|Budget Amount *help
¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1996 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1995 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Adult cardiomyocytes were isolated by Langendorff perfusion method with collagenase solution, and paraquat (PQ) solution of various concentrations were administrated. Morphological changes and extracellular release of intracellular enzyme, both of which are the markers of cell damage, were investigated in vivo. Enzymes used, their combinations, and perfusion conditions were tested. Consequently, it was suggested that the administration of PQ seemed to be harmful to cardiomyocytes.
Cultured cells and the medium solutions, 2 and 18 hours after administration of PQ,were investigated. Concentrations of PQ were from 0.01mM to 1.0.
There was the possibilities that the pH of the perfusion solution has been changing, so it was ascertained to be intact after PQ administration.
Using phase-contrast microscope, photographs of cells were taken.
In high concentrations, cells were damaged as follows ; (1) shrinkage in length, (2) bleb formation, (3) lost of striations, (4) swelling, and so on. These appearances or changes are similar to that when these cells were exposed to hypoxic conditions. But not all these cells were dead, because dead cells could not contact to the bottom of culture dish, nor take up blue pigments.
Mesurement of released enzyme (CPK ; creatine phosphokinase), using supernate of the medium at the end of cell culture, revealed that in 18 hours, CPK level had a tendency to be high in the dish with over 0.1mM of PQ,but in 2 hours, it was low as the control even at 0.3mM.
To explore the participation of apoptosis, electrophoresis of DNA from the cells was done, but there was no typical evidence of DNA fragmentation such as the "ladder".
Free radicals may play a role in these change, so moredetailed research would be needed in the future.