| ObjectiveThe purpose of this study is to study the effect of trehalose on reducing the lowtemperature storage damage of platelets stored at 4℃,explore the mechanism of trehalose reducing the low-temperature storage damage of platelets stored at 4℃ from the perspective of ROS expression level,and establish a storage method that can not only prolong the shelf life of platelets but also ensure the safety and effectiveness of platelet transfusion.MethodsStudy 1 Effect of trehalose on low temperature storage damage of platelets stored at 4℃1.Comparison of in vitro functions of platelets stored at 22℃ and 4℃Fresh ABO platelet concentrates were collected aseptically and randomly divided into two groups: 22℃ group and 4℃ group.Each group was aseptically sampled at different time points,and the platelet parameters,metabolic and functional indexes were measured.2.Comparison of in vitro functions of platelets preserved at 4℃ with different concentrations of trehalose and screening of the best concentration of trehaloseAfter aseptic collection,fresh ABO platelet concentrates were randomly divided into six groups: 4℃ group（control,whole plasma preservation）,M-Sol group（control）,1g/L trehalose group,5g/L trehalose group,15g/L trehalose group and 25g/L trehalose group.All the samples were stored at 4℃.Each group was aseptically sampled at different time points,and the platelet parameters,metabolic and functional indexes were measured.Study 2 Preliminary exploration of the mechanism of trehalose to reduce low temperature storage damage of platelets stored at 4℃1.Correlation between ROS expression level of platelets preserved at 22℃ and 4℃ and functional changes in vitroFresh ABO platelet concentrate samples were collected aseptically and were divided into two groups: 22℃ group and 4℃ group.At different preservation time points,the expression level of ROS and the functional indexes such as apoptosis rate,CD62 p positive rate and maximum aggregation were measured in the two groups.2.To verify the correlation between ROS expression level of platelets stored at 4℃ and functional changes in vitro.The ROS expression level of platelets stored at 4℃ was reduced by adding Nacetylcysteine（NAC）.After aseptic collection,fresh platelet concentrates of ABO were divided into four groups: control group（whole plasma preservation）,5m M NAC group,10 m M NAC group and 15 m M NAC group,all stored at 4℃.At different preservation time points,the expression level of ROS and functional indexes such as apoptosis rate,CD62 p positive rate and maximum aggregation were measured in control group and each concentration NAC group.3.Effect of trehalose on ROS expression level of platelets stored at 4℃After aseptic collection,fresh ABO platelet concentrate samples were divided into two groups: control group（whole plasma preservation）and 5g/L trehalose group,which were stored at 4 ℃.The expression levels of ROS in 5g/L trehalose group and control group at different preservation time points were measured,and the amount of ROS produced in the two groups within 3 days and 5 days after preservation was calculated respectively.ResultsStudy 1 Effect of trehalose on low temperature storage damage of platelets stored at 4℃1.Comparison of in vitro function of platelets preserved at 22℃ and 4℃During the storage,except the difference of platelet count between 4℃ group and 22℃ group was not obvious（P>0.05）,the changes of PDW,MPV and GLU concentration within 14 days in 4℃ group were significantly lower than those in 22℃ group（P<0.05）,and the p H value and maximum aggregation degree of platelets in 4℃ group were significantly higher than those in 22℃ group（P<0.05）2.Comparison of in vitro functions of platelets preserved at 4℃ with different concentrations of trehalose and screening of the best concentration of trehaloseThe positive rate of CD62 p of platelets in all trehalose groups was significantly lower than that in the 4℃ control group from the first day of storage or the third day of storage（P<0.05）.At the same time,the positive rate of CD62 p in 5g/L trehalose group was the lowest,and from the seventh day of storage,the positive rate of CD62 p in 5g/L trehalose group was significantly lower than that in other groups（P<0.05）,which indicated that trehalose could inhibit the activation of platelets at 4℃,and in addition,during storage,the positive rate of CD62 p in 5g/L trehalose group was significantly lower than that in 22℃ group（P<0.05）.During the storage period,there was no significant difference in platelet count,PDW,MPV,GLU concentration within 14 days and LAC concentration within 9 days between trehalose group and 4℃ control group（P>0.05）.At d14,the p H value of trehalose groups with different concentrations was higher than that of 4℃ control group,among which the p H value of 5g/L trehalose group was the highest,which was statistically different from that of 4℃ control group（P<0.05）.When stored for d14 days,there was no significant difference between the maximum aggregation degree of trehalose groups with different concentrations and the control group at 4℃（P>0.05）.The above results indicated that trehalose at 4℃ could better maintain the morphology,metabolism and aggregation function of platelets,and the optimal concentration of trehalose was 5g/L.Study 2 Preliminary exploration of the mechanism of trehalose to reduce low temperature storage damage of platelets stored at 4℃1.Correlation between ROS expression level of platelets preserved at 22℃ and 4℃ and functional changes in vitroDuring storage,the expression level of ROS in 4℃ group was significantly higher than that in 22℃ group（P<0.05）.The expression level of ROS in 22℃ group was correlated with the rate of platelet apoptosis and maximum aggregation（P<0.05）,but not with the positive rate of CD62p（P>0.05）.The expression level of ROS in 4℃ group was correlated with the apoptosis rate of platelets,the positive rate of CD62 p and the maximum aggregation degree（P<0.05）,and the absolute values of correlation coefficients were all higher than those in 22℃ group,which indicated that the expression level of ROS in 4℃ group had a higher correlation with the changes of platelet function in vitro,and ROS expression level might be one of the key influencing factors of platelet cryopreservation damage at 4℃.2.To verify the correlation between ROS expression level of platelets stored at 4℃ and functional changes in vitro.During the storage period,5m M NAC group,10 m M NAC group and 15 m M NAC group gradually decreased the ROS expression level of 4℃ platelets in a concentrationdependent manner.At the same time,the absolute values of the correlation coefficients between ROS expression level of NAC groups with 4℃ platelets apoptosis rate,CD62 p positive rate and maximum aggregation degree also decreased in turn.The results showed that the correlation between ROS expression level in 5m M NAC group,10 m M NAC group and 15 m M NAC group and the changes of platelet function in vitro at 4℃ gradually weakened,which further verified that ROS expression level might be one of the key influencing factors of low temperature storage damage of platelets at 4℃.3.Effect of trehalose on ROS expression level of platelets stored at 4℃Compared with the control group,the 5g/L trehalose group was heated in water bath at 37 ℃ for 2 hours before preservation,and the trehalose was loaded into the platelet cells.This treatment may lead to a difference in the basic level of ROS expression between the two groups before preservation,resulting in a higher level of ROS expression in the 5g/L trehalose group before preservation.Therefore,this study mainly compared the difference of ROS production within 3 days and 5 days between the 5g/L trehalose group and the control group.The results showed that the ROS production within 3 days and 5 days after preservation in the 5g/L trehalose group were significantly lower than those in the control group,indicating that trehalose may reduce the low-temperature storage damage of platelets stored at 4 ℃ by down-regulating the expression of ROS.Conclusion1.Trehalose can significantly reduce the CD62 p positive rate of platelets stored at 4℃,inhibit the activation of platelets,and reduce the low-temperature storage damage of platelets stored at 4℃,among which 5g/L trehalose group has the best storage effect.2.The expression level of ROS may be one of the key factors affecting the-low temperature storage damage of platelets stored at 4℃.3.Trehalose may be one of the ways to reduce the low-temperature storage damage of platelets stored at 4℃ by down-regulating ROS expression level. |
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