贮存过期红细胞的生化功能及形态的恢复研究

Restoration of biochemical function and morphology of stored outdated erythrocytes

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。

Objective To investigate whether the biochemical function and morphology of stored outdated erythrocytes were able to be restored to normal levels.Methods The experiments were carried out with 4 groups of erythrocytes.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.