Apoptosis Events During Thermal Stress In Kidney Cell In Vitro Of Grass Carp(Ctenopharyngodon Idellus)

The thesis studied apoptosis events during thermal stress in kidney cell in vitro of grass carp(Ctenopharyngodon idellus). Four different experimental temperatures were set for cultivating cells:28℃,32℃,36℃, and40℃, of which28℃was the most appropriate temperature for the growth of CIK cell, therefore this group was taken as control group. Under the above temperatures, cells was acute heat shocked for2h and constantly heat shocked for24h, each of which was repeated three times. The cell activity, the LDH leakage rates of CIK, the cell cycles and cell apoptosis rate, DNA fragmentation, the content of polyubiquitinylated proteins and the expression of caspase-3and bcl-2were detected, and the changes in cell morphology were observed. The main contents and results are as below:1. The acute heat shock on CIKThe acute heat shock under different temperatures showed that the cell activity decreased significantly (p<0.05) with the increase of incubation temperature. The LDH leakage rate increased as temperature increased,36℃and40℃groups increased more significantly (p<0.05) than the control group; According to the detection on the expression of relevant multi-ubiquitin protein and relevant apoptosis genes after acute heat shock, it is found that the content of multi-ubiquitin protein increased as heat shock temperature increased. The36℃and40℃groups both increased more significantly (p<0.05) than the control group. And caspase-3expression increase as the incubation temperature increased. And The36℃and40℃groups both increased more significantly (p<0.05) than the control group, the expression of bcl-2decreases as heat shock temperature increased. However, the cell cycles and the cell apoptosis rate had no significant changes. The vacuoles occurred in the cytoplasm and karyopyknosis happened.The results illustrates that acute thermal stress can induce the damage of cell membrane and protein, increase the permeability of cell membranes and cut down the cell activity. The increase of multi-ubiquitin protein expression illustrates that heat stress lead to damage of the cells. The change of the expression of caspase-3and bcl-2proves that the heat stress opens the apoptosis pathway. But the cell apoptosis have no selectivity at different periods.2. Constant thermal stress on the CIKAfter the CIK cells were heated at different temperature for2h,6h,12h,24h. The cell viability decreased with the increase of the temperature, after treated6h the40℃groups decreased significantly(p<0.05), after treated12h or24h each experimental group decreased significantly(p<0.05). However, the LDH leakage rates increased with the temperature at the same exposed time, and increased with the exposed time at the same incubation temperature, the40℃groups at exposed6h and24h are increased significantly(p<0.05). After exposed12h and24h the cell apoptosis rate increased with the incubation temperature. The heat stress can lead to the DNA fragmentation as well. The cytoplasmic vacuoles were more serious after heat stress observed by Transmission electron microscopy (TEM).The results imply that thermal stress have impact on the cell activity, cell membrane and cell organelles, and with the increase of the temperature the cell apoptosis rate and the DNA fragmentation-have increased. And heat shock for longer time can lead to the increase of apoptosis rate, after thermal heat shock DNA Ladder can be detected, that is, the number of small plots of DNA increases, which illustrates thermal heat shock can lead to the damage of DNA, which is one feature of the cell apoptosis too.