【作者】 陈能星；

【导师】 侯连生；

【作者基本信息】 华东师范大学 ， 动物学， 2009， 硕士

【摘要】 盘基网柄菌(Dictyostelium discoideum)是一种低等的真核原生生物,在营养丰富的条件下以阿米巴单细胞形式吞噬细菌为食,行二分裂方式繁殖生长。一旦食物匮乏,单个细胞聚集成多细胞体,细胞历经细胞丘、蛞蝓体、拔顶阶段完成发育和分化过程,最终形成由孢子细胞和柄细胞组成的子实体。在合适条件下,孢子细胞萌发开始新生命周期。盘基网柄菌与后生动物有许多共同的细胞生理生化反应,如胞浆移动、吞噬作用、趋化性、信号转导以及一些发育事件如细胞分类、模式形成和细胞类型决定等。由于其具有单细胞和多细胞的生命过程,生命现象丰富,作为非常好的模式动物,用来研究细胞的运动性,细胞间的信号转导以及细胞类型的特化和细胞发育等。LagC基因编码的gp150蛋白是一种膜蛋白,在细胞与细胞接触区域特别丰富,它通过异嗜性相互作用来调节细胞间的紧密黏着。gp150蛋白是在盘基网柄菌发育聚集期开始后才开始表达,当盘基网柄菌发育到12小时的时候,用Westernblot可以在野生型KAx-3细胞中检测出明显的gp150蛋白条带;而在突变型AK127细胞中,完全检测不到gp150蛋白的表达。gp150缺失的细胞株AK127不能完成多细胞发育,发育只能停留在细胞松散聚集阶段。由此推测,gp150蛋白在盘基网柄菌发育过程中起着重要作用,它可能参与介导细胞分化和凋亡的信号转导途径,但具体的信号通路至今仍未完全清楚。尿囊酸酶(allantoicase)是嘌呤代谢中一种重要的酶,催化尿囊酸生成脲基乙醇酸,后者进一步降解为氨、二氧化碳和乙醛酸。据研究发现,不同物种尿囊酸酶基因都是位于染色体上,但是表达的尿囊酸酶蛋白在细胞内定位不同;大多数鱼类及两栖类动物中尿囊酸酶具有活性,在爬行类、鸟类及哺乳动物中没有活性,但是奇怪的是在鼠、牛和人等哺乳动物组织内却有尿囊酸酶基因的转录表达。为什么尿囊酸酶的活性在生物进化过程中会逐渐消失?为什么在高等哺乳类没有尿囊酸酶活性却还有尿囊酸酶基因的表达?其作用机制尚不清楚,这就引起我们对尿囊酸酶研究的极大兴趣。而在低等的原生动物盘基网柄菌中关于尿囊酸酶蛋白的细胞内定位及其在分子信号传导途径中的作用机制尚不清楚。为此我们利用本实验室表达获得的尿囊酸酶蛋白制备多克隆抗体,用ELISA测定抗体的效价,达到1:64000以上;Western blot检测抗体的特异性和亲和性,抗血清能特异性和目的蛋白结合,而对照血清与抗原没有杂交带。应用获得的抗体进行免疫印迹分析尿囊酸酶在盘基网柄菌细胞内的表达情况,发现尿囊酸酶蛋白在多细胞整个发育过程中都有表达,细胞聚集阶段蛋白印迹灰度值约40-50,到多细胞聚集进入细胞丘和蛞蝓体阶段尿囊酸酶蛋白表达量显著增加,蛋白扫描灰度值达104-109,即细胞丘和蛞蝓体阶段尿囊酸酶蛋白的表达量是细胞聚集阶段尿囊酸酶表达量的一倍,表明尿囊酸酶在野生型KAx-3多细胞发育分化中可能起重要作用;同时用尿囊酸酶抗体免疫印迹还发现分子量为62kD左右的蛋白在多细胞发育过程中也有明显的条带,表达量较尿囊酸酶要高,但是表达变化趋势与尿囊酸酶蛋白变化相似,提示尿囊酸酶蛋白与62kD蛋白可能存在功能相关性;通过免疫荧光技术和激光共聚焦确定尿囊酸酶的细胞内定位,发现尿囊酸酶蛋白在前柄细胞内的表达量较前孢子内高,结果提示尿囊酸酶在前柄细胞的分化中可能发挥作用;同时荧光结果发现尿囊酸酶蛋白在细胞膜附近荧光有所加强,而且发现尿囊酸酶在细胞内存在Capping结构,Capping是膜蛋白典型运动特征,提示尿囊酸酶与盘基网柄菌细胞膜上相关蛋白可能发生作用。通过分子生物学软件分析尿囊酸酶蛋白不具有跨膜结构域,为什么在膜附近该蛋白的表达增加?我们分析尿囊酸酶可能是通过细胞内连接蛋白与相关膜上蛋白锚定,从而在盘基网柄菌的多细胞的发育、分化中起作用。尿囊酸酶蛋白是本实验室人员通过盘基网柄菌野生型KAx-3细胞与gp150蛋白缺失的突变型AK127细胞差异表达获得的,因此本研究为进一步深入探讨盘基网柄菌粘附分子gp150相关的胞内信号通路提供了背景资料,并为进一步研究其他更高等生物体内信号转导的调控及相互关系作出了贡献。更多还原

