The Study Of Residual Water On The Lifespan Of Lyophilized Red Blood Cells

ObjectiveBased on the previous research,this study further explored the correlation between red blood cell lifespan and its membrane surface protein adhesion molecules CD47,CD44,and CD147,and established a detection method for red blood cell lifespan.The expression changes of CD47,CD44,and CD147 on the surface of freeze-dried red blood cell membranes in different residual water content groups were compared during storage,and the effect of residual water on the lifespan of freeze-dried red blood cells was explored.MethodsCollect leukocyte-removed specimens from healthy unpaid blood donors?n=10?,and use Percoll density gradient centrifugation to divide the red blood cells into 5layers to obtain red blood cells of different age groups.The first layer is the youngest red blood cell,and the lower red blood cell age increases sequentially.The fifth layer is the oldest red blood cell.Flow cytometry is used to detect the expression of CD47,CD44,and CD147 on the surface of the red blood cell membrane,and the SPSS statistical software is used to analyze the variance of the layered red blood cell protein expression.In this study,five different freeze-drying procedures were set up to prepare freeze-dried red blood cells with different residual water content groups.The residual water content was detected by Karl Fischer titration?KFT?,and the results were 3.14%,4.63%,6.88%,7.02%,10.25%of the five groups of freeze-dried red blood cells,then stored at room temperature,after 1 day,7 days,14 days,30 days,and 60 days,the lyophilized red blood cells were rehydrated and washed,and then the red blood cell membrane surface adhesion molecules CD47,CD44,CD147 were detected.Expression level,analyze the changes in the lifespan of freeze-dried red blood cells with the prolonged storage time in different residual water content groups.ResultsThe red blood cells stratified by Percoll density gradient centrifugation showed that the expression levels of CD47,CD44,and CD147 protein molecules on the lower?old?red blood cell membrane surface were lower than those on the upper?young?red blood cells.After analysis of variance,the surface protein molecules of each red blood cell membrane were found There are statistical differences between the expression levels.According to the expression of surface adhesion molecules on erythrocyte membrane of different age groups,the correlation between CD47,CD44,CD147 and life span is as follows:The correlation between CD47 molecule?Y₁?and life span?X₁?can be explained by the logarithmic model,Y₁=23.266-3.773ln?X₁?,the correlation coefficient R²=0.991,the analysis of variance shows that F=348.893,P<0.001,indicating that this model is statistically significant;the correlation between the CD44 molecule?Y₂?and life?X₂?can be explained by the logarithmic model,Y₂=36.566-4.814ln?X₂?,the correlation coefficient R²=0.995,the analysis of variance shows that F=566.945,P<0.001,indicating that this model is statistically significant;the correlation between CD147 molecule?Y₃?and life span?X₃?It can be explained by the linear model,Y₃=34.293-0.155?X₃?,R²=0.985,and the analysis of variance shows that F=192.465,P=0.001,indicating that this model is statistically significant.The residual water content of the five groups of freeze-dried red blood cells set in this study were 3.14%,4.63%,6.88%,7.02%,and 10.25%,respectively.The expression levels of CD47,CD44,and CD147 molecules on the surface of the freeze-dried red blood cell membrane showed a downward-regulating trend,The down-regulation trend is obvious after weeks;the expression levels of CD47,CD44,and CD147 on the membrane surface of freeze-dried red blood cells with a residual water content of 3.14%are higher than those of other residual water content groups.The less residual water content in freeze-dried red blood cells,the red blood cell preservation effect The better.ConclusionRed blood cell membrane surface adhesion molecules CD47,CD44,CD147 are related to red blood cell lifespan and can be used as markers for red blood cell lifespan detection.With the increase of red blood cell age,the expression of CD47,CD44,CD147 protein molecules on the red blood cell membrane surface shows a significant downward-regulation trend.The residual water content of freeze-dried red blood cells can affect the life of red blood cells.The red blood cells of the lower residual water content group have better freeze-drying preservation effects.The best residual water content in this study is3.14%.