【摘要】 为了研究辛伐他汀治疗高胆固醇血症过程中肝脏的差异表达基因,我们利用抑制消减杂交(SSH)技术构建了高胆固醇血症小鼠及高胆固醇血症经辛伐他汀治疗小鼠的肝脏双向差异表达基因文库,并采用改进的negative subtraction chain(NSC)方法去除正向文库的绝大部分假阳性背景,使得SSH文库的容量缩小了近50倍,对筛选到14个差异表达克隆再经过斑点杂交验证,最终获得8个阳性克隆.经DNA序列测定和基因功能分析,结果表明,辛伐他汀治疗高胆固醇血症过程中,小鼠肝脏差异表达的已知功能基因主要有以下几类:脂类代谢基因、炎性免疫反应相关基因、细胞粘附基因、逆境胁迫基因.改进的NSC和SSH技术的结合使用,大大地增加了差异表达基因文库的阳性率,提高了获得差异表达基因的效率.更多还原

【Abstract】 Two Suppression Subtractive Hybridization (SSH) libraries were constructed between hypercholesterolemia mice (HC) and simvastatin-treated hypercholesterolemia mice (ST) for identification of the differential expression cDNAs in ST-mice. In order to reduce the false positives, a modified Negative Subtraction Chain (NSC) method was also introduced. By combination of SSH with NSC, we obtained fourteen candidate clones that may differentially express in ST-mice, from which eight positive clones were finally identified by using dot hybridization. The sequence data showed that one of the genes is unknown but the rest seven are all functional genes, which involve in the lipid metabolism, anti-inflammation immune response, cell adhesion and stress-response. The results described above indicated that combination of optimized NSC and SSH may greatly increase the probability to identify the positive clones in differential expression library.更多还原