【作者】 李风铃；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2009， 博士

【摘要】 大多数鱼类从受精卵开始就一直生活在水中,而它们所赖以生存的水环境充满了大量的微生物,无时无刻不对它们进行攻击,产生威胁。成年鱼类在抵御病原物进行自身免疫保护时,先天性免疫系统首先发挥作用,然后是适应性免疫系统发生反应。而作为鱼类的胚胎,各种免疫器官尚未发育成熟,它们是如何成功抵御微生物的侵袭而存活下来的呢?适应性免疫系统又是在什么时期开始具有功能活性的?一直以来,这方面的议题一直是发育免疫学的研究热点。我们实验室之前已经研究了鱼类先天性免疫系统的发生。在此基础上,本文首先在斑马鱼中鉴定了适应性免疫系统标志性基因的表达以及对脂多糖(LPS)的免疫应答,并进行了分析讨论。我们还从重要的养殖鱼类半滑舌鳎中分离出一个免疫相关转录因子Ikaros,并对其进行了分析和表达研究。本论文第一部分主要研究了适应性免疫系统的早期发生和发育。我们选择的适应性免疫系统的关键基因是Rag2、AID、TCRAC、IgLC-1、mIg、sIg和Mznf297,首先对它们在斑马鱼的不同发育时期以及成鱼中的表达模式进行了研究,然后用LPS对斑马鱼的胚胎/幼鱼以及成鱼进行感染,进一步分析上述基因表达水平的变化。根据实验结果,我们发现除了Rag2和Mznf297之外,其余5个基因在胚胎发生早期以及幼鱼时期表达量都非常低,随着斑马鱼胚胎发育的进行,表达量也相应提高。而对于Rag2和Mznf297,在胚胎发育早期即原肠期,我们检测到了异常的高水平表达,推测可能是因为母源性的mRNA在发育早期大量存在于胚胎中导致。而随着幼鱼发育,二者的表达水平逐步稳定升高。当我们用LPS对胚胎/幼鱼进行感染后,发现大多数基因都做出了迅速应答,表达水平产生了明显变化。除IgLC-1和sIg之外,其余基因在受精后8天就发生了表达量的显著性提高,这表明适应性免疫系统在此时期已经可以对病原感染做出应答。而IgLC-1和sIg分别是在受精后23天和13天对LPS的刺激具有了较强的反应,表达量开始显著提升。据此,我们能够初步证明,斑马鱼胚胎/幼鱼的适应性免疫系统在个体发育的早期已经逐步建立起来,并且对抗原的侵袭具有了一定的反应能力,其发育成熟的时间可能会稍早于之前认为的4-6周。在本论文的第二部分,我们从重要的经济鱼类半滑舌鳎中分离出了一个关键的血细胞分化相关因子-Ikaros基因。Ikaros的cDNA编码430个氨基酸,其开放阅读框为1290 bp。用Ikaros基因的氨基酸序列与其他物种的Ikaros以及家族其他成员包括Helios、Aiolos和Eos的氨基酸序列构建系统进化树,发现半滑舌鳎中的Ikaros与包括青鳉、鰤鱼、虹鳟以及斑马鱼在内的鱼类展示了较近的亲缘关系,在进化树中聚为一支。进一步用部分物种Ikaros的保守区进行同源性比对,发现同源性较高的区域主要集中在N、C末端的锌指结构域。这同哺乳动物中的研究结果是一致的,即N末端和C末端的锌指结构在进化过程中变化较小,保守性较强。我们又利用DNAstar中的CLUSTAL W方法对Ikaros的氨基酸进行了结构上的相似度分析,结果与聚类分析一致,与青鳉、鰤鱼、虹鳟以及斑马鱼相似度较高,分别达80.0%、76.7%、66.7%以及57.3%。因为在其他物种早已证明Ikaros是调控免疫系统发生和发育的转录因子,为了验证它是否与免疫相关,我们首先利用实时定量RT-PCR技术检测了其在不同组织的表达情况。结果显示,Ikaros基因在半滑舌鳎中的胸腺、肝脏和脾中表达量较高,在肾脏、精巢和心脏中也有一定量的表达,而在肠、皮肤以及鳃中几乎检测不到或者表达量很低。这与美西螈、晶吻鰩和七鳃鳗中的检测结果基本一致,这表明了半滑舌鳎中的Ikaros基因在主要的免疫器官中均有表达,在表达模式上也具有一定的保守性。这暗示我们,半滑舌鳎中的Ikaros可能与其他物种的Ikaros一样,在免疫防御上具有一定的功能。我们从半滑舌鳎的头肾中分离出巨噬细胞,进行培养,然后用LPS或LTA对其刺激,收集不同处理样品并运用实时定量技术鉴定Ikaros基因的表达变化。结果表明,相比较于对照组,LPS或LTA处理后Ikaros表达量显著提高,这表明Ikaros基因是一种可以在巨噬细胞中表达的受LPS和LTA调控的免疫防御分子。更多还原

【Abstract】 Fish has been living in the water from the stage of fertilized eggs, however, at the same time, all kinds of pathogens also lives around them and attack them all the time. The adult fish will carry out innate immune system firstly when they are attacked, and if pathogens have invaded into body, fish will begin to start the adaptive immune system (AIS). Then how do the embryos resist attack from pathogens successfully and protect themselves? When do the AIS functionally mature and what is the protection mechanism of immune system? The issues have been focused for a long time. To be close to the answers, the following research is carried out. Firstly, the expression of some marker genes involved in the AIS and response to LPS in zebrafish embryos/larvae are detected, and then Ikaros gene which is a transcription factor related to blood cell differentiation is cloned and analyzed about its structure and expression pattern.The first section of this paper dealt with the early manifestation of the AIS in zebrafish. The ontogenetic and differential expressions of seven key genes (Rag2, AID, TCRAC, IgLC-1, mIg, sIg and Mznf297) that are the suitable markers for indicating the maturation of AIS and their responses to the challenge with LPS during development of D. rerio are demonstrated. Apart from