【作者】 李磊；

【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】 卵黄蛋白原(Vitellogenin,Vg)是卵黄蛋白(Yolk protein)的前体,它特异存在于卵生雌性动物中。脊椎动物肝脏在雌激素的刺激下产生Vg,随后Vg被分泌到血液中并随血液循环系统进入卵巢,被卵巢吸收后分解成卵黄脂磷蛋白(lipovitellin)和卵黄高磷蛋白(phosvitin),为胚胎和幼体生长发育提供能量与营养物质。Vg通常情况下只存在于雌性动物中,但是在17β-雌二醇(17-β-estradiol,E2)的诱导下,雄性成体以及未性成熟的幼体也可以表达Vg。另一方面,在抗雌激素如它莫西芬(Tamoxifen, TMX)的诱导下,雌性动物的Vg表达会被抑制。目前,越来越多的研究表明,Vg除了可以为胚胎发育提供营养以外,还具有其它生物学功能。其中,免疫防御是比较受关注的一项功能。体外实验已经证明Vg具有识别病原相关分子模式的功能,并且具有凝血活性和抑菌活性。然而关于Vg在体内是否具有免疫防御的作用,目前还知之甚少。因此,本文从以下几个方面探讨了Vg在体内的免疫防御作用:一、研究病原相关模式分子是否能够诱导雄性斑马鱼表达Vg以及所表达的Vg是否具有抑菌活性;二、研究当斑马鱼受到病原菌侵害时,Vg是否能够对斑马鱼起到一定的保护作用。论文第一部分的研究表明,Vg是一种急性时相蛋白。在脂多糖(Lipopolysaccharides, LPS)和脂磷壁酸(lipoteichoic acid,LTA)的诱导下,雄性斑马鱼能够迅速表达Vg,表达模式类似于急性时相蛋白;并且诱导表达的Vg能够与细菌结合并具有抑菌活性。本文用腹腔注射法向雄性斑马鱼注射LPS和LTA,然后在注射后0h、0.5h、1h、2h、3h、5h、9h和12h分别提取斑马鱼总RNA和血清。通过荧光实时定量PCR(Real-time PCR)和酶联免疫吸附实验(ELISA)检测雄性斑马鱼Vg在mRNA和蛋白水平的表达情况。实验结果表明:注射LPS后,Vg1基因立即开始大量表达,在0.5h达到正常雄鱼的104.1倍,在2h达到峰值,为正常雄鱼Vg1基因表达量的193.2倍,并在此后所检测的12小时内维持在较高的表达水平。在注射LTA0.5h以后,雄鱼Vg1基因表达量达到正常雄鱼的45倍,此后的表达水平与对照组相当。对照组注射生理盐水后,Vg1基因表达量变化不大。在蛋白水平,Vg的表达量也有显著上升,注射LPS0.5小时后,血清Vg表达量即开始显著上升,为对照组的1.3倍;注射LTA 5h后,Vg表达量开始显著上升,9h时血清中的Vg为对照组的1.2倍。由此可以看出,病原相关分子模式LPS和LTA能够有效诱导斑马鱼Vg表达。因此,斑马鱼Vg的表达模式具有急性时相反应的特点,是一种急性时相蛋白。本文应用Western blotting检测了Vg是否能够与细菌结合。将LPS诱导0.5h的雄鱼血清分别与大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)共同孵育,然后洗脱细菌上结合的蛋白,用洗脱下的蛋白进行western blotting,抗体为兔抗斑马鱼Vg多克隆抗体。结果表明,Vg与大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)都能结合。本文用LPS诱导0.5h的雄性斑马鱼血清(Vg含量高)进行抑菌实验,结果表明:它对大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)抑菌能力分别比对照组高6.8%和6.6%;经过加热灭活补体后,对两种菌的抑菌能力比对照组分别高18.3%和11.4%;经过加热灭活补体,然后又加入不同量的Vg抗体以沉淀血清中的Vg,随着Vg抗体量的加大,血清的抑菌活性逐渐降低,在加入过量Vg抗体后,其抑菌活性与对照组无显著差异,与加入抗体之前的血清相比,对两种菌的抑菌活性分别降低18.6%和11.7%。由此可见,Vg的抑菌活性是显著的,而且其对革兰氏阴性菌的抑制率高于对革兰氏阳性菌的抑制率。论文第二部分首先探索了E2和TMX合适的诱导浓度及诱导时间,目的是既能显著调控Vg表达量又不对斑马鱼造成损伤。结果表明:20 nM E2诱导斑马鱼20天后,Vg表达量显著上升,其肝实质细胞形态仍然保持正常,肝脏基本未受影响;500 nM TMX诱导斑马鱼20天后,Vg表达量显著下降,肝实质细胞形态也基本保持正常,肝脏功能未受影响。然后我们又研究了E2和TMX对斑马鱼免疫球蛋白IgM(Immunoglobulin M)和白细胞密度的影响,结果表明:E2和TMX都能够提高斑马鱼血清中IgM的水平,但是两者对IgM的诱导作用差别不大;E2和TMX对白细胞密度都无显著影响。最后我们用鳗弧菌V. auguillarum对20 nM E2和500 nM TMX诱导20天的斑马鱼以及正常斑马鱼进行攻毒,实验结果表明:E2诱导的雌鱼的存活率显著高于正常雌鱼和TMX诱导的雌鱼;E2诱导的雄鱼的存活率与正常雄鱼相仿,但是显著高于TMX诱导的雄鱼。说明当斑马鱼受到病原菌入侵时,Vg对斑马鱼可能具有一定的保护作用,能够提高斑马鱼的存活率。然而关于Vg的作用机制,还有待于进一步研究。综上所述,本文发现斑马鱼Vg是一种急性时相蛋白,病原相关分子模式LPS和LTA能够诱导雄性斑马鱼表达Vg,其表达模式具有急性时相反应的特点;而且所表达的Vg具有抑菌作用。另外,实验表明,当斑马鱼受到病原菌入侵时,Vg对提高斑马鱼存活率可能具有一定作用。更多还原

【Abstract】 Vitellogenin (Vg), a phospholipoglycoprotein, is the precursor of major egg-yolk proteins in all oviparous species from invertebrates to vertebrates. All forms of fish Vgs are synthesized by the liver of females in response to the endogenous estrogen, 17-β-estradiol (E2), secreted into bloodstream and transported to ovary, where they are internalized by growing oocytes via receptor-mediated endocytosis and proteolytically cleaved to form the egg-yolk proteins that are later used as the nutrients by developing embryos and larvae. Normally, Vgs are undetectable in male and immature female fish; however, their synthesis can be induced by