贮存过期红细胞的生化功能及形态的恢复研究

目的研究如何使贮存过期红细胞的生化功能和形态恢复到正常水平。方法恢复前红细胞组为ACD保养液在(4±2)℃贮存3周过期3d洗涤2次的红细胞,将此红细胞分成等量的2组,1组加入由腺嘌呤、肌苷、丙酮酸钠、磷酸氢二钠混合组成的恢复液作为恢复组,另1组加入25ml0.9%NaCl作为盐水对照组,2组均37℃孵育1h,从上清液中离心分离获得囊泡;将采集献血者新鲜全血的同时分离的红细胞作为正常水平组。分别测定4组红细胞ATP、2,3DPG、MCV、形态学积分、渗透脆性程度及Hb浓度。结果ATP、2,3DPG及形态学积分:恢复组比恢复前组和盐水对照组均显著增加(P<0.001),与正常水平组无显著差异(P>0.05)。MCV:恢复组显著低于恢复前组及盐水对照组(P<0.001)。恢复组与盐水对照组红细胞在0.50%和0.55%NaCl处的低渗性溶血度比恢复前组显著减少(P<0.05),与正常水平组无显著差异(P>0.05)。恢复组囊泡内脱落的Hb比盐水对照组显著增多(P<0.05)。结论腺嘌呤、肌苷、丙酮酸钠、磷酸氢二钠组成的恢复液可使ACD(4±2)℃贮存过期3d的红细胞的生化功能和形态都恢复到正常水平;在恢复再生过程中,出胞的微小囊泡引起细胞膜部分脱落,导致红细胞的MCV变小。

Restoration of biochemical function and morphology of stored outdated erythrocytes

Objective To investigate whether the biochemical function and morphology of stored outdated erythrocytes were able to be restored to normal levels.Methods The experiments were carried out with 4 groups of erythrocytes. Group 1 consists of post-rejuvenated erythrocytes, which were harvested by incubating twice washed stored erythrocytes on the 3 rd day after outdate at 37℃ for 1h, with a rejuvenating solution containing adenine 5mmol/L, inosine 100mmol/L, pyruvate 100mmol/L, phosphate 103mmol/L. Group 2 was pre-rejuvenate erythrocytes, obtained by washing stored erythrocytes on the 3rd day after outdate. Group 3 was saline control erythrocytes, which were incubated with 0.9% saline. Group 4 was freshly collected erythrocytes used as normal controls. ATP, 2, 3-DPG, morphology scores, MCV, osmotic fragility, and hemoglobin content of RBC in four groups were measured.Results The post-rejuvenate RBC ATP [(3.41±0.52)μmol/gHb] was significantly higher(P<0.001) than that of the pre-rejuvenate[(1.80 ± 0.34)μmol/gHb] and the control[(1.69±0.12)μmol/gHb],with no significant difference (P>0.05) compared with fresh RBC [(3.45±0.51)μmol/gHb].The post-rejuvenate RBC 2,3-DPG [(14.08±3.10)μmol/gHb] was significantly higher(P<0.001) than that of the pre-rejuvenate [(0.35±0.28)μmol/gHb] and the control[(0.30±0.21)μmol/gHb] , with no significant difference (P>0.05) compared with fresh RBC [(14.87±3.10)μmol/gHb].The post-rejuvenation morphology score(130.00±10.80) was significantly greater (P<0.05) than that of the pre-rejuvenation(258.00±20.80) and the control (265.90±19.40), with no significant difference (P>0.05) compared with fresh RBC (120.00±5.00). The post-rejuvenate mean corpuscular volume [(89.30± 7.20)fl] was significantly lower (P<0.001) than that of the pre-rejuvenate[96.80±7.30)fl] and the control [(109±8.70 )fl].Rejuvenated and saline control RBC were both significantly less susceptible to hypotonic lyses than pre-rejuvenated RBC at 0.50% and at 0.55% NaCl(P<0.05), with no significant difference compared with fresh RBC (P>0.05). The hemoglobin shed in vesicles during rejuvenation was significantly greater than that in the saline control [(0.51±0.33)vs(0.20±0.18)mg/dl RBCs;P<0.05 )].Conclusion The rejuvenating solution containing 5mmol/L adenine, 100mmol/L inosine, 100 mmol/L pyruvate, and 103 mmol/L phosphate could restore biochemical function and morphology of outdated stored erythrocytes to normal levels. The exocytic microvesiculation induces partially membrane loss, which causes the decrease of RBC MCV during rejuvenation.