【作者】 余素红；

【导师】 陈新华； 彭宣宪； 徐洵；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 大黄鱼（Pseudosciana crocea）是我国特有的经济鱼种,但随着养殖规模的扩大及养殖环境的恶化,病害问题日益严重,已成为制约大黄鱼养殖业持续稳定发展的主要因素。因此,开展大黄鱼免疫的分子基础研究,揭示其免疫反应规律及免疫的分子机制,不仅可以深化对以鱼类为代表的低等脊椎动物免疫系统的认识,也为大黄鱼病害防治提供重要的理论依据。本文首先采用双向电泳分析了Poly（I:C）诱导不同时段大黄鱼脾脏组织差异表达的蛋白,结合PMF和MALDI-TOF-TOF质谱技术共鉴定了38个差异点,其中28个差异点表达上调,10个表达下调,且表达变化多集中于诱导后12h-24h之间。为了了解各差异点间的相互关系以及它们在抗病机制中的作用,通过应用Cytoscape软件构建了差异蛋白互作网络图,发现其中有许多差异点如PPRAγ、AnnexinⅣ、ROCK1等同时参与了免疫调节与凋亡调节,尤其是AnnexinⅣ表现出多功能的特性。为了研究大黄鱼AnnexinⅣ的功能,采用重组AnnexinⅣ注射大黄鱼,结果发现AnnexinⅣ的活体过表达能明显提高Caspase 3的酶活力,并促进脾脏细胞的凋亡。同时,它能抑制趋化因子CXC、CC的表达和上调抑炎因子IL-10的表达,表明了大黄鱼AnnexinⅣ具有促进细胞凋亡和调节炎性反应的功能。在PolyI:C诱导后12h,AnnnxinⅣ的上调可能在一定程度上促进了脾脏细胞凋亡并抑制了炎性反应,而诱导后48h,凋亡抑制因子PRDX I、AIP的表达上调则有利于凋亡的抑制,从而调节PolyI:C诱导的免疫反应及维持免疫平衡。通过比较对照组和三联疫苗注射组的双向电泳图谱,找到了24个差异点,其中21个表达上调,3个表达下调。质谱分析鉴定发现其中有许多与免疫应答及炎症调节相关,尤其抗氧化蛋白酶家族成员如PRDXⅠ、PRDXⅡ、PRDXⅣ在三联疫苗诱导前后表达量都发生了改变,提示该家族可能在三联疫苗诱导免疫反应中发挥重要的作用。研究表明,人PRDXⅣ能通过调节IκB的磷酸化来激活核因子NF-κB,但对其功能尚不清楚。为此,本文对大黄鱼PRDXⅣ的分子特征及功能进行了研究:体外实验表明重组的大黄鱼PRDXⅣ具有明显的抗氧化活性;免疫电镜分析发现PRDXⅣ主要分布于脾脏细胞内质网和过氧化物酶体中,并且三联疫苗的免疫刺激促进了PRDXⅣ在内质网上的合成;针对PRDXⅣ的siRNA干扰可上调脾组织TNF-α2、CC趋化因子的表达水平,下调抑炎因子IL-10的表达,同时核因子NF-κB（p65）的表达水平也明显上升;PRDXⅣ的过表达则抑制了TNF-α2、CC趋化因子的表达,上调了IL-10的表达,核因子NF-κB（p65）的表达也受到明显的抑制,说明了大黄鱼PRDXⅣ具有抑制炎性反应的功能。以三种混合活菌感染大黄鱼,发现干扰组脾脏组织TNF-α2、CC及NF-κB（p65）的表达上调更加显著,抑炎因子IL-10的表达明显低于对照组,同时干扰组大黄鱼的死亡率（94%）明显高于过表达组（42%）和对照组（72%、78%）,这些结果表明了大黄鱼PRDXⅣ可能通过抑制NF-κB表达发挥其抗炎作用,从而保护大黄鱼抵抗细菌的感染。本文研究结果揭示了一条鱼类抗细菌感染的新途径。此外,还分析了差异表达蛋白NCCRP-1和β₂-microglobulin mRNA的组织分布及不同诱导条件下的表达调控方式,这些将有助于对大黄鱼免疫反应规律的全面认识。更多还原

【Abstract】 Large yellow croaker（Pseudosciana crocea）is an economically important marine fish species in China.In recent years,with the rapid development of large yellow croaker culture industry,virus and bacteria-caused infectious diseases become more and more severe,while the knowledge about the genetic and immunological basis of large yellow croaker is limited. The lack of knowledge hinders the establishment of effective measures in diseases control and genetic improvement.In this study,proteomics approaches were utilized to identify the differentially expressed proteins in the spleen tissue of large yellow croaker in response to poly I:C induction at four different time points（0,12,24 and 48h after induction）.All together,38 protein spots differentially expressed were identified and further analyzed by PMF or MALDI-TOF-TOF, in which 28 were up-regulated and 10 showed down-regulated.And most changes took place around 12 and 24h post induction.To understand the inter-relationship of these differentially expressed proteins and investigate their roles in disease-resistance mechanism,protein interaction network was build up by Cytoscape software.In this network,PPRAγ,AnnexinⅣand ROCK1 were identified to participate in both immune response and apoptosis control. AnnexinⅣ,for its diverse biological functions and huge amount of binding partners, especially drew our attention and was chosen for further investigation.The over-expressed AnnexinⅣwas found to cause the