【作者】 高巍；

【导师】 顾福康；

【作者基本信息】 华东师范大学 ， 动物学， 2008， 硕士

【摘要】 细胞骨架是真核细胞中的蛋白质纤维网架体系,是当前细胞生物学研究中最活跃的领域之一。广义的细胞骨架包括细胞外基质、细胞膜骨架、细胞质骨架和细胞核骨架,它们在结构上相互连接、形成贯穿于细胞的网架体系。细胞骨架在维持细胞形态,保持细胞内部结构的有序性中起到重要作用,而且与细胞运动、物质运输、能量转换,信息传递、细胞分裂、基因表达、细胞分化等生命活动密切相关。目前细胞骨架的研究已从形态观察为主迅速推进到分子水平。原生动物由一个单细胞构成,并行使生命活动的全部功能,因此原生动物细胞的复杂程度是多细胞生物中的一个细胞远不能及的。以腹毛目纤毛虫为材料研究原生动物的细胞骨架,对了解原生动物的细胞功能、揭示种属之间的进化关系有重要的意义。在原生动物中不同类群纤毛虫的细胞微管骨架排列和分化程度不同。腹毛目纤毛虫具有复杂的微管骨架结构。包括纤毛和纤毛器骨架,皮层微管骨架以及其他胞质微管骨架。这些骨架结构对细胞形态和功能的维持都具有重要作用。异毛虫(Allotricha curdsi)和尖毛虫(Oxytricha sp.)分别隶属于侧毛虫科(Pleurotrichidae)和尖毛虫科(Oxytrichidae),均是腹毛目内分化较高的类群,两者在亲缘进化关系上存在一定差异。本文以这两种纤毛虫为材料,采用Flutax直接荧光标记与免疫荧光标记相结合的方法,对其形态和形态发生及其休眠细胞纤毛结构的分化进行探索,显示腹毛目尖毛虫和异毛虫在不同生命周期中皮层微管骨架,揭示它们的发生、分化及定位的规律。并利用生化抽提结合SDS-PAGE电泳的方法对其蛋白组分进行了初步分析。结果如下:1、异毛虫无性生殖周期中微管胞器的形态及形态发生FLUTAX直接荧光标记和抗α-微管蛋白抗体免疫荧光标记显示,异毛虫细胞微管胞器由口围带、波动膜、额腹横棘毛、左右缘棘毛、背触毛等皮层纤毛器、纤毛器基部附属微管组成。其中腹面皮层纤毛器基部附属微管包括小膜托架、小膜附属微管、波动膜骨架;额腹横棘毛基部围棘纤维篮、额腹横棘毛和左右缘棘毛基部前纵微管束、后纵微管束和横微管束,他们以各自的纤毛器基部为中心,分别向皮层不同方向发射,在细胞皮层内形成一个复合三维结构微管网;并且,左、右缘棘毛基部横微管显示非镜像对称的图像,横棘毛基部微管前纵微管束向前伸展后其前端并非聚集在一起。无性生殖过程中,后口仔虫口围带原基在表膜下首先发生,此后突出表膜形成相应的纤毛器原基,经历一系列的发育分化过程而成为新仔虫的纤毛器。腹部纤毛器发育包括口围带、波动膜、额腹横棘毛发生瓦解,新纤毛器在细胞皮层的特定位置发生,从相应纤毛器原基发生、分组、迁移,至最终形成前、后两套新的纤毛器,新结构的形成和老结构的瓦解是一个相伴相随的过程。2、尖毛虫皮层微管类细胞骨架的组成和形态发生利用FLUTAX直接荧光标记和抗α-微管蛋白抗体免疫荧光标记显示,尖毛虫(Oxytricha sp.)细胞微管胞器由口围带、波动膜、额腹横棘毛、左右缘棘毛、背触毛等皮层纤毛器及纤毛器基部附属微管组成。纤毛器微管和基部附属微管按照“口围带-额腹横棘毛-左右缘棘毛”的皮层模式定位,所有这些结构对于支持细胞运动,维持细胞背腹面的分化具有重要作用。无性生殖过程中,额腹横棘毛原基分别在老的口围带和新口围带的右侧出现两套,之后逐步分化发育形成前后仔虫的相应结构,部分老的额棘毛和横棘毛在新的结构出现之后才最终瓦解。3、异毛虫与尖毛虫细胞蛋白组分的比较采用异毛虫和尖毛虫为实验材料,利用生化方法抽提处理得到两种纤毛虫的骨架蛋白(cytoskeleton protein,ck)、不可溶性蛋白(insoluble protein,c)和可溶性蛋白(soluble protein,s),进行电泳分析,用以研究两种淡水类纤毛虫骨架蛋白的差异。结果发现:异毛虫和尖毛虫骨架蛋白的表达主要集中在43-66kDa区间,充分证明α-微管蛋白在纤毛虫微管类细胞骨架中具有一定的保守性。而两种纤毛虫不可溶性蛋白的表达具有一定差异。更多还原

