知识与财富的链接
探讨动脉粥样硬化（Atherosclerosis，AS）病人LDLR基因启动子区12个CpG二核苷酸结构甲基化的改变。改进并联合应用巢式、Touchdown—PCR，甲基化敏感单链构象分析法（methylationsensitive—single—stand conformation analysis，MS—SSCA），对61例AS病人及28例健康对照者的LDLR基因启动子区CpG岛的甲基化状态进行检测。在AS患者中发现三种不同的甲基化模式，即完全甲基化、部分甲基化和非甲基化。61例AS患者中有2例完全甲基化，13例部分甲基化，其余为非甲基化．甲基化率为24．59％；28例正常对照者中仅1例发生部分甲基化，甲基化率为3．57％。两组甲基化率差异有显著性（P〈0．05）。AS病人LDLR基因启动子区甲基化程度增高，在LDLR基因调控区的高甲基化修饰可能参与了AS的发病。同时，改进并联合应用的巢式PCR、Touchdown—PCR．MS—SSCA法降低了对DNA模板严格度的要求，提高了灵敏度和特异性，在检测基因甲基化中具有方便、经济、效率高的优点，有良好的应用前景。
To investigate the methylation pattern of 12 CpG dinucleotide sites in promoter region of LDLR gene in atherosclerosis patients, peripheral blood DNA samples were prepared from 61 atherosclerosis patients and 28 healthy subjects. The methylation status of CpG islands in LDLR gene promoter region was measured by using a modified coordinative method with nested-PCR, Touchdown-PCR and methylation-sensitive-single-strand conformation analysis (MS-SSCA). Three types of methylation patterns were detected in promoter region of LDLR gene in peripheral blood genome of atherosclerosis patients: full-methylation, part-methylation and none-methylation. 2 cases were full-methylation and 13 cases were part-methylation, the methylation frequency was 24.59% in atherosclerosis patients. While in 28 healthy control, only 1 half-methylation sample was found, the methylation frequency was 3.57%, which is much lower than that in atherosclerosis patients (P < 0.05). A significantly higher methylation degree in promoter region of LDLR gene was found in peripheral blood genome of atherosclerosis patients compared with the controlled healthy subjects, which may suggest an involvement of aberrant methylation of LDLR gene in initiation and development of AS. The modified coordinative method with nested-PCR, Touchdown-PCR and MS-SSCA manifests merits of convenience, cheap and high efficiency, which lowers the requirement for the DNA template and increases the sensitivity and specificity.
