【作者】 王志平；

【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】 多数鱼类卵子体外受精,胚胎在体外充满大量各种微生物的环境中发育。此外,鱼类胚胎在孵化时,淋巴系统仍在发育之中,它合成免疫相关成分的能力有限。鱼类胚胎是如何抵抗各种微生物感染而存活下来的?这是鱼类生殖和发育免疫学的中心议题之一。已有证据表明:鱼类卵子中存在母源性免疫因子,如IgM、溶菌酶、凝集素和补体C3等。然而,关于这些母源性免疫成分特别是补体成分的功能尚不清楚。本论文主要针对母源性补体因子的传递及其功能进行研究。另外,还研究了补体系统的个体发育和成熟。本论文第一部分主要研究了斑马鱼母源性补体的免疫功能及其作用机制。我们首次通过体外实验证明了革兰氏阴性菌大肠杆菌能够被斑马鱼受精卵的胞浆(即卵子提取物,也叫无细胞体系)所杀灭。这些结果都表明,斑马鱼母源性补体系统在卵子的溶菌活性中起重要作用。首先,在斑马鱼卵无细胞体系中加入一定浓度的C3抗体使补体C3沉淀失活能够显著降低其抑菌活性,而C3正是所有补体激活途径中的关键因子。另外,将斑马鱼卵无细胞体系在45℃水浴30 min后,其抑菌活性也显著降低,而该温度足以使鱼类的补体灭活。通过补体途径的抑制实验证明,斑马鱼早期胚胎主要是通过替代性补体激活途径来发挥溶菌作用。首先,在斑马鱼卵无细胞体系中分别加入适量C1q(经典途径关键成分)抗体和C4(同时参与经典途径和凝集素途径)抗体分别沉淀C1q和C4后,卵子胞浆的溶菌活性并未发生明显变化,而加入Bf(仅在替代途径中起重要作用)抗体使Bf沉淀却能够显著降低卵子胞浆的溶菌活性。其次,在斑马鱼卵无细胞体系中加入EGTA螯合Ca2+而抑制经典途径和凝集素途径对卵子胞浆的抑菌活性并无显著影响,而加入EDTA同时螯合Ca2+和Mg2+使替代途径受到抑制后,溶菌活性则显著降低。再次,在EDTA处理过的斑马鱼卵无细胞体系中补充足量Mg2+可以在一定程度上恢复卵子胞浆的溶菌活性,而补充Ca2+却不能使溶菌活性得到恢复。最后,在斑马鱼卵无细胞体系中加入经典途径特异性抑制剂L-赖氨酸对卵子胞浆的抑菌活性几乎没有影响,而加入替代途径的特异性抑制剂酵母聚糖则能够在很大程度上降低卵子胞浆的溶菌活性。本论文第二部分主要研究了母源性补体成分的传递及其对后代的免疫保护作用。实验表明,斑马鱼母源补体因子可以传递给胚胎并在胚胎的抗感染作用中起重要作用。首先,以灭活的嗜水气单胞菌免疫雌鱼不仅能提高亲鱼体内补体关键成分C3和Bf的含量,而且其所产生的卵子中相应蛋白含量也显著升高,说明母源性补体可以传递给后代。其次,雌鱼免疫后不仅亲鱼体内的补体活性升高,其所产卵子中的补体活性也相应升高,说明传递给后代的母源性补体具有功能活性。再者,胚胎攻毒实验表明免疫亲鱼所产后代的死亡率显著低于未免疫亲鱼所产后代的死亡率,说明母源传递因子具有免疫功能。最后,向正常斑马鱼胚胎中注射补体C3和Bf抗体后,胚胎的抗感染能力明显降低,这就从另一侧面说明补体C3和Bf参与早期胚胎免疫。本论文第三部分主要研究斑马鱼补体系统的发生与成熟。除补体因子C6外,所有目的基因在孵化前的表达量都很低,孵化后则明显升高;而补体C6(参与溶解途径)在新受精卵中的大量表达,之后其表达水平迅速降低,孵化后逐步回升。此外,参与替代途径的Bf表达水平一直高于参与经典途径的C1r/s和C4,说明在斑马鱼胚胎发育和早期胚后发育过程中替代途径比经典途径更为重要。以LPS感染胚胎/幼鱼后,参与替代途径的C3和Bf在孵化后迅速上调并超过对照组;参与经典途径和凝集素途径的C1r/s和MASP的表达水平却没有升高,甚至还有所降低;同时参与经典途径和凝集素途径的C4在胚胎发育早期呈下调趋势,之后其表达水平维持在与对照组同等水平并略有波动;参与凝集素途径的MBL的表达水平在18 dpf之前与对照组无显著差异,但此后却开始显著上调。由此我们推测斑马鱼的替代性补体系统可能从胚胎孵化后逐渐发育成熟并获得免疫活性。另外,参与溶解途径的C6基因的调控模式比较特殊,在孵化前其表达水平显著下调,孵化后不久又开始迅速上调,可能意味着LPS感染促进了卵中母源性C6 mRNA的大量消耗。从胚胎到3周大小的幼鱼,C1r/s和C4的表达水平几乎不受LPS感染的影响,说明补体经典途径可能仍在发育之中,而关于凝集素途径的发育还有待于深入研究。更多还原

【Abstract】 Fish larvae are exposed to pathogens long before their lymphoid organs mature and immunocompetence is attained. It appears that fish embryo and larvae mainly rely on the maternal immune factors transferred from parents. The presence of humoral non-specific factors like lysozymes, lectins and complement components has been reported in the eggs of different teleost species. However, little information is available on the maternal immunity in fish. The main objectives of this study were to examine the transfer of maternal complement components in zebrafish (Danio rerio), their roles in early development, and the ontogeny and maturation of complement system in D. rerio.The paper first dealt with the bacteriolytic activity of maternal complement in the fertilized eggs of zebrafish and its mode of action. We showed for the first time that the Gram-negative bacterium E. coli was sensitive to the lysis by the cytosol prepared from the fertilized eggs of zebrafish D. rerio, and all the findings point to complement system being one of the most important factors involved in the bacteriolytic activity. First, the bacteriolytic activity was abolished by pre-incubation of anti-C3 antibody with the egg cytosol, a process that would cause the precipitation of the central component of all known complement pathways, C3. Second, the lytic activity was depleted by heating at 45℃, a temperature known to inactivate fish complement.To determine which pathway of complement activation might be involved in the bacteriolytic activity of the egg cytosol, the antibodies against C1q (a key component of CP), C4 (a key component of both CP and LP) and Bf (a key component of AP) were utilized to block the CP, LP or AP, respectively. It is found that precipitation of C1q and C4 causes little loss of the bacteriolytic activity of the egg cytosol, whereas precipitation of Bf results in a significant reduction of the lytic activity. Furthermore, addition of EGTA to remove Ca2+ from the egg cytosol, which can inhibit both CP and LP, induces little decrease in the bacteriolytic activity. In contrast, pre-incubation of EDTA with the egg cytosol leads to a substantial reduction of the bacteriolytic activity, and saturation of the chelator with Mg2+ is capable of