【Abstract】 Cytoskeleton is the protein fibre network in eukaryotic cell and becomes a focus of attention in cell biology research. In general the cytoskeleton which contains cytomatrix, membrane skeleton, cytoplasm skeleton and nuclear skeleton, forms network system running through the whole cell. The cytoskeleton plays an important role in keeping the shape of cell and holding the order of cell inner structure, also closely relates with cell movement, material transportation, energy conversion, information transmission, cell division, gene expression, cell differentiation, and other life activities. Currently the cytoskeleton research has rapidly advanced from the morphology observation to the molecular level. The protozoa ciliate which consists of a single cell, exercise all features of life activities. So it is much more complex than the one cell from multi-cell organisms. It has important significance to investigate the cytoskeleton of protozoa, understand the function of the cell and reveal the evolution relationship between different genus by using hypotrichous ciliate as the materials. Different groups of ciliates have different cytoskeleton arrangement and differentiation. The hyportrichous ciliate has complex microtubule structure, including the cilia and the ciliature-skeleton, cortical microtubule skeleton and other cytoplasm microtubule skeleton. These skeleton have an important role in maintenance the cells form and function. Allotricha curdsi and Oxytricha are advanced species in hyportrichous ciliates. In this paper, we use these two kinds of ciliate as materials to detect their cilia-structure differentiation in the term of morphology, morphogenesis and cyst through the method of Flutax direct fluorescent-labeled and anti-αtubulin antibody. We found that the cortical microtubule skeleton is different in the different lifecycle. This revealed the law of its morphogenesis, tubulin differentiation and positioning. And we also use the biochemical extraction and SDS-PAGE to analyze its protein components. The results are as follows:1. Morphology and morphogenesis of Allotricha curdsiThe microtubular organelles in the hyportrichous ciliate Allotricha curdsi were analyzed with fluorescence labeling of FLUTAX and anti-αtubulin antibody. These microtubular organelles consisted of adoral zone of membranelles (AZM), undulating membranes (UM), frontal-ventral-transverse cirri (FVTC), left and right marginal cirri (L- & RMC), dorsal kineties (DK) and the base-associated microtubules of the ciliatures. The base-associated microtubules in the ventral cortex of ciliature comprised membranelle brackets and its base-associated microtubules, UM cytoskeleton, fibrillar cirrral basket (FCB) located at the base of FVTC, anterior longitudinal microtubules (ALM), posterior longitudinal microtubules (PLM) and transverse microtubules (TM) situated at the base of FVTC, LMC, and RMC. Sending from the base of ciliatures and extending to different directions in the cortex, these microtubules formed a complex three-dimensional microtubular net underneath of cortex. In addition, the arrangement of transverse microtubules (TM) at the base of the LMC and RMC displayed a non-mirror symmetrical image, and anterior longitudinal microtubules (ALM) at the base of the TC extend forwards and just did not converge together. During the term of morphogenesis, the oral primordium of the opisthe would originated underneath the cell ellicle, protruding from the cell pellicle to form corresponding ciliature primordium, which then developes into new ciliature through the process of differentiation. The development of ventral ciliature consists the collapse of AZM, UM, FVTC. new ciliature originated in the given cortex of cell groups and moves form certain ciliature primordium, and finally becomes new ciliature.2. The consistence of microtubule cytoskeleton and morphogenesis in Oxytricha sp.Microtubular organelles of protozoa Oxytricha n.sp. was analyzed with anti-α-tubulin antibody and FLUTAX. The results show that the microtubular organelles consisted of adoral zone of membranelles(AZM), undulating membranes(UM), frontal-ventral-transverse cirri(FVTC), left-and-right-marginal cirri(L-RMC), dorsal kinetics(DK) and their base-associated microtubules. The ciliature microtubule and its base-associated microtubule which oriented in such cortical form: AZM-FVTC-LRMC, plays an important role in supporting the movement of ciliary organelles and sustaining the dorsoventral differentiation. During the term of binary fission, two sets of FVTC primordia occurs beside the old and the new AZM, then form certain structures of opisthe and proter. Some old FC and TC finally disappeared after the new structure formed.3. Analysis on the protein components of Allotricha curdsi and OxytrichaThe differences of cytoskeleton protein components between Allotricha curdsi and Oxytricha was investigated using biochemical extraction and electrophoresis. The results are as follows: the cytoskeleton protein components of two kinds of ciliate express between 43kDa to 66kDa. This showed thatα-tubulin is conservative in microtubule cytoskeleton of ciliate. However the insoluble protein has some differences between two kinds of ciliate.更多还原