exogenous E2. On the other hand, synthesis of both Vg mRNA and proteins can be inhibited by tamoxifen (TMX), a E2 antagonist. Recent studies have shown that in addition to being involved in egg-yolk protein formation, Vg appears to have evolved pleiotropic functions. A novel function of Vg is linked with immune defense. In the protochordate amphioxus (Branchiostoma belcheri) as well as the bony fish rosy barb (Puntius conchonius), Vgs have been demonstrated to possess both hemagglutinating and antibacterial activities. In vitro experiments have shown that fish Vg acts as a multivalent pattern recognition receptor with an opsonic activity. However, the potential immunologic role of vitellogenin in vivo remains obscure. This study demonstrated that serum Vg in zebrafish D. rerio is an acute phase protein with bacterial-binding and inhibiting activities. It also bolsters the notion that factors normally involved in control of female reproduction are linked with immunity in organisms that rely on Vg for oocyte development. This study also indicated that Vg may enhance the survival rates of D. rerio following challenge with V. anguillarum.This paper examined whether the injection of pathogen-associated molecular patterns (LPS and LTA) to male D. rerio could trigger Vg synthesis in vivo. Male D. rerio were injected with LPS, LTA or 0.9% NaCl solution (control) and were sampled at 0h, 0.5h, 1h, 2h, 3h, 5h, 9h and 12h post injection (hpi) for total RNAs and serums. Real-time PCR showed that LPS could trigger Vg1 gene expression in vivo immediately after injection. Vg1 gene expression of LPS-induced male D. rerio was about 104.1-fold of the control at 0.5 hpi, reached the peak of about 193.2-fold at 2 hpi and kept in high levels at the following time points. Vg1 gene expression induced by LTA increased to 45-fold at 0.5 hpi but quickly decreased to the control level at the following time points. The results of ELISA showed that serum Vg levels increased significantly to 1.3-fold of the control at 0.5 hpi after injection with LPS while it increased to 1.2-fold at 9 hpi after injection with LTA. It revealed that LPS as well as LTA could significantly trigger Vg synthesis in vivo in both mRNA and protein levels in male D. rerio. The response of Vg induced by PAMPs revealed that Vg was an acute phase protein. We applied Western blotting to detect whether Vg can bind to E.coli and S.aureus. The serum of LPS-induced male D. rerio at 0.5 hpi was incubated with E.coli and S.aureus respectively. Then the protein binding to bacteria were eluted carefully. The elution was carried out for western blotting with rabbit anti-Vg polyconal antibody. The results showed that Vg was capable of binding to both E.coli and S.aureus. Inhibition of bacterial growth by serum Vg induced by LPS was also detected. The sera from control and LPS-injected male D. rerio all exhibited conspicuous antibacterial activities against E. coli and S. aureus, with the antibacterial activities of the sera collected at 0.5 hpi from LPS-injected