应用抑制消减杂交技术筛选动脉粥样硬化相关基因

Screening of atherosclerosis related genes using suppression subtractive hybri dization technique

目的 筛选动脉粥样硬化 (AS)相关基因。方法通过菌落原位杂交技术筛选用抑制消减杂交法构建的传代培养前后牛主动脉内皮细胞差异表达基因消减cDNA文库 ,用聚合酶链反应方法进一步筛选出有插入片段的阳性克隆 ,将阳性克隆进行DNA测序和同源性比较分析。建立兔AS模型 ,采用RNA点杂交技术鉴定部分筛选到的cDNA序列的AS相关性。结果 从消减文库中随机挑取的 75 0个白色克隆中筛选出 88个阳性克隆 ,DNA测序获得 6 1个cDNA序列 ,其中 2 6个为已知牛基因序列 ,19个与已知人基因具有高度同源性 ,其他 16个是未知基因片段。通过基因同源性分析 ,选择部分cDNA序列进行组织表达差异及其与AS病变的关系分析 ,结果表明 ,转录调节因子DEAD box ,Ⅻa因子抑制蛋白 ,淋巴调适蛋白和转位复合蛋白 β基因为AS相关基因。结论 这 4种基因为本文首次报道的AS相关基因。

AIM To screen the atherosclerosis related genes. METH OD S The differentially expressed subtracted cDNA library of freshly isolate d adult bovine endothelium and cultured new-born bovine endothelium constructed by suppression subtractive hybridization technique was screened by colony in situ hybridization, the positive clones were further screened with PCR amplification. The positive clones were sequenced and analyzed for homology in the GenBank databases with basic local alignment search tool. The atherosclerosi s related genes were analyzed by RNA dot-blot analysis on rabbit atherosclerosi s model. RESULTS Eighty-eight positive clones were obtained, and 6 1 cDNA sequences were identified. Sequences of 61 cDNA showed that 26 cDNA were known as bovine gene sequences, 19 cDNA were homologous with the human g enes published in GenBank and 16 cDNA were unknown genes. Through gene homologou s analysis, some differentially expressed cDNA sequences were selected for tissu e differential and atherosclerosis related analysis. We found that DEAD-box pro tein, factor Ⅻa inhibitor, lymphocyte adaptor protein and protein translocation complex β are all atherosclerosis related genes. CONCLUSION This is the first time to report four genes are related to atherosclerosis.