摘要
目的筛选并分析传代培养后血管内皮细胞上表达下调基因。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(PCR)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,用反转录聚合酶链反应(RT-PCR)方法对部分cDNA序列进行初步鉴定。建立兔动脉粥样硬化模型,采用RNA点杂交技术鉴定部分筛选到的cDNA序列的动脉粥样硬化相关性。结果从消减文库中随机挑取的750个白色克隆中筛选出88个阳性克隆,DNA测序获得61个cDNA序列,其中26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,选择4个已知基因采用RT-PCR验证了基因筛选的可靠性。5个新基因具有动脉粥样硬化相关性。结论用抑制消减杂交法构建的传代培养前后主动脉内皮细胞差异表达基因消减cDNA文库富含动脉粥样硬化相关基因。
Objective To screen and analyze of differentially expressed down-regulated genes when the aortic endothelium were cultured. Methods The differentially expressed subtracted eDNA library of freshly isolated endothelium and cultured endothelium constructed by suppression subtractive hybridization（SSH） 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 （BLAST）. Some of the cDNA sequences were used for experimental ensuring by RT-PCR analysis. The atherosclerosis related genes were analyzed by RNA dot-blot analysis on rabbit atherosclerosis model. Results Eighty-eight positive clones were obtained, and 61 eDNA sequences were identified. Sequences of 61 eDNA showed that 26 eDNA were known as bovine gene sequences, 19 cDNA were homologous with the human genes published in Genbank and 16 eD- NA were unknown genes. RT-PCR indicated that 4 known eDNA were only expressed in freshly isolated endothelium. It was found that DEAD-box protein, factor -Ⅻa inhibitor, lymphocyte adaptor protein, protein translocating complex β and H-cadherin are all atheroscierosis related genes. Conclusion The subtracted eDNA library constructed by SSH technique contains the atherosclerosis related genes when the aortic endothelium were cultured.
出处
《中国药物与临床》
CAS
2006年第1期11-17,共7页
Chinese Remedies & Clinics
基金
国家自然科学基金资助项目(30300420)
关键词
血管内皮细胞
抑制消减杂交
CDNA文库
Vascular endothelial cell
Suppression subtractive hybridization
cDNA library
