【作者】 施佳乐；

【导师】 鲁心安；

【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】 社会变形虫盘基网柄菌(Dictyostelium discoideum)是一种单细胞真核生物，以细菌为生。在营养丰富情况下，以二分裂方式繁殖后代；当食物耗尽时，进入多细胞发育阶段，其发育过程类似于多细胞的胚胎发育，有着严格的时间和空间的发育程序，并且受到精密的调控。当多细胞聚集形成细胞丘时，cAMP的浓度升到了一定水平，调节细胞分化发育相关的基因开始表达，产生相关的蛋白，其中涉及很多细胞组分(如粘附分子、分化诱导因子、细胞骨架蛋白等等)，传递细胞分化必需的信号，导致细胞分化成前柄细胞和前孢子细胞两种类型。最终，形成由柄细胞和孢子细胞组成的子实体，而柄细胞是死亡细胞；因此，该细胞分化的过程实际上是细胞凋亡的过程。目前对盘基网柄菌两类细胞蛋白组分研究很少，对发育分化过程中是否依赖Caspase了解的也不是很清楚。 本文以野生型Kax-3细胞株为材料，每隔2小时收集16—22小时发育阶段蛞蝓体细胞，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-PAGE电泳，对比两类细胞不同发育时期全蛋白组成、数量及相对百分含量的变化，发现前柄细胞和前孢子细胞在蛋白含量和组分上存在一定差异。在蛞蝓体时期，101.2kD、85.9kD、68.2kD、49.8kD、42.4kD、40.6kD、33.0kD、24.4kD和21.5kD在两类细胞中变化基本一致，其中101.2kD、68.2kD、49.8kD、42.4kD和33.0kD含量较高。101.2kD蛋白可能是gp100，主要作用于发育早期细胞间粘着；68.2kD蛋白可能是肌动蛋白结合蛋白，加强细胞间粘着作用；49.2kD蛋白与微管蛋白分子量接近，参与细胞形态的维持、细胞运动。42.4kD蛋白可能是肌动蛋白，对细胞迁移运动有重要作用。在前柄细胞中出现了37.5kD蛋白和31.5kD蛋白，且含量随细胞分化进程增加，提示两者与细胞凋亡有密切关系。其中31.5kD与Caspase-3分子量32.0kD接近，有待进一步证实。36.7kD蛋白仅在细胞丘时期出现，在蛞蝓体时期却消失了，说明该蛋白可能是发育早期的一种蛋白。而33.0kD、24.4kD和21.5kD这几种蛋白在发育分化中的作用还有待于深入研究。 lagC基因编码gp150分子，一个97kD蛋白被高度糖基化后的产物，是一种异嗜性细胞表面粘附分子。如果缺失lagC基因，gp150突变细胞的多细胞发育就只能停留在细胞疏松结合阶段，说明不仅调节细胞间的粘着，也调节细胞类型的分化。在以往的研究中发现，发育中的盘基网柄菌细胞开始分化后，出现了两种分子量的gp150分子，一个是150kD，另一个是145kD。为什么会出现两种分子量的gp150蛋白呢?是两个都与柄细胞的分化有关，还是其中一个?以前柄细胞和前孢子细胞为材料，Western blot分析gp150蛋白在两类细胞的表达情况。结果发现gp150蛋白仅出现在前柄细胞中，且随发育进程，含量增加，且gp150和gp145都在前柄细胞中表达。推测gp145可能是gp150分子的mRNA的变位更多还原

【Abstract】 The social amoeba Dictyostelium discoideum which phagocytes soil bacteria for food is a unicellular eukaryote. When food is plentiful, it multiples by equal mitotic division. Cells are triggered to undergo multicellular development under starvation conditions. The development is similar to mutilcellular embryogenesis regulated strictly by spatial-temporal process. Cells gather to form a mound under cAMP concentration, and begin to express some genes regulating cell differentiation and related proteins involving many cellular components, such as adhesion molecule, differentiation inducing factor, cytoskeleton proteins and so on. They transfer signals between cells, and cells differentiate into two distinct cell-types, prestalk and prespore cells. Eventually, cells form a fruiting body consisting of spores and a stalk. The latter is a result of cell death. Therefore, the cell differentiation is actually a process of apoptosis. We don’t know clearly that the difference of protein components between these two types and whether Caspase is necessary for apoptosis.We took Kax-3 (wild type) cell as experiment material and collected cells every two hours during slug stage. Prestalk and prespore cells obtained from mechanically dissociated slugs were separated by the centrifugation on preformed Percoll density gradients. Proteins were separated using SDS-PAGE and scanned by BIO-PROFILE of VILBER LOURMAT. The datum showed the lands 101.2kD、 85.9kD、 68.2kD、 49.8kD、 42.4kD、 40.6kD、 33.0kD、 24.4kD and 21.5kD were shared by prestalk and prespore cells. Among these proteins, 101.2kkD、 68.2kD、 49.8kD、 42.4kD and 33.0kD were the most stable. 101.2kD may be gp100, which is required for the cell-cell adhesion. 68.2kD may be actin-link protein, whose function strengthens the adhesion of cells. 49.2kD is close to tubulin, which is involved in maintain of cell morphogenesis and movement. 42.4kD may be actin, which plays an important role in cell migration. The most different existed in some proteins, for example, 37.5kD and 31.5kD, these proteins were only expressed in prestalk cells, whose content were raised with the cell differentiation. This result indicated these proteins had a relationship with apoptosis. Moreover, 31.5kD is close to Caspase-3, whether it is or not, should be proved in further research. 36.7kD was only existed during mound stage, which showed that was required for early development. And the functions of 33.0kD、 24.4kD and 21.5kD should be further investigated.更多还原