体外转运富含GPI-蛋白的红细胞囊泡改善PNH溶血的探索

Protection of PNH Red Blood Cells from Lysis by Transfering GPI anchored Protein Vesicles of Normal Human RBCs in vitro

目的 在体外观察是否能将富含ＣＤ５９ 的红细胞囊泡转运到缺失ＣＤ５９的阵发性睡眠性血红蛋白尿症（ＰＮＨ）红细胞膜上，以改善ＰＮＨ 溶血，为ＰＮＨ的治疗提供新的线索。方法 采用免疫亲和层析分离技术分离ＰＮＨ ＣＤ５９－ 红细胞，采用Ｗｅｓｔｅｒｎ 印迹分析、流式细胞仪及蛇毒因子溶血实验等检测和分析红细胞与囊泡的结合。结果 Ｗｅｓｔｅｒｎ 印迹分析显示，红细胞囊泡含有ＣＤ５９；流式细胞仪测定表明，ＰＮＨ ＣＤ５９－ 红细胞与囊泡结合后，ＣＤ５９ 的荧光标记率明显增高（Ｐ＜ ０００１）；蛇毒因子溶血实验显示，加入囊泡后ＰＮＨ ＣＤ５９－ 红细胞溶血度也显著下降（Ｐ＜ ００５）。结论 红细胞囊泡上的ＣＤ５９ 可以在体外转运到ＰＮＨ ＣＤ５９－ 红细胞上，并产生抑制补体溶血的作用，使ＰＮＨ ＣＤ５９－

Objective To observe the transfer of GPI anchored protein CD59 from vesicles of normal RBCs to paroxysmal nocturnal hemoglobinuria(PNH) RBCs can correct the susceptibility to complement attack in vitro. Methods Vesicles released from normal RBCs under ATP depletion or storage, are rich in CD59. PNH CD59 - RBCs were separated by elution from immunoaffinity column bound with monoclonal antibody CD59,and then incubated with normal RBCs vesicles.The content of CD59 in vesicles and RBCs was detected by Western blot and flowcytometric analysis respectively and hemolysis of PNH CD59 - RBCs was detected by cobra venom factor hemolysis test. Results The fluorescence intensity of PNH CD59 - RBCs were increased from ( 1 68± 0 57)% to (58 42±8 25)% and the hemolysis was significantly decreased from (27 13±9 69)% to (19 49±7 61)% after incubation with vesicles prepared from normal donors ( P <0 05),while normal RBCs had no obvious change before and after the same treatment.( P >0 05).Conclusions It seems that CD59 protein molecules could be transfered from normal RBCs vesicles to PNH CD59 - RBCs and retaining the complement regulatory function. CD59 - RBCs could be rendered less liable to hemolyze during complement attack.$$$$