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题名	浦肯野细胞退化小鼠分子机制的初步研究
作者	肖瑞
学位类别	硕士
答辩日期	2005-06
授予单位	中国科学院研究生院
授予地点	北京
导师	张亚平
关键词	浦肯野细胞退化小鼠 Agtobp1基因 DNA微阵列 酵母双杂交 基因表达谱 小脑 蛋白相互作用
其他题名	Study of molecular mechanism in Piirkinje cell degeneration mice
中文摘要	浦肯野细胞退化小鼠的主要特征是在出生后15到18天的时候浦肯野细胞开始退化，到出生后30天几乎所有的浦肯野细胞都己死亡。在22到24天左右pcd＿小鼠表现为中等程度的共济失调。同时在pcd小鼠中还伴随着视网膜光感受体细胞和嗅球僧帽细胞的缓慢退化。另外雄性的pcd小鼠精子发育异常。尽管已经发现该小鼠的表型特征是由ATP／GTPbindingprotein1（Agtpbp1）基因的突变引起的，但是引起浦肯野细胞退化的分子作用机制和途径仍然不是很清楚。在本研究中我们运用DNA微阵列技术对出生后20天的浦肯野细胞退化小鼠和野生型小鼠的基因表达谱进行了分析，处于该发育时期的pcd小鼠还没有表现出明显的表型异常现象。结果显示在野生型和突变型小鼠中有3的个表达水平显著不同的基因，其中大多数基因都与新陈代谢和细胞的生理过程相关。同时根据基因表达的方式将野生型和突变型小鼠分为两个不同的类型。我们用semi-quantitatiyeRT-PCR的方法验证了DNA微阵列的结果。在本实验中发现的基因将为我们今后的研究提供方向和分子目标。另一方面，为了探索可能引起浦肯野细胞退化小鼠表型的遗传途径，我们运用酵母双杂交的方法以AgtP如1基因的三个不同长度的片断为诱饵筛选小鼠脑的cDNA文库，试图找到与七切如湘互作用的蛋白。结果筛选到57个阳性克隆，其中22个是不同的cDNAs。共转化实验和信息学分析揭示6个蛋白可能与Agtpbp1发生相互作用。在57个阳性克隆中有15个经测序和共转化分析都属于同一个蛋白，即SNAP25-associatedproteins（Snapin）。它是神经递质释放过程中的一个功能蛋白。为了阐明pcd，J、鼠的突变机制我们需要进一步在哺乳动物中验证可能发生的蛋白相互作用和进行详尽的分析。
英文摘要	Purkinje cell degeneration (pcd) mice are characterized by the postnatal death of virtually all cerebellar Purkinje cells beginning at 15 to 18 days of age, and all the Purkinje cells are almost gone by 30 days. A moderate ataxia appears by 22 to 24 days in pcd mutant. In addition, pcd mutant exhibits a slow degeneration of retinal photoreceptor cells and mitral cells in the olfactory bulb. And the male mutant has a sperm abnormality. Although pcd phenotypes are caused by mutations in ATP/GTP binding protein 1 (Agtpbpl) gene, the molecular pathway involved in Purkinje cell degeneration is poorly understood. In this study, we investigated the gene expression profiling using DNA microarray analysis between wildtype and pcd mutant mice at the age of postnatal day 20 which is before the appearance of clear phenotypic abnormalities. As a result, we identified 300 differentially expressed genes between wildtype and pcd mutant mice. Majority of them were involved in metabolism and cellular physiological process. Wildtype and mutant animals were classified into two different groups based on the expression patterns. To confirm the results, semi-quantitative RT-PCR was carried out. The genes identified in this study will provide directions and target molecules for future studies. On the other hand, in order to explore the possible molecular pathways underlying pcd phenotypes, we performed yeast two-hybrid system using three different length fragments of Agtpbpl as baits to screen mouse brain cDNA libraries to identify the interacting proteins with AgtpbpL 22 different cDNAs of 57 positive clones were identified from the screening result. ^transformations and bioinformatics analysis revealed that 6 proteins might interact with AgtpbpL 15 of 57 clones were sequenced and confirmed with cotransformation as same protein named SNAP25-associated proteins (Snapiri), which is involved into neurotransmitter release process. Further study and comprehensive analysis have to be done to confirm the interactions between Agtpbpl and 6 possible proteins in mammals to clarify the mutation mechanism inpcd mice.
语种	中文
公开日期	2010-10-15
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6163]
专题	昆明动物研究所_分子进化基因组学
推荐引用方式 GB/T 7714	肖瑞. 浦肯野细胞退化小鼠分子机制的初步研究[D]. 北京. 中国科学院研究生院. 2005.

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