Target Marker Screening Of Foam Cell By Aptamer

Objective To obtain target protein of foam cell through the aptamer specifically binding the cells .Methods The SELEX ssDNA random library was constructed to screen and enrich repeatedly in vitro and then obtained aptamers, which characterized with specific binding to macrophage-derived foam cells. One of the aptamers was selected and incubated with different type of lipid-laden foam cells. Oil red O staining was used to observe the intracellular lipid droplets and cholesterol inside cells was detected through HPLC. Then the specification of the aptamer binding to the cells and histological sections were detected by fluorescence microscopy. Biotin-streptavidin system was used to extract the protein of foam cell. 1D-SDS-PAGE and LC-ESI-TRAP were performed to separate protein and analysis the protein components. Lastly, protein levels were detected by Western blotting.Results The ssDNA, NO.23 aptamer, was obtained through SELEX procedure to bind with foam cells derived from macrophage. The model of foam cells were identified by Oil red O and HPLC. The ability of the aptamer which binding to the foam cells derived from THP-1 cell and RAW264.7 cell and aorta atherosclerotic plaque was specific. The protein extracted from foam cell was separated into three components, and the identification of three components was performed via tandem mass spectra, then part of sequence information results were tested through NCBInr database. Twelve proteins, including vimentin were evaluated. Target protein 2 and vimentin were selected and detected by Western blotting at protein level. The result indicated that vimentin expression in THP-1 macrophage-derived foam cell is much higher than in untreated THP-1 macrophage by ox-LDL, and target protein 2 only expresses on foam cell.Conclusions1. The aptamer (number 23 aptamer, 81nt and 5'-biotinylated single-stranded DNA) characterized specifically binding macrophage-derived foam cell and atherosclerotic lesion.2. 12 candidate target proteins were identified to specifically bind to aptamer.3. Target protein 2 was specially expressed on THP-1 macrophage induced by ox-LDL, and may be a new target for diagnosis and therapy.