| Red blood cells(RBCs)are important components in blood,an indispensable medical resources.Its long-term preservation is one of the important subjects in the area of transfusion medicine.Compared with current protective methods of RBCs,Lyophization might be a potential,effective and efficient approach for its long term preservation.However,membrane damage has become one of the obstacles hindering the development of RBCs lyophilization.Therefore,how to reduce membrane damage or repair damaged membrane is still a world problem to be resolved in the field of RBCs preservation.Aim:Previous studies showed that the residual water had an important effect on lyophilized RBCs during freeze-drying and the structural water,its components,played a vital role in the integrity of RBC membrane.Therefore,our research takes structural water as starting point and be performed from three aspects 'as follow:in the freeze-drying process,to explore whether trehalose protect the membrane through the water replacement mechanism;form the aspect of membrane phase transition temperature,to explore the optimal temperature for the self-repairing of RBCs during rehydration.Finally,to study the effects of liposomes on the membrane of freeze-dried RBCs in the stages of freeze-drying,rehydration and elution.Method:RBCs were incubated with different concentration of trehalose solution,their intracelluar trehalose were determined and recovery rate and residual water content of lyophilized RBCs were analyzed.Meanwhile,lyophilized red cell ghost and RBCs were prepared based on our previous study on the optimization of lyo-protective agents and freeze-drying procedures and their content of residual water were determined.In the second part,lyophlized RBCs were rehydrated under different temperatures to analyze their recovery rate.Phase transition temperature of lyophilized RBC ghost were determined by differential scanning calorimetry and the volume change of lyophilized RBC was observed by environmental scanning electron microscope.Finally,liposome made of DOPC were added into lyo-protective agents,rehydration solution,and elution solution respectively or simultaneously,which were designed in six groups,analyzing the recovery rate and function of elutropic RBCs.Results:1.When the extracellular trehalose concentration were 100,200,400,600,800,1000mM,the trehalose uptake(mmol/L)were 5.2±0.8,7.6± 1.1,11.9± 1.0,25.1 ±3.9,42.8 ± 2.5 and 55.9 ± 4.3,respectively and the last three groups had statistically difference with each other(P<0.05);the recovery rate(%)were 31.2 ±2.5,32.4 ± 1.5,34.1 ± 2.1,45.0 ± 2.1,22.4 ± 2.5,respectively and the highest recovery at 800mmol/L was statistically different from other groups(P<0.05);the residual water content(%)were 5.7±0.3?5.7±0.4?5.6±0.4?4.7±0.3?3.5±0.2 ? 2.4±0.2,respectively.There was no statistical difference in the first three group,while all of them had statistical differences with the other three(P<0.05).2.The recovery rate of RBCs increased after rehydration as the temperature increased except at 42 ?,and the highest recovery rate,89.8 ±6.7%,was obtained at 37 ?(P<0.01).The recovery rate was significantly different from that at other temperatures.The phase transition temperatures of the three samples were 37.6 ?,35.4 ?,36.5 ? respectively and the average phase transition temperature was 36.5 ±1.2 ?.The slight difference among the three samples was probably due to individual differences.The diameter and thickness of the cells first increased and then decreased.At the time of 45min,the cell thickness and diameter reached 1.53 m and 6.15 m respectively.Compared with the original volume,it increased by 1.54 times and 1.04 times respectively.3.The highest elutropic recovery rate of RBCs was in the experimental group with liposomes in protective agent,rehydration solution and normal saline simultaneously and it reached 50.7 ±1.1%,which was statistically different from that of other groups(P<0.05).Compared with the elutropic recovery rate of 29.0 ± 1.4%in the control group,there were statistically difference between it and other group respectively(P<0.05).But there was no statistically difference between the 4 groups(P>0.05).The ATP level and SOD activity of elutropic freeze-dried RBCs in five experimental groups were not significantly different from that in the control group.Conclusion:1.With the increase of the concentration of trehalose solution,the recovery rate of red blood cells was first stable and then increased;the residual water content of the freeze-dried was first stable and then decreased;Combined with the results that lyophilized RBCs and ghost cells had similar water content and our previous study,the distribution of residual water on freeze-dried erythrocyte membrane was preliminarily proved.And it is preliminarily confirmed that trehalose protects freeze-dried RBC membrane through water replacement mechanism.2.The rehydration temperature has an important effect on the recovery rate of freeze-dried RBCs and 37 ? may be the optimal temperature at which the membrane can repair itself.In addition,osmotic injury is also an important factor in rehydration injury.The changes of cell diameter and thickness first increase and then decrease,and the ability to change the diameter of red blood cells is much larger than that of its thickness.3.Compared with other groups,liposomes play a better protective role when they are added into lyoprotective agents,rehydration solution and eluent,simultaneously.While their effects on the cell function of elutropic RBCs have not been observed. |
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