抑制性差减杂交结合cDNA芯片筛选家兔高脂血症及动脉粥样硬化相关基因

发布时间：2018-04-24 13:49

本文选题：动脉粥样硬化 + 高脂血症　；参考：《南京医科大学》2005年博士论文

【摘要】：目的:探寻与AS发生发展相关的调节脂质代谢、氨基酸代谢、糖代谢、炎症反应、免疫应答、变态反应等的肝脏差异表达基因,明确肝脏在高脂血症和AS发生中的作用。方法:通过给新西兰白兔喂饲高胆固醇饮食复制高脂血症-As动物模型。根据其血清胆固醇含量分为敏感组和不敏感组。分别取两组动物的肝脏,抽提总RNA,并分离mRNA,以造型敏感组为受检者(tester)、造型不敏感组为驱动者(driver),进行正向SSH;在反向SSH中则相反。经两轮杂交及两次抑制性PCR,将产物与T/A载体连接构建cDNA差减文库,并转化大肠杆菌进行文库扩增。随机挑取800个阳性克隆,以插入片段两侧载体序列PinPoint和Sp6为引物PCR扩增,PCR产物经浓缩、变性处理,点于尼龙膜上,制成cDNA芯片。提取造型敏感组和不敏感组兔肝mRNA,反转录后经同位素~(33)P标记,分别与两张相同的芯片杂交,获取量化的杂交信号。将差异表达克隆测序,所得结果通过Blastn软件与GenBank中的已知核苷酸序列进行查询和同源比较。选取6条基因,通过荧光半定
[Abstract]:Aim: to explore the differentially expressed genes related to the regulation of lipid metabolism, amino acid metabolism, glucose metabolism, inflammation, immune response and allergic reaction, and to clarify the role of liver in hyperlipidemia and as. Methods: hyperlipidemia-as animal model was established by feeding New Zealand white rabbits with high cholesterol diet. According to its serum cholesterol content, it was divided into sensitive group and insensitive group. The total RNAs were extracted from the liver of the two groups, and the mRNAs were isolated. The positive SSH was carried out in the sculpt sensitive group and the modeling insensitive group as the driver. The cDNA subtractive library was constructed by two rounds hybridization and two inhibition PCR. The product was linked with the T / A vector and transformed into E. coli to amplify the library. Eight hundred positive clones were randomly selected. The PCR products were condensed and denatured with the primers of PinPoint and Sp6 on both sides of the inserted fragments. The cDNA microarray was made by placing them on the nylon membrane. The rabbit liver mRNAs were extracted from the sensitive and insensitive groups, and then labeled with the isotopic mRNAs, then hybridized with two identical microarrays, respectively, to obtain the quantitative hybridization signals. The differentially expressed clones were sequenced. The results were queried and homologous compared with known nucleotide sequences in GenBank by Blastn software. Six genes were selected, and the results were determined by fluorescence semidefinite.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346

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