Selection Of Aptamers To Early Gastric Adenocarcinoma Primary Cells And Preliminary Extraction Of Cell Membrane Molecular Markers

Objective:Separate and extract early gastric adenocarcinoma primary cells as target cells by collagenase solution IV;screen out the aptamers to the target cells by Cell-SELEX;by analyzing the aptamers,select the ones with high affinity or specificity for preliminary extraction of target cell membrane molecular markers,setting a molecular target foundation for diagnosis and treatment of early gastric adenocarcinoma.Methods:Collagenase solution Ⅳ was applied to separate and extract early gastric adenocarcinoma primary cells and normal gastric mucosa cells,and then the purity of cells was analyzed by immunohistochemistry with epithelia cytokeratin antibody CK8and CK18.A synthesized88nt ssDNA library containing a random sequence of52nucleotides flanked by5’and3’fixed regions of18nucleotides was used,early gastric adenocarcinoma primary cells were used as target cells,and normal gastric mucosa cells were as counter-target cells in the cell-SELEX.After several rounds of screening,the last round secondary ssDNA library was amplified by PCR, proliferation products were spliced with pGEM-T vector after purification. After transduction and blue-white screening,30white clones were randomly selected to be sequenced.Predication of secondary structure and primary structure homology analysis to aptamers were made by DNAMAN7.0,then the aptamers was amplified by PCR using primer marked by fluorescein, and the binding affinity and specificity of aptamers were determined. Biotin-streptavidin magnetic beads system was used to select aptamers with high affinity or specificity,and these aptamers was used to preliminary extract early gastric adenocarcinoma cell membrane molecular markers.Results:1. The purity of early gastric adenocarcinoma and normal gastric mucosa cells isolated by0.1%collagenase solution IV was above90%,which could meet the need of cell-SELEX screening. 2. PCR condition for amplification was optimized before screening,and the optimal annealing temperature was46℃and the number of cycles was15.After12round screening, binding rate between the secondary ssDNA library and early gastric adenocarcinoma cells reached higher levels.After blue-white screening and sequencing,in the primary structure of aptamers,random sequence of C15and C23aptamers was a nucleotide shorter than predicted, while C29random sequence was one longer.The sequence length of other27aptamers was the same as predicted,within which aptamer C17has identified sequence with C27.By homology analysis,all sequences undergone sequencing shared no conservative sequence.By secondary structure analysis,aptamers formed into different stem-loop structures,which were the structural basis for binding of aptamers and target cells.3. By binding specificity analysis,binding fluorescence intensity between aptamers and gastric adenocarcinoma primary cells were significantly different with that between normal gastric mucosa cells and blank control(P<0.05),and with that between cultured SGC-7901and BGC-803cells(P<0.05). By affinity analysis,dissociation coefficient between aptamers and early gastric adenocarcinoma primary cells reached nmol/L level,indicating a high affinity.4. Aptamer with high specificity and biotin-streptavidin magnetic beads system were used in the extraction of target substances.Target substances were under SDS-PAGE electrophoresis, then molecular weight of aptamer-binded cell membrane molecular markers were around100kDa with a relative clear band were observed. After cells were treated with protease, fluorescence intensity between aptamers and cells were significantly different before and after binding(P<0.01),which can be used to determine that protein existed in substances binded with aptamers.Conclusion:1. Primary cells can be isolated by collagenase solution Ⅳ with high affinity,which can be applied in further studies as a cell dissociation method.2. Aptamers with high specificity to early gastric adenocarcinoma primary cells can be screened out by cell-SELEX with complete structure;predication of secondary structure indicated that stem-loop structure could be the binding basis of aptamers and target cells.3. The method that fluorescence was used to modify aptamers successfully determined the binding specificity and affinity between aptamers and early gastric adenocarcinoma cells.The flurorescence intensity between cultured cells and cells isolated from tissues binding with aptamers was statistically different.4. Applying aptamers modified by biotin and combined with biotin-streptavidin magnetic beads system,molecular markers with high specificity was isolated from cell membranes of early gastric adenocarcinoma,the molecular weight of the markers was get,and the protein in the molecular markers was determined.