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目的:探讨甲氧基聚乙二醇(mPEG)遮蔽修饰的红细胞RhD血型抗原的有效性和稳定性以及被修饰红细胞结构和功能的变化。方法:用mPEG-SPA修饰RhD血型阳性人红细胞,观测修饰和未修饰红细胞的抗-D凝集反应性、mPEG结合产物稳定性、电镜下细胞形态、变形指数、渗透脆性、自身溶血率、2,3-二磷酸甘油酸含量、膜Na+,K+-ATP酶、乙酰胆碱酯酶活性和胆固醇含量。结果:0.5 mmol/L、1.0 mmol/L、2.0 mmol/L、2.5mmol/L和5.0 mmol/L的mPEG-SPA修饰红细胞与抗D的凝集反应情况依次为50%凝集、可见凝集、无凝集、无凝集、无凝集,在4℃保存14 d和在37℃保存2 d的修饰红细胞与抗-D均无凝集反应,而未修饰红细胞则全凝集。电镜下修饰和未修饰红细胞均为双凹圆盘状形态,细胞大小均一。修饰和未修饰红细胞的各观测指标分别为:2,3-二磷酸甘油酸含量(71.00±12.88)mmol/L和(65.13±13.98)mmol/L,红细胞沉降率(2.75±2.05)mm/h和(8.00±3.82)mm/h,细胞变形指数(0.98±0.18)和(0.98±0.29),自身溶血率(2.27±0.28)%和(1.32±0.32)%,渗透脆性(0.44±0.06)%和(0.44±0.03)%,膜胆固醇含量(0.10±0.03)g/L和(0.10±0.06)g/L,Na+,K+-ATP酶活性(4.83±1.27)U/mg和(5.41±1.32)U/mg,乙酰胆碱酯酶活性(27.88±5.09)U/mg和(29.68±4.16)U/mg。修饰红细胞的沉降速率低于未修饰红细胞,自身溶血率高于未修饰红细胞(P<0.05),但是修饰红细胞沉降速率和自身溶血率都在参考值范围。结论:2.0 mmol/L的mPEG-SPA能够有效并且稳定地遮蔽红细胞RhD血型抗原。修饰红细胞具有未修饰红细胞的双凹圆盘状形态、细胞膜结构、变形性和运送氧气能力。
Objective: To investigate the effectiveness and stability of mPEG masked modified erythrocyte RhD blood group antigen and the changes of structure and function of modified erythrocytes. Methods: RhD blood group positive human erythrocytes were modified with mPEG-SPA. The anti-D agglutination reactivity, the stability of mPEG binding products, cell morphology, deformation index, osmotic fragility and autologous hemolysis of modified and unmodified erythrocytes were observed. 3-diphosphoglycerate content, membrane Na +, K + -ATPase, acetylcholinesterase activity and cholesterol content. Results: The agglutination reaction of 0.5 mmol / L, 1.0 mmol / L, 2.0 mmol / L, 2.5 mmol / L and 5.0 mmol / L mPEG-SPA modified erythrocytes with anti-D followed by 50% agglutination, , No agglutination, no agglutination, no change in agglutination of modified erythrocytes and anti-D preserved at 4 ℃ for 14 days and at 37 ℃ for 2 days, while unmodified erythrocytes were aggregated. Electron microscopy modified and unmodified erythrocytes are biconcave disk-shaped morphology, uniform cell size. The observed indexes of modified and unmodified erythrocytes were as follows: the content of 2,3-diphosphoglycerate (71.00 ± 12.88) mmol / L and (65.13 ± 13.98) mmol / L, the erythrocyte sedimentation rate (2.75 ± 2.05) mm / (0.99 ± 0.18) and (0.98 ± 0.29)%, (2.27 ± 0.28)% and (1.32 ± 0.32)% respectively, and the osmotic fragility (0.44 ± 0.06)% (0.10 ± 0.03) g / L and (0.10 ± 0.06) g / L and Na + and K + -ATPase activities (4.83 ± 1.27) U / mg and (5.41 ± 1.32) U / mg, acetylcholinesterase activity (27.88 ± 5.09) U / mg and (29.68 ± 4.16) U / mg. The rate of erythrocyte sedimentation was lower than that of unmodified erythrocytes, and the rate of hemolysis was higher than that of unmodified erythrocytes (P <0.05). However, the rate of erythrocyte sedimentation and the rate of autologous hemolysis were within the reference range. Conclusion: 2.0 mmol / L mPEG-SPA can effectively and stably mask red blood cell RhD blood group antigen. Modified Erythrocytes Biconcave disk-shaped morphology with unmodified erythrocytes, cell membrane structure, deformability, and ability to deliver oxygen.