我国内蒙古东北部三种泡状棘球绦虫感染引起宿主体内基因差异表达的研究
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摘要
唐崇惕等对我国内蒙古东北部呼伦贝尔草原泡状棘球蚴的病原情况进行调查时,在沙狐(Vulpes corsac)体内发现多房棘球绦虫的成虫,在布氏田鼠(Microtus brandti)体内发现了泡状棘球蚴。随后的研究发现,流行于此地区的泡状棘球蚴病原应包含独立的三种,分别为:欧洲的多房棘球绦虫(Echinococcus multilocularis),西伯利亚棘球绦虫(Echinococcus sibiricensis)以及苏俄棘球绦虫(Echinococcus russicensis)。
本论文主要对这三种泡状棘球绦虫引起的宿主体内基因差异表达变化进行研究,包括以下三个部分:
1.三种泡状棘球绦虫棘球蚴核糖体基因(rDNA) ITS(Internal TranscribedSpacer,内转录间隔区)序列比较分析:
通过DNA聚合酶链式反应对三种泡状棘球绦虫棘球蚴核糖体内转录间隔序列(ITS序列)进行扩增,将扩增产物进行克隆测序,所得序列结果进行比对和聚类分析。发现同种泡状棘球蚴的ITS序列有至少两种不同序列,序列复杂多变,尤其体现在ITS2序列部分。然而西伯利亚泡状棘球蚴的序列明显与另2虫种不同,尤其体现在ITS1序列部分。聚类分析显示来源于同种泡状棘球蚴的不同克隆序列大多能类聚在一起,表明这三种泡状棘球绦虫ITS序列间存在差异。
2.三种泡状棘球蚴引起宿主体内基因差异表达的研究:
本部分内容主要是运用基因芯片技术和蛋白质组学技术,结合生物信息学分析,对三种泡状棘球蚴感染的小鼠肝脏内基因表达的变化予以研究。按照结果分析的侧重点不同,共分为两章:
第一章主要分析与讨论在三种泡状棘球蚴感染的宿主体内均有差异表达的基因(共性部分):通过基因芯片试验结果我们发现,在三种泡状棘球蚴感染的小鼠肝脏内表达变化均有显著差异的基因一共有64个,进一步的GO功能分析结果显示它们与几个功能组相关,其中包括了防御反应和免疫功能,氧化还原酶活性功能和细胞代谢功能类别。而蛋白质组学实验结果显示表达变化差异显著的73个蛋白也多与这几大功能类别相关,更为重要的是防御反应和免疫功能相关的蛋白表达值都升高,而具氧化还原酶活性功能以及和细胞代谢功能相关的蛋白表达值都降低,支持了基因芯片的实验结果。
第二章进一步比较与分析感染不同种泡状棘球蚴的各个实验组间基因表达情况的差异(个性部分):发现有278个基因仅在感染了西伯利亚和苏俄泡状棘球蚴的感染组A和B中表达差异显著,而在感染欧洲泡状棘球蚴的感染组C中表达变化不明显。在这278个基因当中,205个上调的基因与多个生物学过程相关,包括防御与免疫应答反应,细胞凋亡,细胞分化,细胞周期,细胞分裂,DNA复制等。同时我们还发现在感染苏俄泡状棘球蚴的小鼠体内与细胞分裂、细胞骨架构建与生成、DNA损伤应答的基因表达值升高的幅度明显高于另两种泡状棘球蚴感染的小鼠,而且后两个GO功能类别未在感染组A或C中被显著富集。表明宿主适应性程度、宿主免疫应答反应差异会造成这三种泡状棘球蚴感染的小鼠体内基因表达变化的不同。
3.感染三种泡状棘球蚴的小鼠体内细胞因子动态变化的研究:
对不同感染组小鼠血清中的Th1/2型细胞因子动态表达变化差异进行研究。结果发现Th1型细胞因子TNF-α和IFN-γ在三种泡状棘球蚴感染感染的小鼠血清中的表达值均在感染初期就略有升高,接种后80到100天时,升高幅度达到峰值;而Th2型细胞因子IL-4在感染初期变化并不显著,在感染约100天之后表达值才显著提高。此外,IL-10也是在感染后期的小鼠血清中被检测到有表达。
同时,三种泡状棘球蚴感染的小鼠血清中这几种细胞因子动态变化存在差异。西伯利亚泡状棘球蚴感染的TNF-α和IFN-γ表达在接种80-100天后达到峰值,之后仍相对维持在较高水平,而感染苏俄泡状棘球蚴和欧洲泡状棘球蚴小鼠血清中这两种细胞因子含量达到峰值后迅速下降,而且幅度更为显著。提示在西伯利亚泡状棘球蚴感染的小鼠体内,TNF-α和IFN-γ高表达的时间更长些,作用也更为持久。此外,从整体上来看,感染欧洲泡状棘球蚴的小鼠体内这两种细胞因子的表达水平均比另两个感染组要低。
At the end of last centry, Tang Chongti et al found there were three types of alveolar echinococcus in the Northeast Inner Mongolia, China. Subsequent studies indicated that they should be regarded as independent species: Echinococcus multilocularis, Echinococcus sibiricensis and Echinococcus russicensis.
In this dissertation, the differential host gene expression during the infection with three alveolar echinococcus was explored.
There are three parts in the paper.
1. The comparative analysis of ITS sequences from three alveolar echinococcus:
