Preliminary Study On Cold-stress Induced Cell Death Of Cells From Colossoma Brachypomum

A cell line of Colossoma brachypomu/n(CB1), which has nontolerant of cold-stress,was used to study the death mechanism under low temperatures at the level of cytobiology.Method: CBT cultured in 37 were used as control. For the experimental cells,was CBT exposured to 10癈 or 3'C for 3h, 6h, 12h,24,48h or 72h respectively. The adult fishes of Colossoma brachypomum were also raised in different temperatures for at least 6hrs, then were dissected for a detailed examination or analysis.The approaches used in the study included: Observing the microstructure and ultrastructure of the cell line of Colossoma brachypomum (CBT) and the cell line of Carp (CP) stressed low temperatures under fluoroscopy and TEM; Analysis of DNA damage in the cultured cells under temperatures stress by DNA gel electrophoresis. DNA content, intracellular Ca2+ and mitochondrial membrane potential in cultured CBT or CP were analyzed. 02~content was useing NBT method , activity of SOD was determined based on auto-oxidation of pyrogallol.Results showed: (1) CBT cell death in low tempratures is accompanied by characteristic changes, such as, reduced cell size, distorted nucleus,chromatin condensation and margination and cell (cytoplasmic) vacuolization; cell mortality and Ca2* concentration increase along with time passed in low temperature. Mitochondrial membrane potential and 02 increased at first, and then decreased. Activities of SOD decreased at first, followed by significant increasing and finally depressed. No DNA ladder which denote apoptosis showed. (2) The mortality of CP cell in 10 were increasing directly related with the time in the low temperature. After 120d some CP cells died in the way of apoptosis. Most nucleus of cells were condensed, and chromosome was marginated beside the nucleus inner membrane, cell sizes were reduced, DNA ladder showed in DNA gel electrophoresis. (3) Activity of SOD in brain, liver, kidney, muscle and branchia of cultured fish Colossoma brachypomum. The total level of activities of SOD in the fish rised with temperature rising. The data indicated that SOD activity in brain become stronger at early time when the temperature fall down, and then decreased. (4) the color of the fishes in low temperature become darker, then changed bright when we transferred the fishes in high temperature. After three days, the color of these fishes recovered. The lipoid in the fishes brain had been viscid when the temperature at 17. And become more solid with the temprature went dowra unceasingly.Conclusions: (1) The results indicate that cell death of the cell line from Colossoma brachypomum under cold-stress is likely to be a non-apoptotic program cell death. (2) The CP cells conserved in 10癈 fail to reculture because of apopotosis of CP. (3) There are different mechanisms of death in low temperatures. (4) The activity of SOD in the fishes went highher when the culture temperature rose. It indicated metabolism of the fishes was increased with temperature rising. Low temperatures may resulted in fish hypoxia in brain, showed enhanced SOD activity.