深低温贮存实验犬红细胞适宜甘油浓度的选择  

Selection of suitable glycerol concentration for experiment canine red blood cells at deep-low temperature storage

作　　者：于建 李丽玮[1] 李兴龙 钟宁[1] 严赛 杨燕[1] 严钱勤[1] 丁在咸[1] 李志强[1] YU Jian;LI Liwei;LI Xinglong;ZHONG Ning;YAN Sai;YANG Yan;YAN Qianqin;DING Zaixian;LI Zhiqiang(Shanghai Jiao Tong University Affiliated Sixth People's Hospital,Shanghai 200233,China)

机构地区：[1]上海交通大学附属第六人民医院输血科,上海200233

出　　处：《中国输血杂志》2020年第6期561-564,共4页Chinese Journal of Blood Transfusion

基　　金：上海市“科技创新行动计划”实验动物研究领域项目指南项目(16140900301)。

摘　　要：目的探讨深低温贮存实验犬红细胞适宜的甘油浓度。方法 16只比格犬应用戊巴比妥钠(35 mg/kg)静脉麻醉后,颈静脉放血3袋/只(200 mL/袋),共600 mL,分别标记为冰冻前组、20%复方甘油溶液组和40%复方甘油溶液组。取标记为20%、40%复方甘油溶液组各2袋犬血,以无菌导管连接器将血袋与200 mL转移袋相连接,离心移除血袋中血浆与血小板;将20%、40%复方甘油溶液160 mL缓慢滴注入只含红细胞的血袋中,轻振荡混匀,在室温中水平静置后离心挤出上清液,并各分装成5袋(25 mL/袋)并标记,分别置于-80℃冰箱贮存。冰冻前组、20%复方甘油溶液组和40%复方甘油溶液组在冰冻后d1、d35、d60、d90、d180各取红细胞解冻,分别加无菌蒸馏水裂解红细胞后,应用全自动生化仪测定犬红细胞裂解液中血钾(K+)、钠(Na+)、氯(Cl-)、钙(Ca2+)及乳酸脱氢酶(LDH)、葡萄糖(Glu)、游离血红蛋白(FHb)、乳酸(LA)变化;应用酶标仪测定2,3二磷酸甘油酸(2,3-DPG)、衰变加速因子(CD55)、反应性溶血膜抑制物(CD59)、Na+-K+-ATP酶和6-磷酸葡萄糖酶(G-6-PD)变化。结果 20%和40%甘油-80℃保存红细胞后各贮存时间组血K+、Na+、Cl-、Ca2+、LDH、Glu、CD55分别与贮存前相互比较有明显变化(P<0.05);而FHb、LD、2,3-DPG、Na+-K+-ATP酶、G-6-PD、CD59分别相互比较无明显改变(P>0.05)。另外,应用20%甘油比40%甘油行深低温贮存红细胞,对血K+、Na+、Cl-及LDH、Glu影响程度较小,2组间分别相互比较有明显差别(P<0.05),而对血Ca2+影响程度较大(P<0.05);但对FHb、LD、2,3-DPG、Na+-K+-ATP酶、G-6-PD、CD55和CD59的影响2组无明显差别(P>0.05)。结论实验犬红细胞甘油深低温(-80℃)贮存的适宜浓度为20%;贮存期≤180 d反映红细胞质量的相关参数变化相对较小。Objective To explore the appropriate glycerol concentration for experiment canine RBCs at deep-low temperature storage. Methods Beagle dogs were subjected to jugular vein bloodletting after intravenous anesthesia with sodium pentobarbital(35 mg/kg,),and 3 bags of blood(200 mL/bag,600 mL in total) were collected,and then marked as pre-freezing group, 20% glycerol group and 40% glycerol group. 2 bags of canine blood(marked as 20% and 40% glycerol groups) were connected to transfer bags(200 mL) using sterile catheter connectors, 20%(160 mL) and 40%(160 mL) glycerol were injected slowly to replace the plasma and platelets removed through centrifugation, along with gently vibration and blending of glycerol and remaing RBCs in bags. After standing horizontally at room temperature and subsequent removal of the supernatants through centrifugation, each of the 2 bags were divided into 5 aliquots(25 mL/aliquot) and marked, then stored in a deep-low temperature blood storage refrigerator at-80℃. Aliquots in pre-freezing group, 20% glycerol group and 40% glycerol group were thawed on the 1 st, 35 th, 60 th, 90 th, and 180 th days after freezing. After the RBCs were lysed with sterile distilled water, changes in serum potassium(K+), sodium(Na+), chloride(Cl-), calcium(Ca2+), lactate dehydrogenase(LDH), glucose(Glu), free hemoglobin(FHb), and lactic acid(LD) were determined using Hitachi Automatic Biochemical Analyzer(model 7600—120);changes in 2,3 diphosphoglycerate(2,3-DPG), decay accelerating factor(CD55), reactive hemolytic membrane inhibitor(CD59), Na+-K+-ATPase and 6-phosphoglucose(G-6-PD) were measured using a microplate reader(Infinite F50).Results Significant differences in blood K+, Na+, Cl-, Ca2+, LDH, GLU, and CD55(P<0.05), but not in FHb, LD, 2,3-DPG, Na+-K+-ATPase, G-6-PD and CD59(P>0.05) were notable by storage with 20% and 40% glycerol at-80℃(vs. before storage). In addition, 20% glycerol, rather than 40% glycerol, applied for RBCs storage in deep-low temperature, tended to demonstrate less impact on bloo

关 键 词：犬红细胞 甘油浓度 深低温贮存 血液参数 

分 类 号：R457.12[医药卫生—治疗学] Q952.5[医药卫生—临床医学]