Fluorescence Labeling, Analysis And Applied Research Of Nucleoside Transporter On The Membrane Of Tumor Cell

A simple, rapid and sensitive fluorescence method for the determination of dipyridamole bound on cell membrane in acetonitrile-water (2:1, v/v) solution was developed. Pancreas-1 tumor cell was cultured in Dulbecco's modified Eagle medium under appropriate conditions to a final density of (3-5)×105 cells ml-1. The cell plasma membrane was separated from the ultrasonically dissolved cell mixture by gradient centrifugation. After dipyridamole was bound to the cell membrane, the test sample was obtained by ultrasonically dissolving the labeled membrane in 10 mmol L-1 tris-HCl membrane lyses solution. Under selected experimental conditions, the fluorescence intensity of dipyridamole determined in acetonitrile-water (2:1, v/v) solution at λem of 295 nm and λem of 485 nm was proportional to its concentration ranging from 1.0(10-12 to 1.0(10-9 mol L-1 with a detection limit of 2.8(10-13 M (0.14 pg ml-1) at 3σ. The value of affinity constant of the cell membrane to dipyridamole was 4.7((0.53)×1010.The determination of intracellular concentration of 6-thiopurine was established with fluorimetry. The fluorescence spectral character of 6-thiopurine was investigated and the influences of pH, temperature and delay time on the determination of fluorescence intensity of 6-thiopurine were discussed. Under the optimum experimental conditions, the concentration of 6-thiopurine was directly proportional to its fluorescence intensity ranging from 4.0×10-11 to 4.0×10-6 M with a detection limit of 8.2×10-12 M at 3σ. The intracellular concentration of 6-thiopurine was then calculated to be 2.38±0.088 nM. The presented method was precise and accurate. It can be used as a reference method for the determination of intracellular concentration of 6-thiopurine in clinical medicine.The action of dipyridamole conjugating on Panc-1 cell membrane on the intracellular concentration of 6-thiopurine was studied. When Panc-1 cell was incubated with 6-thiopurine, its intracellular concentration reached equilibrium after 60 min. After adding 200 μM dipyridamole, the intracellular concentration of 6-thiopurine increased about 0.49 fold comparing to without adding dipyridamole. The results showed that there existed a statistic difference between with and without dipyridamole.