【作者】 王光锋；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 博士

【摘要】 文昌鱼属于脊索动物门,头索动物亚门,是与脊椎动物亲缘关系最接近的无脊椎动物,一直被认为是研究脊椎动物起源和进化的模式生物。近年来,人们开始在文昌鱼中探索免疫系统的起源和进化。本文通过细菌感染文昌鱼,研究了文昌鱼清除体内细菌的免疫机制,并通过荧光实时定量PCR技术对文昌鱼的补体关键基因表达模式以及是否存在免疫致敏作用作了研究。首先,通过用绿色荧光蛋白标记的大肠杆菌感染文昌鱼,将文昌鱼切成小段把体液涂抹于载玻片上,在荧光显微镜下观察体液中细菌,证明文昌鱼能有效清除入侵的异源微生物。这是在头索动物中关于体内细菌清除的首次报道。血细胞的吞噬作用被认为是无脊椎动物清除细菌的最重要途径。我们通过组织切片法在文昌鱼体内寻找包含大肠杆菌的细胞,至少在2 000条文昌鱼中都没有发现任何吞噬有大肠杆菌的细胞,表明文昌鱼既使具有自由循环的巨噬细胞,也不是其清除入侵大肠杆菌的主要因素。而体液可能是清除细菌的主要成分,为了确定文昌鱼体液中哪些成分参与了细菌清除作用,我们通过体外杀菌来分析。提取文昌鱼体液,把细菌加入其中,然后于不同时间取样涂布平板,进行菌落计数。发现体液对大肠杆菌具有较强的杀灭活性。通过特异性抗体抑制发现补体和溶菌酶是参与文昌鱼体液杀菌作用的重要体液因子。相比之下,补体较溶菌酶作用更强。为进一步确定哪些补体激活途径参与了文昌鱼体液的杀菌作用,通过分别抑制特定的补体途径,进而比较体液杀菌活性变化的方法,发现主要是通过替代途径激活参与细菌清除作用。Western blot结果显示大肠杆菌感染后,文昌鱼体液中C3和溶菌酶水平均有明显升高,进一步证明,补体和溶菌酶确实是参与文昌鱼体内细菌清除的主要因子。其次,用大肠杆菌和鳗弧菌持续感染文昌鱼,在感染后的不同时间段取材,分别提取去肠鱼和肠组织的RNA,运用荧光实时定量PCR技术研究补体系统关键基因的表达模式。研究发现文昌鱼对大肠杆菌和弧菌两种革兰氏阴性菌的反应性基本一致,补体基因的表达规律非常类似。两种细菌感染文昌鱼后,与正常对照组相比,Bf、C3及C6三基因都存在不同程度的上调。大肠杆菌感染后,肠中的表达在3 h就达到最高值,而去肠鱼中的表达在感染后的6 h达到峰值。鳗弧菌感染后,肠中的表达在6 h时上调最为显著,而去肠鱼中的表达在感染后的12 h达到最高。这表示这些基因在文昌鱼感染的急性阶段可能发挥重要作用。有趣的是,MASP在感染后表达降低,这可能是细菌逃避机体补体系统杀伤,维护自身的一种机制。Bf、C3及C6三基因在肠中的总体表达水平,均高于在去肠鱼中的表达,达到峰值的时间均比在去肠鱼中的提前。MASP在感染后,肠中表达的下调倍数也比在去肠鱼中的高。这说明文昌鱼的消化系统可能是参与免疫的重要的器官,在清除外来微生物的免疫应答中起着重要作用。最后,分别用大肠杆菌和鳗弧菌对文昌鱼进行二次感染或交叉感染,运用real time RT-PCR技术对补体四个基因的表达模式进行分析,以研究文昌鱼的免疫系统是否具有免疫致敏作用。发现用同一种细菌二次感染后补体基因Bf、C3和C6的表达上调均比首次感染后显著,达到峰值的时间都明显提前,而且表达水平能较长时间维持在一个较高的水平。同样,在两种细菌交叉感染实验中,二次感染后基因表达上调明显,但与同种细菌感染两次相比,基因的上调倍数还是略低。所有这些特点与脊椎动物的免疫致敏作用类似,证明文昌鱼免疫系统可能存在免疫致敏作用。总之,本实验表明缺乏自由循环的血细胞的文昌鱼能够有效清除入侵的大肠杆菌。补体和溶菌酶是参与体内细菌清除的主要因子,但补体的作用较溶菌酶更强。这是关于文昌鱼清除体内细菌机制及其相关因子的首次报道。通过荧光实时定量PCR技术证明文昌鱼存在免疫致敏作用,这在头索动物中亦属首次报道。更多还原

【Abstract】 Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. Previously, amphioxus is widely used as the model organism for studying evolution and development. Recently, it has been used to explore the origin and evolution of immune system. This paper, deals with the bacterial clearance and immunological priming in amphioxus Branchiostoma belcheri.First, amphioxus was challenged with E.coli with transgenic green fluorescent protein (GFP) gene, and the numbers of E. coli in the humoral fluids were counted under a fluorescence microscope. Results indicated that amphioxus B. belcheri was able to efficiently clear the invading bacteria like E. coli from its humoral fluids. Phagocytosis by blood cells is regarded as an important way to remove bacteria in invertebrates. To find out the major elements responsible for the clearance of infected invaders, tissue sections were tested to search for the cells which may engulf E. coli in amphioxus. Not a single haemocyte-like cell has ever been seen among at least 2 000 samples examined, suggesting that free circulating macrophages not the main elements for the elimination of invading bacterium E. coli in amphioxus. Then the humoral fluids were tested for screening the factors attributable to the removal of invaders in amphioxus. The findings suggest that the