ИСТИНА

Firefly luciferase is a perfect label to use in different cellular processes monitoring. It is easy to detect and to deliver into cell with help of genetic engineering. The aim of this work was to study the effect of different agents on cell membrane permeability and integrity using firefly luciferase reporter. The gene of luciferase was inserted into the plasmid, intended for eukaryotic cells transfection. HEK-293 cells were transfected using lipofectamine with high efficiency. Procedures of cell lysis and luciferase activity measurements were optimised. The effects of concentration and exposition time on cell membrane integrity were studied by measuring luciferase signal in cell supernatant. The system also allowed to measure ATP rate inside and outside cells, which is also an important cell physiology marker. It was found that unmodified luciferin is unable to get inside cells under physiological conditions, which makes it clear, that obtained signals come from reactions, catalyzed by luciferase released from damaged cells. Kinetic curves of cell membrane destruction were obtained for different effectors. It was studied, whether they affect the enzyme, to exclude the effect of luciferase inactivation. The method was optimized to be used in compounds screening and membrane studies. It was proven with different types of membrane affecting compounds – saponins, antibiotics and substances, which cause oxidative stress. Digitonin was shown to cause fast destruction of cell membrane due to interaction with intramembrane cholesterol. Polymyxin, which is often used to treat infections, caused by gram-negative microorganisms, showed slower kinetics of membrane destruction. Both effectors had linear dependence on concentration in the studied interval. Agents of oxidative stress, which are reported to damage membranes, demonstrated no active effect nor in short, neither in long periods of time.