细胞核内肌动蛋白与染色体构建关系的研究

【作者】 荆玉起；

【导师】 曾宪录；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2002， 硕士

【摘要】 肌动蛋白是首先在肌肉细胞中发现的一种重要的收缩蛋白。肌动蛋白主要存在于细胞质中，是细胞骨架微丝的重要组成成分，受多种肌动蛋白结合蛋白的调控，参与与细胞形态和结构相关的多种生理过程。近20年来，许多学者相继报道在细胞核和染色体中也存在肌动蛋白。细胞核中的肌动蛋白可能参与了核内的一些重要的生理活动，如核骨架维持、染色质集缩和RNA转录等。 本实验以多头绒泡菌原质团为材料，采用同步化培养、细胞核提取、SDS-PAGE、免疫印迹、非细胞体系构建、光学显微镜观察等方法，研究了有丝分裂前期核内肌动蛋白对染色体构建的影响。主要结果如下： 一．建立了多头绒泡菌自然同步化细胞分裂的实验体系。Westernblotting分析结果表明，Cyclin B存在于S期、G₂期、前期和中期的原质团中，分子量约为62kDa，Cyclin B的含量从S期到中期逐渐增加，特别是有丝分裂前期增加明显，中期原质团Cyclin B含量最高，后末期原质团中没有Cyclin B。细胞核中Cyclin B的含量变化可以作为有丝分裂前期进程的标志。 二．鉴于以往研究核内肌动蛋白功能时，很难避免细胞质肌动蛋白的干扰，本实验构建了非细胞体系以研究核内肌动蛋白对染色质构建过程的影响。我们自同步化生长的多头绒泡菌中获得了有丝分裂中期提取物，Western blotting分析表明提取物中含有Cyclin B。将G₂晚期细胞核温育于中期提取物中，免疫印迹结果表明随着温育时间的延长，细胞核中Cyclin B的含量逐渐增加，而在没有中期提取物的对照组细胞核中Cyclin B含量并不随着温育时间的延长而增加，说明多头绒泡菌中期提取物具有促进有丝分裂前期进程的作用，同时也说明我们构建了具有MPF活性的非细胞体系。 三．将经细胞松弛素B处理后的G₂晚期细胞核在有丝分裂中期提取物中温育，经洗涤后对细胞核中CyCli＊ B的含量进行分析，发现随着温育时间的延长，细胞核中Cyclin B的含量没有明显的变化。但如果q晚期的细胞核不用细胞松弛素B处理，则随着温育时间的延长，细胞核中Cyclin B的含量逐渐增加。上述结果说明，细胞松弛素B处理直接影响细胞核中 Cyclin B的含量。因为 Cyclin B是有丝分裂前期进程的标志性蛋白，所以我们认为细胞松弛素B破坏细胞核内肌动蛋白聚合后，直接影响了有丝分裂前期的进程，进而悦明核内肌动蛋白在有丝分裂前期染色体构建过程中起着重要作用。更多还原

【Abstract】 Actin is an important protein in cytoplasm, playing numerous cytoplasmic roles. Recently, A great number of biochemical analyses and immunoctochemical experiments have proved that actin presents in nuclei and chromosomes in plants and animals, showing that actin is involved in the nuclear processes such as chromosome condensation, RNA transcription and so on. In order to avoid the possible contamination of the cytosolic actin, we ultilized a cell-free system to study the effect of nuclear actin on the process of chromosome construction.In this paper, the effect of nuclear actin on the process of chromosome construction has been studied by utilizing the precise natural synchrous plasmodium of Physarum polycephalum, SDS-polyacryl amide gel electrophoresis (SDS-PAGE), western blotting, the cell-free system and optics microscopy. The major results and conclusions are as follows:1. A synchronous mitotic experiment system that was in an unchanged culture condition was established. SDS-PAGE and western blotting of plasmodia of Physarum polycephalum showed that cyclin B was present in the plasmodia of S phase, G2 phase, prophase, and metaphase. The molecular weight of Cyclin B in Physarum polycephalum was around 62kDa. Cyclin B was gradually accumulated from S phase to metaphase, and reached the maximal level at metaphase, however disappeared in the plasmodia of anaphase and telophase. Cyclin B can be a kind of mark protein which represents the process of prophase.2. Mitotic extracts were prepared from the synchronous metaphase plasmodia of Physarum polycephalum. The results of western blotting indicated that cyclin B was a component of the mitotic extracts of Physarum polycephalum. After the nuclei of late G2 phase were incubated in the mitotic extracts harvested from metaphase plasmodia, the cyclin B in the nuclei gradually accumulated with the prolongation of incubating time. While, in the control group, the content of cyclin B changed little. This indicated that the mitotic extracts of metaphase plasmodia had the activity of accelerating the process of prophase, and the cell free system with MPF activity was established.3. The nuclei of the late G2 phase treated by cytochalasinB were incubated in the mitotic extracts harvested from the metaphase plasmodia. Western blotting analysis showed that the content of cyclin B in the nuclei changed little. In contrast, in the control group, the content of cyclin B gradually increased with the prolongation of incubating time. Our results showed that the treatment of cytochalasin B affected the increase of the cyclin B content in the nuclei directly. As cyclin B is the mark protein representing the process of prophase, we infered that cytochalasin B destroyed the polymerization of the nuclear actin and directly affected the process of prophase. Nuclear actin played an important role in the process of the chromosome construction.更多还原