Studies On The Effect Of Cryopreservation And Resuscitation On Rat Primary Hepatocytes Apoptosis

Objective: To explore the effect of cryopreservation and resuscitation on apoptosis of hepatocytes and the protection of preincubation on murine primary hepatocytes. to study a new method for increasing the viability of resuscitation cell after cryopreservation,to provide theory evidence for improveing the cryopreservation pool of hepatocytes and promote and spread therapy application of hepatocyte transplantation.Methods: To isolate hepatocytes from SD male rats weighing 150 ~ 200g by modifying Seglen collagenase perfusion in situ. The viability (MTS) of freshly isolated hepatocytes and hepatocytes directly recovered from cryopreserved and preincultured hepatocytes were detected. Oxidative stress index such as SOD and MDA were detected .apoptosis of hepatocytes were detected by flow cytometry. The expression of apoptosis-related genes caspase-3 was detected by western-plot.Results: (1)The apoptosis of each group showed: comparing with control group, the apoptosis of direct resuscitation group from cryopreservation group was significantly increased, there were statistical significance (P<0.05). Comparing with direct resuscitation group, the apoptosis of preincubation group was significantly decreased, there were statistical significance (P<0.05). Comparing with control group, the apoptosis of preincubation group was significantly increased, there were statistical significance (P<0.05).(2)The MTS of each group showed: comparing with control group, the MTS of direct resuscitation group was significantly decreased, there were statistical significance (P<0.05).there were no statistical significance between preincubation group and direct resuscitation group (P>0.05). Comparing with direct resuscitation group, the MTS of preincubation group was significantly increased, there were statistical significance (P<0.05).(3)â‘ The comparison of SOD and MDA of each group: there were differences among control group,preincubation group and direct resuscitation group. Comparing with control group, the SOD of direct resuscitation group was significantly reduced, there were statistical significance (P<0.05). Comparing with direct resuscitation group, the SOD of preincubation group was significantly increased, there were statistical significance (P<0.05). Comparing with control group, the SOD of preincubation group was significantly decreased, there were statistical significance (P<0.05).â‘¡On the effect of MDA, there were differences among control group, preincubation group, direct resuscitation group (P<0.05). Comparing with control group, the MDA of direct resuscitation group was significantly improved, there were statistical significance (P<0.05). Comparing with direct resuscitation group, the MDA of preincubation group was significantly decreased, there were statistical significance (P<0.05). Comparing with control group, the MDA of preincubation group was significantly decreased, there were statistical significance (P<0.05). Oxidative stress was one damage factor of hepatocytes during cryopreservation and resuscitation. Preincubation could ruduce oxidative stress responses.(4)The expression of Caspase-3 among three groups: the difference of hepatocytes Caspase-3 protein in gray scale scanning among three groups was significantly (P<0.05). Compared with the control group, the expression of Caspase-3 in direct cryopreservation group increased, and the difference was significantly (P<0.05). Compared with the control group, the expression of Caspase-3 in Preincubation group also increased, and the difference was significantly (P<0.05). Compared with the direct cryopreservation group, the expression of Caspase-3 in Preincubation group decreased, and the difference was significantly (P<0.05).Conclusion: 1. Resuscitation from cryopreservation could increase the level of oxidative stress responses in rat hepatocytes.2. Apoptosis of rat liver cell resuscitation from Cryopreservation was increased.3. The expression of protein caspase-3, an effect enzyme of Apoptosis in rat hepatocytes resuscitation from Cryopreservation was high.4. Preincubation could reduce apoptosis of liver cell resuscitation from Cryopreservation and enhance the viability of hepatocytes. 5. Preincubation could decrease the expression of protein caspase-3 which is an effect enzyme of Apoptosis.