increase of the activity of caspase-3 and apoptosis in spleen tissue,also,it up regulated the expression of pro-inflammatory inhibitor IL-10,and down regulated expression of chemokines CXC and CC.These results indicated that up-regulation of AnnexinⅣat 12h after induction with PolyI:C could cause the spleen cell apoptosis and regulate the inflammatory response.On the other hand,some anti-apoptosis proteins such as PRDXⅠand AIP were up-regulated at 48h post-induction.These differential expressed proteins ensured a dynamic balance between cell proliferation and apoptosis so as to maintain the stable inner environment and control the immune response in large yellow croaker.In the differential proteomics analysis of large yellow croaker spleen tissue in response to trivalent bacterium vaccine induction,24 differential expressed proteins spots were identified and further analyzed by PMF or MALDI-TOF-TOF.Three altered proteins,PRDXⅠ,PRDXⅡ,PRDXⅣ,were found belong to PRDXs superfamily.This indicated PRDXs may play an important role in immune response to the trivalent bacterium vaccine-induction in large yellow croaker.A differential expressed protein（Spot 7）homologous to a mammalian PeroxiredoxinⅣattracted our attention,for it is reported to mediate the activation of NF-kB in the cytosol by modulating IkBαphosphorylation in mankind.The recombinant large yellow croaker PRDXⅣexhibited activities of oxidation resistance.Intracellular localization of PRDXⅣin the spleen cells by immune colloidal gold electron microscopic analysis showed that most of the gold particles exist in the peroxisome,cytoplasm and rough endoplasmic reticulum,and more gold particles in rough endoplasmic reticulum were found in the spleen of trivalent bacterium vaccine-induced fish than that of normal fish.Furthermore, the suppression of PRDXⅣby siRNA caused the up-regulated expression of TNF-α2,CC, and NF-kB（p65）and down-regulated expression of IL-10.But the over-expression of PRDXⅣcaused the down-regulated expression of TNF-α2,CC,and NF-kB（p65）and up-regulated expression of IL-10.To further corroborate the anti-inflammation activity of PRDXⅣ, twelve hours after the siRNA or protein injection,fish were infected by mixed pathogenic bacterium The result shows that after the PRDXⅣexpression was suppressed,the fish mortality was significantly higher than that of control group,while the enhanced PRDXⅣcould protect large yellow croaker from the bacterium induced death.All these results indicate that PRDXⅣcan inhibit the inflammation and protect large yellow croaker from bacterial infection by inhibiting the expression of NF-kB.Our research reveals a new way of anti-bacterium infection in fish.Two other genes,NCCRP-1 andβ₂-microglobulin were also cloned,their tissue distribution and expression patterns upon stimulation with polyI:C and inactivated trivalent bacterial vaccine were analyzed.These results will help us to further stand immune response and disease-resistance mechanism in large yellow croaker.更多还原