By using PCR technology, we amplified ITS sequences from three alveolar echinococcus. The results showed that there were at least 2 different kinds of sequences from the same type of alveolar echinococcus, especially in the ITS2 part. However, the ITSl sequences of Echinococcus sibiricensis were quite different from those of Echinococcus russicensis and Echinococcus multilocularis. As such, the clustering result showed that most of the clone sequences from the same type of alveolar echinococcus can be distinguished from another one, implying the genomic differences in these types of alveolar echinococcus.
2. The study on the differential host gene expression during the infection with three alveolar echinococcus:
In this part, microarray, proteomic and bioinformatic technology were used to explore the differential gene (protein) expression induced by three alveolar echinococcus. The results and discussion part are shown as in two chapters:
Chapter 1: A total of 64 genes were common to infection in the liver of all three alveolar echinococcus with 73 common proteins found. GO analysis of the microarray data showed that up-regulated genes included those involved in immune response and the depressed genes were related to oxidoreductase activity and energy metabolism, which was strongly supported by the proteomic data.
Chapter 2: Furthermore, analysis of the gene expression data indicated 278 genes were significantly changed in the mice infected with Echinococcus sibiricensis and Echinococcus russicensis, but not in those infected with Echinococcus multilocularis. In the 278 genes, 205 were induced including those related to immune response, apoptosis, cell differentiation, cell cycle, cell division and DNA amplification, etc. Moreover, a number of genes were only significantly up-regulated in the mice infected with Echinococcus russicensis, including those related to cell division, cytoskeleton organization and biogenesis, and DNA damage response. These genes in this infected group have a much higer expression ratio than those in the other two groups as well as the GO functional categories "cytoskeleton organization and biogenesis" and "DNA damage response" were only enriched in Echinococcus russicensis infected group.
3. Developmental changes in the serum Th1/2 cytokine expression of mice infected with three alveolar echinococcus:
Th1/2 cytokine expressions were examined in the serum of mice infected with three alveolar echinococcus. The results showed that TNF-αand IFN-γwere slightly induced at early stage of the infection and then remarkably up-regulated at later stage (80-100 day post infection) in all of the infected groups. Contrarily, IL-4 and IL-10 were induced only at the later stage (100 days post infecion).
