【作者】 彭建涛；

【导师】 侯连生；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 盘基网柄菌(Dictyostelium discoideum)是一种“土壤阿米巴”，以吞食细菌为生。在营养丰富条件下，以单细胞形式存在；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；而柄细胞无论用什么条件都不能萌发，因此柄细胞是死亡细胞，柄细胞的形成是细胞凋亡的过程。目前认为线粒体不仅是细胞内的能量工厂，而且在细胞分化和凋亡过程中也起重要的调节作用，线粒体膜通透性改变可能是细胞凋亡的关键性机制。线粒体通透性转运孔(PTP)的开放，能够导致线粒体跨膜电位的下降，Caspases活化和凋亡诱导因子(如细胞色素c、AIF等)的释放，ATP生成减少等等。对于盘基网柄菌细胞不同发育时期线粒体蛋白组分、膜电位的变化情况的研究很少，对凋亡诱导因子细胞色素c在柄细胞凋亡中的作用了解不是很清楚。 本实验以Kax-3野生型细胞和AK127突变型细胞为材料，提取不同时期细胞线粒体。另外用Percoll密度梯度分离技术获得蛞蝓体中前柄和前孢子两种类型细胞，用SDS-聚丙烯酰胺凝胶电泳法，分析16小时前柄细胞和前孢子细胞线粒体和细胞质中蛋白的变化，研究比较与前柄细胞和前孢子细胞分化有关的蛋白。发现前柄细胞和前孢子细胞的线粒体和细胞质蛋白存在一定差异。野生型细胞发育过程中，线粒体蛋白也存在一些差异。整个细胞发育时期，54kD、47kD、41.2kD、38.4kD等蛋白具有基本一致的变化。另外，83kD在细胞聚集阶段和蛞蝓体拔顶阶段含量都有增高，而在细胞丘阶段和蛞蝓体形成阶段含量又有所降低。而细胞聚集期75kD、19.5kD蛋白的含量也有增加，到细胞丘阶段后这两者含量开始明显减少。所以75kD、19.5kD蛋白可能是线粒体膜蛋白，参与某些蛋白向线粒体内转运，对于盘基网柄菌细胞聚集过程线粒体的能量参与分化、运动等过程。随细胞发育到细胞丘阶段，65.6kD蛋白的含量有所增加，其它蛋白的含量比较稳定。蛞蝓体形成阶段，66kD、56.8kD、23.8kD、16.1kD、14.3kD等蛋白含量增加。而16小时前柄细胞和前孢子细胞中：50kD、26kD蛋白仅存在于前孢子细胞的线粒体中，可能为前孢子细胞线粒体中的特异性蛋白；在前孢子细胞的细胞质中没有检测到79kD蛋白，而在前柄细胞的细胞质中该蛋白有一定量的存在；在前柄细胞的细胞质中没有检测到53kD蛋白存在，而是存在于前孢子细胞的细胞质中；51kD、31.4kD蛋白在前柄细胞的细胞质中的含量远远高于其前孢子细胞质中的含量，而48kD、33kD、21.6kD蛋白在前柄细胞的细胞质中的含量又远远低于前孢子细胞质中的含量；59.7kD、44kD、42kD、39.7kD、39kD、29.7kD、27kD等蛋白的含量基本相差不大。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. However, the formation of stalk cells is a process of cell death. Current evidences suggest that the mitochondria not only provide energy for the cell, but also play a crucial role in cell differentiation and cell apoptosis. The change of mitochondrial membrane permeability may cause the start of apoptosis. The opening of permeability transition pore (PTP) would induce the decline of membrane potential and the release of some apoptosis inducing factors (such as cytochrome c, AIF) and the decrease of ATP production. The differences of mitochondria components, the variety of transmembrane potential during the development and the role of cytochrome c in the process of apoptosis were not clear yet.Kax-3 cells (wild type) and AK127 cells (mutant type) were taken as experimental materials and the mitochondria were got at different developmental stages. Prestalk and prespore cells obtained from mechanically dissociated slugs were separated by the centrifugation on preformed percoll density gradients. Proteins of mitochondria and cytoplasm were separated by SDS-PAGE. Proteins during differentiation of prestalk cells and prespore cells were investigated and some different proteins were found in mitochondria and cytoplasm between prestalk and prespore cells. Different mitochondrial proteins were found in the development of Kax-3 cells. During the whole development, 54kD、 47kD、 41.21kD、 38.4kD proteins were shared consistent change. The content of 83.0kD protein ascended at aggregation and culmination stages, and declined at mound and slug stages. The content of 75kD and 19.5kD rose at aggregation stage, but decreased obviously at mound stage. So 75kD and 19.5kD may be mitochondrial membrane proteins, and participate in cell differentiation and movement at aggregation stage. The content of 65.6kD protein increased at cell mound stage, but other protein kept changeless. 50kD protein and 26kD protein only occurred in prespore mitochondria, they might be prespore mitochondrial specific proteins. 79kD protein wasn’t found in prespore cytoplasm, but there was a little amount in prestalk cytoplasm. 53kD protein wasn’t found in prestalk更多还原