【Abstract】 The social amoeba Dictyostelium discoideum which phagocytosis soil bacteria for food is a unicellular eukaryote.Under favorable condition,it multiplies as a unicellular organism.Upon starvation,a pathway involving aggregation,mound,slug, culmination stages induces the formation of a fruiting body consisting of a head of spores supported on a stalk of vacuolated cells.Spores await dispersal and germinate in favourable conditions for amoeboid growth.A wide range of fundamental cellular processes are common to D.discoideum and metazoans.These processes include cytokinesis、motility、phagocytosis、chemotaxis、signal transduction and aspects of development such as cell sorting、pattern formation and cell-type determination. Dictyostelium discoideum is an excellent system in which to study cell mobility,cell-cell signal transduction,cell type differentiation,development and so on for its unicellular and multicellular life stages and rich life phenomena.gp150 is encoded by lagC gene,and it is a membrane glycoprotein.High concentrations of gp150 are present at cell-cell contacts,they regulate cell-cell adhesion by heterophilic interactions,gp150 is expressed on cells in the early post-aggregation stage,it can be detected in the wild type KAx-3 cells but not in the mutant type AK127 cells by Western blot.The mutant type strain AK127,as a result of absence of gp150,can’t complete multicellular development and the development is arrested at the loose aggregate stage.The obtained data suggested gp150 proteins play an important part in Dictyostelium discoideum development,it may take part in the signaling pathway for regulating cell differentiation and cell apoptosis.So far,our knowledge of the signaling pathway is still very limited. Allantoicase is an important enzyme in the degradation of purines.It catalyzes the conversion of allantoic acid into ureidoglycolate and urea.And the former was resolved into ammonia、CO2、glyoxylate.According to the research discovery,the different species allantoicase gene all is located in the chromosome,but expresses the oprotein locates differently in the cell;The different living creature purine metabolism end product is dissimilar,therefore in pathways each kind of enzyme activity is dissimilar,in the majority fish and the amphibians the allantoicase has activity,in reptiles,birds and the mammal does not have aUantoicase activity,however in the mouse,mammal,and human organizations has the allantoicase gene duplication and expression.Why can allantoicase activeness vanish gradually in the biological evolution process? Why in higher mammalia doesn’t have the allantoiase enzyme activity but have the allantoicase gene expression actually? Its mechanism still not clear,this caused us enormous interest in the allantoicase research.But in the Protozoa Dictyostelium discoideum about the localization of allantoicase and the molecular signal conduction mechanism are still not clear.So we use the allantoicase which is obtains by our laboratory to prepare the polyclonal antibody,The titer of the antiserum measured by ELISA could achieve 1:64000.;Western blotting examinate anti-body specificity and appetency,the antiserum can specifically combine with goal protein,compared serum is not have the mixed with antigen.Using the obtained anti-body analysis allantoicase in cell expression situation by Western blot,found that allantoicase have expression in all multicellular development.Aggregation stage the protein gray value of allantoicase is about 40-50,in the stage of mound and slug allantoicase expression have a significant increase and gray value is 104-109.Thats mean in the stage of mound and slug allantoicase expression quantity is double than aggregation.prompts that the allantoicase play a important role in multicellular development and differentiation of KAx-3 cells;at the same time found that its still having 62kD protein expression by allantoicase polyclonal antibody in multicellular development.The expression quantity is higher than allantoicase but the protein expression change trend is similar to allantoicase.suggesting that allantoicase may have some relations with 62kD protein.Analysising the location of allantoicase in Dictyostelium discoideum cell through the immunity fluorescence technology and the laser,discovered the allantoicase expression in prestalk cells is higher than prespore cells,suggest allantoicase may have a role in differentiation of prestalk cells;fluorescence also found that allantoicase in the membrane fluorescence be enhanced and found Capping of allantoicase,Capping structure is a typical characteristic of membrane protein.suggesting that allantoicase may play a role with membrane-associated protein of Dictyostelium discoideum.By the analysis of molecular biology softwares found that allantoicase have no transmembrane-domain but have high expression of allantoicase around the cell membrane.We analyzed the allantoicase may be anchoring with the cell membrane protein through connexin and then play a important role in multicellular development of Dictyostelium discoideum.allantoicase is found by members of my laboratory through DDRT-PCR of wild type KAx-3 cells and mutant type strain AK127(knockout lagC gene),so research might be considered as the background for the research on intercellular signaling pathway involving adhesion molecule gp150 in Dictyostelium discoideum, as well as favor the further study on regulation of signaling transductions and their relationships in other more advanced organisms.更多还原