The expression of TNF-αand IFN-γin the mice infected with Echinococcus sibiricensis reached at the maximum at 80-100 days post infection and then remained at a relatively high level, however, it signicantly reduced in the other two groups. It implied that the Th1 type cytokines TNF-αand IFN-γfunctioned in the Echinococcus sibiricensis-induced infection with a stronger activity. As such, the expression levels of TNF-αand IFN-γwere lower in the mice infected with Echinococcus multilocularis.
本论文主要对这三种泡状棘球绦虫引起的宿主体内基因差异表达变化进行研究,包括以下三个部分:
1.三种泡状棘球绦虫棘球蚴核糖体基因(rDNA) ITS(Internal TranscribedSpacer,内转录间隔区)序列比较分析:
通过DNA聚合酶链式反应对三种泡状棘球绦虫棘球蚴核糖体内转录间隔序列(ITS序列)进行扩增,将扩增产物进行克隆测序,所得序列结果进行比对和聚类分析。发现同种泡状棘球蚴的ITS序列有至少两种不同序列,序列复杂多变,尤其体现在ITS2序列部分。然而西伯利亚泡状棘球蚴的序列明显与另2虫种不同,尤其体现在ITS1序列部分。聚类分析显示来源于同种泡状棘球蚴的不同克隆序列大多能类聚在一起,表明这三种泡状棘球绦虫ITS序列间存在差异。
2.三种泡状棘球蚴引起宿主体内基因差异表达的研究:
本部分内容主要是运用基因芯片技术和蛋白质组学技术,结合生物信息学分析,对三种泡状棘球蚴感染的小鼠肝脏内基因表达的变化予以研究。按照结果分析的侧重点不同,共分为两章:
第一章主要分析与讨论在三种泡状棘球蚴感染的宿主体内均有差异表达的基因(共性部分):通过基因芯片试验结果我们发现,在三种泡状棘球蚴感染的小鼠肝脏内表达变化均有显著差异的基因一共有64个,进一步的GO功能分析结果显示它们与几个功能组相关,其中包括了防御反应和免疫功能,氧化还原酶活性功能和细胞代谢功能类别。而蛋白质组学实验结果显示表达变化差异显著的73个蛋白也多与这几大功能类别相关,更为重要的是防御反应和免疫功能相关的蛋白表达值都升高,而具氧化还原酶活性功能以及和细胞代谢功能相关的蛋白表达值都降低,支持了基因芯片的实验结果。
第二章进一步比较与分析感染不同种泡状棘球蚴的各个实验组间基因表达情况的差异(个性部分):发现有278个基因仅在感染了西伯利亚和苏俄泡状棘球蚴的感染组A和B中表达差异显著,而在感染欧洲泡状棘球蚴的感染组C中表达变化不明显。在这278个基因当中,205个上调的基因与多个生物学过程相关,包括防御与免疫应答反应,细胞凋亡,细胞分化,细胞周期,细胞分裂,DNA复制等。同时我们还发现在感染苏俄泡状棘球蚴的小鼠体内与细胞分裂、细胞骨架构建与生成、DNA损伤应答的基因表达值升高的幅度明显高于另两种泡状棘球蚴感染的小鼠,而且后两个GO功能类别未在感染组A或C中被显著富集。表明宿主适应性程度、宿主免疫应答反应差异会造成这三种泡状棘球蚴感染的小鼠体内基因表达变化的不同。
3.感染三种泡状棘球蚴的小鼠体内细胞因子动态变化的研究:
对不同感染组小鼠血清中的Th1/2型细胞因子动态表达变化差异进行研究。结果发现Th1型细胞因子TNF-α和IFN-γ在三种泡状棘球蚴感染感染的小鼠血清中的表达值均在感染初期就略有升高,接种后80到100天时,升高幅度达到峰值;而Th2型细胞因子IL-4在感染初期变化并不显著,在感染约100天之后表达值才显著提高。此外,IL-10也是在感染后期的小鼠血清中被检测到有表达。
同时,三种泡状棘球蚴感染的小鼠血清中这几种细胞因子动态变化存在差异。西伯利亚泡状棘球蚴感染的TNF-α和IFN-γ表达在接种80-100天后达到峰值,之后仍相对维持在较高水平,而感染苏俄泡状棘球蚴和欧洲泡状棘球蚴小鼠血清中这两种细胞因子含量达到峰值后迅速下降,而且幅度更为显著。提示在西伯利亚泡状棘球蚴感染的小鼠体内,TNF-α和IFN-γ高表达的时间更长些,作用也更为持久。此外,从整体上来看,感染欧洲泡状棘球蚴的小鼠体内这两种细胞因子的表达水平均比另两个感染组要低。
At the end of last centry, Tang Chongti et al found there were three types of alveolar echinococcus in the Northeast Inner Mongolia, China. Subsequent studies indicated that they should be regarded as independent species: Echinococcus multilocularis, Echinococcus sibiricensis and Echinococcus russicensis.