fish being significantly higher than those from saline-injected fish. When the complement activities of the sera were inactivated by heating, their antibacterial activities against E. coli and S. aureus were remarkably reduced. However, some antibacterial activities remained in all the heated sera. In particular, after heating, the antibacterial activities of the sera collected at 0.5 hpi from LPS-injected fish were still significantly higher than control, suggesting the presence of additional antibacterial molecules in these sera. Notably, the antibacterial activities of the heated sera against E. coli and S. aureus were significantly inhibited by the pre-incubation of the sera with anti-zebrafish Vg antibody, but not by the pre-incubation with anti-actin antibody; and this inhibitory effect showed a clear dose-dependence. Moreover, consistent with higher Vg levels in the sera collected at 0.5 hpi, the antibacterial activities in these sera, after heating, were also greater than control. These data together implicated that Vg was an important factor responsible for the antibacterial activities in the sera.In the second part, prior experiments were firstly applied to determine the appropriate concentrations and time of the exposure. The results showed that Vg synthesis in D. rerio exposed to 20 nM E2 at 20 dpe (days after exposure) was significantly up-regulated while it was significantly down-regulated by 500 nM TMX at 20dpe. However, histological examination revealed that exposure to 20 nM E2 and 500 nM TMX cause only slight hepatocytic changes in D. rerio. Western blotting showed that both E2 and TMX were able to significantly trigger IgM synthesis in D. rerio. However, no marked difference of IgM levels was detected between E2- and TMX-exposed fish. We also found that exposures to E2 and TMX had little influence on the density of leucocytes in zebrafish blood. Finally, after exposure to 20 nM E2 or 500 nM TMX, for consecutive 20 days, the exposed fish as well as normal fish were injected intraperitonealy with Vibrio auguillarum (V . auguillarum) and the survival rates were calculated at different time points, respectively. The results showed that the survival rates of the E2-exposed females were significantly higher than those of normal females and TMX-exposed females. In males, E2-exposed group exhibited significantly higher survival rates than TMX-exposed group but similar to those of normal group. The results indicated that Vg may improve the survival rates of D. rerio following challenge with V. auguillarum.In summary, this paper revealed that Vg in zebrafish D. rerio is an acute phase protein with bacterial-binding and inhibiting activities. The PAMPs (LPS and LTA) can induce Vg synthesis in vivo in male D. rerio, and the response of Vg revealed an acute-phase response. Serum Vg induced by PAMPs showed antibacterial activities to both E.coli and S.aureus. In vivo experiments also showed that, Vg may improve the survival rates of D. rerio following challenge with V. auguillarum.更多还原