humoral fluids can readily lyse the bacterium E. coli in vitro. The complement and lysozyme are both involved in the elimination in vivo of the invading bacterium in amphioxus, but the complement appears playing a stronger role than the lysozyme. Pre-incubation of amphioxus humoral fluids with the specific antibodies and inhibitor, results showed that the complement operating via the alternative pathway. Western blot showed that both C3αand lysozyme levels increase with the challenge with E. coli. This was further showed the complement and lysozyme were both involved in the elimination in vivo of the invading bacterium in amphioxus. Quantitative real-time PCR was employed to quantify the complement key gene expression in the gut and gut-free body of B. belcheri challenge with E.coli and V.anguillarum. Results showed that the patterns of the gene expression were similar after challenge with the both bacteria. Compared with the controls, Bf, C3 and C6 genes showed a similar up-regulation profile after infection. Their expression in the gut peaked at 3 h, while the expression in the gut-free body reached the highest levels at 6 h after E.coli challenge. Similarly, the expression of the genes in the gut maximized at 6 h, while their expression in the gut-free body reached maximum at 12 h after V.anguillarum challenge. The results indicated that the complement components play an important role in the acute phase of infection. In contrast, MASP expression decreased markedly after infection. This may be due to the bacterial counter-measure to avoid the activation of the complment. All these indicate that amphioxus digestive system rich in mucosa may be the major immune tissue responding to bacterial challenge and play an important role in the immune response.Finally, amphioxus was re-infected with E.coli and V.anguillarum in different ways, and quantitative real-time PCR was employed to study if there is immunological memory or priming in amphioxus Branchiostoma belcheri. Results indicated that the expression of Bf, C3 and C6 genes were significantly up-regulated and peaked earlier after secondary infection with the same bacteria. What’s more, the gene expression kept at high levels for a longer time compared with the primary infection. In contrast, the expression levels of the genes in the animals infected with the different bacterium were lower than those in the animals infected with the same bacterium. These properties appear bearing resemblance to the immunological priming in vertebrate, suggesting the presence of some immunological priming in amphioxus.In summary, this study demonstrates that the circulating haemocyte-free amphioxus is able to eliminate invading bacteria like E. coli efficiently, both the complement and lysozyme are the main factors acting in concert to eliminate the invaders in vivo. Also, amphioxus appears to have immunological primed ability, the first such data in the protochordates.更多还原