导　　师： 卜碧涛

学科专业： J0204

授予学位： 博士

作　　者： ;

机构地区： 华中科技大学

摘　　要： 本文主要从Ataxin-3敲除导致基因表达谱改变、EphA3基因启动子区域定位表达及活性分析、HDAC3和NCoR介导了ataxin-3对EphA3基因的转录调控三个方面进行了论述。　　第一部分:Ataxin-3敲除导致基因表达谱改变　　目的:采用基因表达芯片技术(Microarray)观察ataxin-3敲除对基因表达变化的影响。　　方法:选取ataxin-3敲除的小鼠胚胎成纤维细胞(ataxin-3 KO MEF)和正常小鼠胚胎成纤维细胞(ataxin-3 WT MEF)为研究对象,提取RNA并用基因表达谱芯片技术观察ataxin-3敲除对基因表达的影响,所得实验结果数据经ThomsonReuters/GeneGo公司的生物信息学软件进行分析,并采用逆转录实时定量PCR技术证实基因表达芯片结果的可靠性。　　结果:与正常细胞相比,ataxin-3敲除导致410个基因表达明显异常(基因表达信号上调或下调超过2倍及以上),其中260个基因表达上调,而150个基因表达下调。GeneGo软件分析这410个基因的生物学功能,并对其功能进行分类。发现ataxin-3敲除导致多个细胞通路转录异常,尤其是炎症免疫和细胞粘附相关基因。　　结论:Ataxin-3分子丢失导致多种细胞通路相关基因转录异常。　　第二部分:EphA3基因启动子区域定位表达及活性分析　　目的:构建含有不同长度EphA3基因启动子片段的报告基因载体,研究其在293T细胞和MEF细胞中的转录活性。　　方法:以Balb/C小鼠基因组DNA为模板,扩增不同长度的EphA3基因启动子片段,并克隆进入荧光素酶报告基因质粒pGL3-Basic真核表达载体内。酶切鉴定及基因测序无误后,将重组质粒和pRL-CMV内对照质粒共转染293T或MEF细胞,分析不同长度的EphA3基因启动子片段的转录活性。　　结果:酶切鉴定和基因测序结果提示表达载体构建成功,EphA3基因的核心启动子区域位于-279～+110bp之间,在293T细胞和MEF细胞中启动子片段的转录活性相似。　　结论:成功构建了� PartⅠ. Loss of ataxin-3 induces transcriptional dysfunction Objective:To investigate the difference of gene expression between ataxin-3 KO MEF cells and ataxin-3 WT MEF cells. Methods:RNA was isolated from ataxin-3 WT and ataxin-3 KO MEF cells, and Microarray was applied to analyse the gene expression. RT-PCR was performed to confirm Microarray data. Results:In comparison with ataxin-3 WT control, loss of ataxin-3 induced the upregulation of 260 genes and downregulation of 150 genes. These genes were categorized based on different biological process, and several pathways including immune response pathway and cell adhesion were altered due to loss of ataxin-3. Conclusion:Loss of ataxin-3 induces transcriptional dysfunction in MEF cells. PartⅡ. Promoter regions of EphA3 and their transcriptional activities in 293T and MEF cell lines Objective:To construct reporter vectors containing EphA3 promoter region in different lengths and test their transcriptional activities in 293T and MEF cells. Methods:Genomic DNA from Balb//C mice was used as a template to synthesize various PCR products in different lengths, and then the PCR products cloned into pGL3-Basic vector to develop a series of reporter constructs. These constructs were cotransfected into 293T or MEF cells with pRL-CMV vector, and luciferase activities were measured. Results:All constructs were verified by restriction enzyme digestion and sequencing. The basic promoter region of EphA3 was from -279bp to transcription start site /(+110bp/), and the promoter activities of EphA3 in 293T and MEF cells were similar. Conclusions:EphA3 promoter region in different lengths are successfully cloned into reporter vector and the basic promoter region is confirmed. PartⅢ. Loss of ataxin-3 leads to transcriptional dysfunction of EphA3 gene via repressing the expression of HDAC3 and NCoR Spinocerebellar ataxia type 3 /(SCA3/) is an autosomal dominant neurodegenerative disease and one of the nine known polyglutamine /(PolyQ/) neurodegen

关 键 词： 脊髓小脑共济失调 型 病理学 缺失诱导 基因转录 芯片技术

领　　域： [医药卫生—神经病学与精神病学] [医药卫生—临床医学]