The Protective Effects Of Perfluorocarbon On The Injury Of Pulmonary Alveolar Epithelial Cells Induced By Ischaemia-reperfusion Injury And The Investigation Of Probable Mechanism

ObjectionThis paper establishes the cell model of LIRI, intervenes with different measures respectively, explores apoptosis mechanism in LIRI, seeks a deeper understanding and interpretation upon PFC's biological protective effect and possible mechanism, and provides relatively objective and stronger theoretical foundation for clinical application of PFC and treatment of LIRI; so as to improve effectively protection of donor lung, research upon and improve new preservation solution of donor lung and provides on a larger scale the protection when applying clinical lung transplant operation and cardiopulmonary bypass operation.Method1. Establish the cell model of LIRI. A549 cells, a human pulmonary epithelial cell line, were preserved in 100% O2 at 4℃for varying periods in low-potassium dextran glucose solution, simulating ischemia, followed by the introduction of warm (37℃) DMEM plus 10% fetal bovine serum to simulate reperfusion.2. The third and/or fourth generation A549 cells were used in our research.The cells were divided into four groups:①control group:cells did not receive any intervention,②PFC group:PFC was added to the cell culture medium to a final volume concentration[vol/vol,PFC:culture media]of 30%.After vortexing,the mixed liquor containing PFC and culture media Was transfered into the culture flask.As PFC is not miscible,vim the medium,cells exposed to PFC were constantly shaken(60 times/min).③IRI group:cells were preserved in 100% O2 at 4°C for varying periods in low-potassium dextran glucose solution, followed by the introduction of warm (37℃) DMEM plus 10% fetal bovine serum.④IRI+PFC group:cells were went through the stimulated process of ischemia- reperfusion injury and PFC added to the cell culture medium to a final volume concentration[vol/vol,PFC:culture media]of 30%.The samples were collected at 6,12h after intervention for FCM.Result1.The result of FCM: The early apoptosis rates of A549 cells went through the IRI,6h and 12h after stimulated,are respectively 10.01% and 14.21%. The late apoptosis and necrosis rates are respectively 4.93% and 5.49%;and the viable cell rates were respectively 84.61% and 79.59%. That is to say, with the elapse of time, the apoptosis injury of A549 cells induced by IRI was elevated at dose dependent,whereas the viable cell rate was decreased at dose dependent.In comparison with control group,PFC and PFC+IRI groups,the early apoptosis rate and the late and necrosis rate in IRI groups were significantly increased at 6 and 12h,the viable cell rates were markedly reduced.And the injury degree at 12h was more severe than that at 6h group.There was no statistical difference of apoptosis and viable cell rate between the control and PFC groups at both 6 and 12h and PFC groups each other.Compared with control and PFC group,the early apoptosis rate of PFC+IRI group at 12h was obviously increased and the viable rate was decreased.2. results of the expression of caspase-3 protein level detected by western blotting:In IRI groups,activated caspase-3(17 kDa and 11 kDa subunits)was significantly increased at 0.5,2,6,12h;There was no obvious effect of PFC alone on the proteins.But in the PFC+IRI groups,the proteins of activated caspase-3 were markedly decreased.Conclusion1. IRI can induce apoptosis injury in AECs in vitro,and the injury degree is at time dependant.The expression of caspase-3 protein level were markedly increased after stimulated by IRI. As Caspase-3 is known as the apotosisexecutioner,which suggests that IRI induces apoptosis in AECs maybe via mediating the pathway of Fas→caspases activation in proper order apoptosis.2.PFC alone does not induce apoptosis injury in AECs in vitro and has no obviously effects on the expression of caspase3 protein level of the signal molecules.In the PFC+IRI groups,however,the apoptosis rates of the AECs cell populations were significantly decreased,the viable cells were markedly increased.Meanwhile,the up-regulation of the expression of caspase-3 protein level induced by IRI were significantly reduced.This means that PFC is able to protect AECs from IRI-induced apoptosis injury via blocking the initiation of IRI signal pathway, but cannot completely block apoptosis effect that IRI do to AECs.