosis. Objective to observe the apoptosis of macrophages (Raw264.7) infected with two isolates of Toxoplasma gondii (TgCtwh3 and RH) and to observe the transcriptional expression of various apoptosis related factors in order to explore the difference of apoptosis mechanism between TgCtwh3 strain and type I strain of Toxoplasma gondii in China, so as to provide theoretical and experimental basis for the prevention and treatment of Toxoplasma gondii in China. Methods 1) the subjects were divided into three groups: normal control group, TgCtwh3 infection group and RH infection group, treated with (staurosporine), an apoptosis inducer, respectively, and the apoptosis of macrophages in three groups was detected by flow cytometry. 2) the transcription levels of apoptosis family members (Bax,Bad), anti-apoptosis family members (Bcl-2,Bcl-xl) and endoplasmic reticulum stress marker CHOP in the three groups were detected by qRT-PCR. 3) the expression of apoptosis promoting protein (Bax,Bad), anti-apoptosis protein (Bcl-2,Bcl-xl), endoplasmic reticulum stress marker CHOP and apoptosis key enzyme Caspase9 in macrophages of three groups were detected by Western blotting. Results 1) the early apoptosis rate of macrophages in the infected group was significantly lower than that in the normal control group. 2) qRT-PCR results showed that the transcriptional levels of Bcl-2,Bcl-xl and CHOP in the infected group were significantly increased, but the transcriptional levels of Bax and Bad were not significantly changed. In addition, the transcription level of Bcl-xl in TgCtwh3 group was significantly higher than that in RH group, but there was no significant difference in Bcl-2 transcription level between the two groups. 3 the results of) Western Blot showed that the expression levels of Bcl-2,Bcl-xl and CHOP protein in macrophages of infected group were significantly increased, but the protein expression levels of Bax and Bad had no significant change. The expression level of Bcl-xl protein in TgCtwh3 group was significantly higher than that in RH infection group, but there was no significant difference in Bcl-2 protein expression level between the two groups. Conclusion in the early stage of Toxoplasma gondii infection, TgCtwh3 and RH isolates significantly inhibited the apoptosis of host cells by up-regulating the expression of Bcl-2,Bcl-xl, and the inhibitory effect of TgCtwh3 strain on apoptosis was stronger than that of RH strain, although it was both virulent, but the toxicity of TgCtwh3 strain was slightly weaker. There was no significant change in Bax,Bad,Caspase9 except CHOP. The effect of inhibiting apoptosis in the early stage of infection was stronger than that of promoting apoptosis.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R531.8

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