In this dissertation, the differential host gene expression during the infection with three alveolar echinococcus was explored.
There are three parts in the paper.
1. The comparative analysis of ITS sequences from three alveolar echinococcus:
By using PCR technology, we amplified ITS sequences from three alveolar echinococcus. The results showed that there were at least 2 different kinds of sequences from the same type of alveolar echinococcus, especially in the ITS2 part. However, the ITSl sequences of Echinococcus sibiricensis were quite different from those of Echinococcus russicensis and Echinococcus multilocularis. As such, the clustering result showed that most of the clone sequences from the same type of alveolar echinococcus can be distinguished from another one, implying the genomic differences in these types of alveolar echinococcus.
2. The study on the differential host gene expression during the infection with three alveolar echinococcus:
In this part, microarray, proteomic and bioinformatic technology were used to explore the differential gene (protein) expression induced by three alveolar echinococcus. The results and discussion part are shown as in two chapters:
Chapter 1: A total of 64 genes were common to infection in the liver of all three alveolar echinococcus with 73 common proteins found. GO analysis of the microarray data showed that up-regulated genes included those involved in immune response and the depressed genes were related to oxidoreductase activity and energy metabolism, which was strongly supported by the proteomic data.
Chapter 2: Furthermore, analysis of the gene expression data indicated 278 genes were significantly changed in the mice infected with Echinococcus sibiricensis and Echinococcus russicensis, but not in those infected with Echinococcus multilocularis. In the 278 genes, 205 were induced including those related to immune response, apoptosis, cell differentiation, cell cycle, cell division and DNA amplification, etc. Moreover, a number of genes were only significantly up-regulated in the mice infected with Echinococcus russicensis, including those related to cell division, cytoskeleton organization and biogenesis, and DNA damage response. These genes in this infected group have a much higer expression ratio than those in the other two groups as well as the GO functional categories "cytoskeleton organization and biogenesis" and "DNA damage response" were only enriched in Echinococcus russicensis infected group.
3. Developmental changes in the serum Th1/2 cytokine expression of mice infected with three alveolar echinococcus:
Th1/2 cytokine expressions were examined in the serum of mice infected with three alveolar echinococcus. The results showed that TNF-αand IFN-γwere slightly induced at early stage of the infection and then remarkably up-regulated at later stage (80-100 day post infection) in all of the infected groups. Contrarily, IL-4 and IL-10 were induced only at the later stage (100 days post infecion).
The expression of TNF-αand IFN-γin the mice infected with Echinococcus sibiricensis reached at the maximum at 80-100 days post infection and then remained at a relatively high level, however, it signicantly reduced in the other two groups. It implied that the Th1 type cytokines TNF-αand IFN-γfunctioned in the Echinococcus sibiricensis-induced infection with a stronger activity. As such, the expression levels of TNF-αand IFN-γwere lower in the mice infected with Echinococcus multilocularis.
引文
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