| Objective:To establish a cell model of platelet hypoxia-reoxygenation(H/R)injury in order to further study platelet ischemia-reperfusion injury.Methods:(1)Human megakaryoblastic leukemia cells(Meg-01)and human umbilical vein endothelial cells(HUVEC)were cultured.The cells were incubated in a three-gas chamber for 1h,2h,4h,8h,16 h,and 24 h.The cell proliferation activity was detected by CCK-8 method and the generation level of Reactive oxygen species(ROS)was determined by dichlorodihydrofluorescein acetoacetate method,and the optimal hypoxia time was selected.Reoxygenation was performed for 1h,2h,4h,8h,16 h,and 24 h at normal temperature and normoxia.The optimal reoxygenation time was determined by CCK8 method and dichlorodihydrofluorescein acetoacetate method.(2)The optimal time window for Meg-01 cells and HUVEC cells was 8 hours of hypoxia and reoxygenation.The experiment was divided into 6 groups: HUVEC normal control group,HUVEC hypoxia 8h group,HUVEC hypoxia 8h reoxygenat-ion 8h group,Meg-01 normal control group,Meg-01 hypoxia 8h group,Meg-01 hypoxia 8h reoxygenation 8h group.The changes of mitochondrial membrane potential in Meg-01 cells and HUVEC cells were detected by fluorescence microscopy,the apoptosis of Meg-01 cells and HUVEC cells was detected by flow cytometry,and the expression of apoptosis-related proteins and platelet activating protein in Meg-01 cells was detected by Western Blot.(3)Platelets were obtained from healthy volunteer donors and washed by gradient centrifugation.The experiment was divided into three groups: normal control group;In hypoxia group,platelets were exposed to hypoxia for 20 min in a three-gas chamber;In the H/R group,platelets were subjected to hypoxia for 20 min in a three-gas chamber followed by reoxygenation for 5min at room temperature and normoxia.Western Blot was used to detect the expression of platelet apoptosisrelated proteins and platelet activation proteins in each group.Results:(1)HUVEC cells and Meg-01 cells were treated with hypoxia for different durations.CCK-8 results showed that: Compared with the normal control group,the proliferation activity of HUVEC cells and Meg-01 cells was significantly decreased at 8h,16 h and 24 h of hypoxia(p < 0.05),but the cell death rate of Meg-01 cells at16 h and 24 h of hypoxia(48.91%,36.22%)was too high,which was not conducive to the subsequent experiments.The results of ROS showed that the immunofluor-escence intensity of HUVEC cells and Meg-01 cells increased significantly after 8h of hypoxia and reached the highest value compared with the normal control group(p< 0.05).Therefore,8h of hypoxia was chosen as the optimal duration of hypoxia.(2)HUVEC cells and Meg-01 cells were treated with different reoxygenation time after hypoxia for 8h.CCK-8 results showed that: Compared with the 8h hypoxia group,the proliferation activity of HUVEC cells and Meg-01 cells decreased significantly at 8h,16 h and 24 h of reoxygenation(p < 0.05),but the cell death rate of Meg-01 cells at 16 h and 24 h of reoxygenation(63.37%,33.14%)was too high,which was not conducive to the subsequent experiments.The results of ROS showed that the immunofluorescence intensity of HUVEC cells and Meg-01 cells increased significantly at 8h of reoxygenation and reached the highest value compared with the 8h of hypoxia group(p < 0.05).Therefore,8h of reoxygenation was selected as the optimal reoxygenation time.In conclusion,8 hours of hypoxia and 8 hours of reoxygenation were selected as the best time window for the model in this experiment.(3)HUVEC cells and Meg-01 cells were treated with hypoxia for 8h and reoxygenation for 8h.The results of fluorescence microscopy showed that:Compared with the control group,the mitochondrial membrane potential of HUVEC cells and Meg-01 cells in the hypoxia 8h group and the hypoxia 8h reoxygenation 8h group were significantly decreased(p<0.05),and the decrease was more significant in the hypoxia 8h reoxygenation 8h group(p<0.05).Flow cytometry results showed that compared with the control group,the apoptosis rates of HUVEC cells and Meg-01 cells in the 8h hypoxia group and the 8h reoxygenation group were significantly increased(p<0.05),and the increase was more significant in the 8h hypoxia reoxygenation group(p<0.05).(4)WB results of platelets and Meg-01 cells treated with hypoxia/reoxygenation showed that: Compared with the control group,the expression levels of proapoptotic proteins Bax,Cyto-C,Caspase-9 and platelet activation protein CD62 p in human platelets and Meg-01 cells in the hypoxia and reoxygenation groups were significantly increased(p<0.05),while the expression level of anti-apoptotic protein Bcl-2 was significantly decreased(p<0.05).The H/R group had significantly higher expression of pro-apoptotic proteins Bax,Cyto-C,Caspase-9 and platelet activating protein CD62 p and significantly lower expression of anti-apoptotic protein Bcl-2(p<0.05).Conclusion:(1)8 hours of hypoxia and reoxygenation for 8 hours is the best time window for the ischemia-reperfusion injury model of platelet cell line Meg-01,and ischemia-reperfusion injury may induce mitochondrial damage and apoptosis in Meg-01 cells.(2)The 8-hour hypoxia/reoxygenation model of Meg-01 cells may be used as a cell model of platelet I/R injury in vitro. |
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