Selection Of DNA Aptamers Recognizing MMP14 Protein Using Living Cell-SELEX And Development Of MR-fluorescent Dual Modality Aptamer Probe For Targeted Imaging Of Pancreatic Cancer

Objective:1.To construct a transfected 293 T cell line of the MMP14 gene,and to use this cell line to screen DNA aptamers that can specifically recognize MMP14 protein based on cell SELEX;2.To construct aptamer MR-fluorescent dual modality molecular probe for targeted imaging of MMP14 positive cells.Methods:1.Constructed the lentiviral vector of MMP14 gene by gene synthesis method,and to construct 293T-MMP14 cells that specifically express MMP14 protein by using this lentiviral vector to transfect 293 T cells.2.Constructed the ss DNA library consisted of a central random ized sequence of 40 nucleotides flanked by two constant sequences of 20 nucleotides for polymerase chain reaction(PCR)amplification,and then to screen DNA aptamers recognizing MMP14 Protein by using living cell-SELEX.293T-MMP14 cells were used as positive cells,and MMP14 negative 293 T cells were used as control cells.After 12 rounds of selection,the resulting ss DNA pool was PCR-amplified and cloned,and monoclonal colony was selected for sequence analysis by Sangon Biotech,thus to obtain aptamer candidates.Flow cytometry was used to verify the affinity of the DNA aptamers with 293T-MMP14 cells.The highest affinity of aptamer was selected to test its Kd value by flow cytometry to assess its binding specificity.Cellular immunofluorescence and flow c ytometry were performed to test the ability of the aptamer to bind to pancreatic cancer cell lines.3.Linked the selected aptamer that was labeled Cy5 and amino in 5' and 3'end was linked to the magnetic Fe3O4 nanoparticle(PEG@Fe3O4)to construct the aptamer MR-fluorescent dual modality molecular probe which include d the fluorescein Cy5 and magnetic contrast agent Fe3O4.The fluorescence imaging function was detected by cellular immunofluorescence and nude mice bearing the tumor,and the magnetic resonance imaging function was detected at the cell level.Results:1.The MMP14 gene lentiviral vector was successfully transfected into 293 T cells,and 293T-MMP14 can specifically expressed MMP14 protein.2.After 12 rounds of cell SELEX,24 effective DNA aptamer sequences were obtained.The M17 DNA aptamer has the highest affinity to 293T-MMP14 cells among 24 aptamer candidates by flow cytometry and its Kd value was 4.98±1.26 n M,which was in the nmol level.Cellular immunofluorescence showed that M17 specifically bound to the surface of 293T-MMP14 cell membrane.And M17 was found to specifically bind to pancreatic cancer cell lines MIA Pa Ca-2 and PANC-1 by flow cytometry.3.MR-fluorescent dual modality molecular probe PEG@Fe3O4-M17-Cy5 was constructed via condensation reactions.Cellular immunofluorescence demonstrated that the molecular probe could specifically perform fluorescence imaging of pancreatic cancer cell lines MIA Pa Ca-2 and PANC-1.MRI showed that the molecular probe could significantly reduce the signal intensity of T2 WI in cells lines MIA Pa Ca-2 and PANC-1.The molecular probe can achieve targeted fluorescence imaging of subcutaneous xenograft tumors of PANC-1 cells in nude mice in vivo by whole-body fluorescent imaging system.Conclusion1.The aptamers screened by cell SELEX can specifically bind to MMP14 positive cell lines and specifically detect pancreatic cancer cell lines MIA Pa Ca-2 and PANC-1.The DNA aptamer is expected to be used as a targeted molecular for the early diagnosis and targeted therapy of the MMP14 positive tumours.2.MR-fluorescent dual modality molecular probe PEG@Fe3O4-M17-Cy5 can achieve fluorescence staining of pancreatic cancer cell lines MIA Pa Ca-2 and PANC-1.The molecular probe can effectively reduce the signal intensity of T2 WI of both pancreatic cancer cell lines.And they can effectively target to subcutaneous xenograft tumors of MIA Pa Ca-2 cells in nude mice and have good fluorescence imaging of the tumor.The molecular probe has a potential application in the early targeted diagnosis in pancreatic cancer and other MMP14 positive tumors.