Rag2 and Mznf297, the remaining five genes (AID, TCRAC, IgLC-1, mIg and sIg) display generally similar trends of expression with the expression levels being relatively low at early stages and then rising subsequently. Rag2 and Mznf297 are both expressed at relatively high levels at the gastrula stage, decrease dramatically later, followed by a slight steady rise. In contrast, all these genes are capable of responding to the challenge with LPS in the embryos/larvae by up-regulating their expression levels at least from 23 dpf (day post fertilization) onward in a fashion similar to that of adult D. rerio, which has a mature function in AIS. It is therefore suggested that the AIS in D. rerio larvae has been founded during the course of ontogeny development, slightly earlier than considered previously. The cDNA of Ikaros from half-smooth tongue sole codes for a protein of 430 amino acids. To investigate the relationship between Ikaros from half-smooth tongue sole and other species, a phylogenetic tree is constructed using the amino acid sequence of Ikaros and that of other representative Ikaros from 14 species and other family members including Helios, Aiolos, Eos. It is found that Ikaros from half-smooth tongue sole formed a cluster together with that of some fish including Japanese medaka, Japanese amberjack, trout and zebrafish. Further comparison between some species and Ikaros reveals that the regions that show higher homology focus zinc finger domains at N and C-terminal. This is consistent with previous results. Using the method of CLUSTAL W in DNAstar, the identity in structure between half-smooth tongue sole and other species is analyzed, and the result is similar to that of phylogenetic tree. Ikaros from half-smooth tongue sole shows higher identity with Japanese medaka, Japanese amberjack, trout and zebrafish, the percent identity is 80.0%, 76.7%, 66.7% and 57.3%, respectively. In many species, Ikaros has been proved to be a transcription factor related to immune system, and to test this, a series of experiments are carried out. Firstly, the expression of Ikaros gene in different organs is detected. According to the result of QRT-PCR, Ikaros gene shows higher expression levels in thymus, liver and spleen, and moderate expression levels in kidney, testis and heart; however, in intestine, skin or gill, we hardly detected expression of Ikaros gene. This is consistent with that of skate, lamprey and axolotl. Ikaros gene shows different expression levels in organs related to immune and conservative expression pattern, which imply that Ikaros from half-smooth tongue sole probably has the same function with that of other species. Then, macrophage cells from the head kidney of half-smooth tongue sole are isolated and have been cultured for 7 days. At the seventh day, the cells are challenged by LPS or LTA, and then detected by Q-PCR technology. Compared to the control, the expression levels of Ikaros gene are up-regulated significantly in cells post LPS and LTA challenge. This indicates Ikaros gene which has showed expression in macrophage cells from head kidney of half-smooth tongue sole is a factor related to immune.更多还原