restoring the lytic activity, but not by addition of Ca2+. Moreover, selective inhibition of the AP by zymosan A induces marked loss of bacteriolytic activity, while addition of L-lysine, an inactivator of the CP, is not inhibitory. Taken together, all these undoubtedly indicate that activation of the AP is responsible for the bacteriolytic activity of the egg cytosol, but the CP and LP have little contribution to the lytic activity.Second, this paper addressed the maternal transfer of complement components in zebrafish and the role of the components in offspring. The in vivo experiments showed that the maternal complement components can be trasfer from mother to offspring and they play key roles in protecting the embryos against Aeromonas hydrophila challenge. The immunization of female zebrafish with formalin-killed A. hydrophila resulted in the elevation of the alternative complement activity (ACH50) and the content of complement C3 and Bf in the vaccinated mother fish and their eggs, and both ACH50 and the complement content increased more significantly when the mother fish were vaccinated a second time, which indicated the transfer of maternal complement components and their immunocompetence in the eggs of D. rerio. Moreover, the eggs released by the vaccinated fish were more resistant to the infection by A. hydrophila than the eggs released by the non-vaccinated fish, and this proved that the maternally transferred immune factors can enhance the immunity of zebrafish offspring. Furthmore, microinjection of anti-C3 and anti-Bf antibodies into the zebrafish eggs led to a significant increase in the motality of the eggs challenged with H. hydrophila, providing additional evidence for the participation of complement components in the eggs of D. rerio.Finally, this paper examined the ontogenic and expression of complement key component genes (C3, C1r/s, C4, C6, Bf, MBL and MASP) of the CP, AP, LP and lytic pathway, and their responses to challenge with LPS during development. The findings showed that all components, except C6, show a similar trend of low expression level before hatch and increasing expression after hatching; Expression of C6 was detected at very high level just after fertilization, then it significantly droped in the early time-point samples and steady increase followed. Moreover, Bf transcript representing the AP is expressed at much higher levels than C1r/s and C4 involved in the CP. Thus it is likely that the AP may be more significant than the CP during embryonic and post-hatching development of D. rerio.When the embyos/larvae of zebrafish was challenged with LPS, it was found that: The contents of Bf transcript representing the AP and C3 transcript important in all the activation pathways are both markedly increased by challenge with LPS soon after hatching, while the expression levels of C1r/s and MASP transcripts representing the CP and LP, respectively, are not elevated, or even decreased by the same challenge throughout the study period; The expression of C4 mRNA involved in both the CP and LP is first down-regulated by LPS challenge and then fluctuates; The expression of MBL mRNA associated with the LP is up-regulated by LPS challenge only after 18 dpf; and C6 transcript representing the lytic pathway is peculiarly expressed in response to LPS challenge, which is significant decreased before hatching, followed by a significant drop post hatching. Taken together, these results hints at the clue that the complement operating via the AP is already competent by responding to challenge with LPS in the hatched larvae of D. rerio. Moreover, C6 expression is relatively high in early embryos, but it is significantly suppressed following LPS challenge. It is probable that there exists a large amount of maternal C6 mRNA stored in the eggs, which is rapidly consumed by challenge with LPS. On the other hand, none of C1r/s and C4 expression is fully responsive to challenge with LPS in the 3-week old larvae, implicating that the CP is still developing. However, the developmental status of the LP remains to be further determined at present.更多还原