人类红细胞囊泡蛋白组分在保存中的规律研究

The protein composition of human erythrocyte-derived vesicles during storage

目的比较不同保存阶段红细胞及囊泡上相关蛋白组分的表达差异,积累红细胞老化、凋亡的相关信息。方法以10 d为1个周期,对保存红细胞制备红细胞血影和红细胞囊泡,SDS-PAGE电泳和双向电泳分析血影和囊泡的电泳图谱格局差异,免疫印迹检测GPA、GPC、stomatin、CD47、flotillin-1、flotillin-2、annexinⅦ和caveolin-1在血影及红细胞上的表达量。结果红细胞保存d11、d21、d31和d41时的囊泡的总蛋白量分别为180、483、1 055和1 357μg;血影和囊泡在PAGE电泳上的格局存在明显差异;d1、d21血影及d41囊泡的双向电泳图上分别可检出305、144和293个点;免疫印迹检测显示,GPC含量和保存时间之间无明显规律,其他几种蛋白的含量变化具有类似的趋势,即随着保存时间的增加,相应蛋白的含量在红细胞上不断减少,在囊泡上不断增加。结论随着保存时间的延长,红细胞多种蛋白表达量下调,囊泡是这些蛋白离开红细胞的1个重要载体。老化红细胞的清除机理可能是涉及多种表达下调膜蛋白的复杂机制。

Objective To compare the protein composition between erythrocyte and vesicle during different storage periods,and gain information of erythrocyte aging and apoptosis. Methods Isolation erythrocyte ghosts and vesicles every 10 days. SDS-PAGE electrophoresis and 2-dimensional（ 2-D） gel electrophoresis were used to analyze the protein composition differences between erythrocyte and vesicle. The expression of GPA,GPC,stomatin,CD47,flotillin-1,flotillin-2,annexin Ⅶ and caveolin-1 on erythrocyte and vesicle was detected by western blot assay. Results The trend of erythrocyte to secrete vesicle was progressively increased over time. The vesicular protein content for day 11,21,31,41 was 180,483,1 055 and 1 357 μg,respectively. The SDS-PAGE Electrophoresis pattern for erythrocyte and vesicle had significant differences. There were 305,144,293 spots could be detected on 2-D electrophoregram for day 1,21 ghosts and day 41 vesicle. The results of western blot showed that the GPC expression on erythrocyte did not change obviously during storage. Other proteins on erythrocyte decreased over time,while increased on vesicle. Conclusion Many kinds of proteins were decreased on aged erythrocyte. The vesicle was the important carrier that carried proteins leave the erythrocyte. The clearance of apoptosis erythrocyte might be a complicated mechanism which concerned many decreased membrane proteins.