Screening and analysis for cDNA of differentially expressed down-regulated genes in cultured aortic endothelium

Objective To screen and analyze of differentially expressed down-regulated genes when the aortic endothelium were cultured. Methods The differentially expressed subtracted cDNA 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 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 genes published in Genbank and 16 cDNA were unknown genes. RT-PCR indicated that 4 known cDNA 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 atherosclerosis related genes. Conclusion The subtracted cDNA library constructed by SSH technique contains the atherosclerosis related genes when the aortic endothelium were cultured.