光学法检测血小板体外保存期间跨膜电位的变化

Optical method to detect variation in the transmembrane potential of platelets during storage in vitro

目的建立使用流式细胞仪检测血小板跨膜电位的方法,观察血小板在体外保存期间跨膜电位变化。方法使用电压敏感染料Di BAC4(3)对20(人)份血小板(20 m L/份)染色后,用流式细胞仪测定血小板在静息状态下和完全去极化时的荧光强度,根据能斯特方程计算血小板跨膜电位,并利用此方法观察血小板在体外保存期间跨膜电位的变化情况。结果 20人份新鲜血小板跨膜电位平均值为(-56±4)m V,CV=5.5%,与膜电位钳法得到的结果(-50-60)m V相似,保存1、3、5 d的血小板跨膜电位分别为(-56±4)、(-54±8)和(-46±6)m V,其中血小板跨膜电位在血小板保存1 d时明显高于5 d(P<0.05)。结论所建立起的方法用于测定血小板跨膜电位准确、可靠。

Objective To establish a method for detecting transmembrane potential of platelet with flow cytometry and to investigate changes in platelet transmembrane potential during storage in＇ vitro. Methods Platelets were stained by the voltage sensitive dye DiBAC4 （3） , and then the fluorescence intensity of platelets were determined with flow cytometry in a resting and complete depolarization state. Platelet transmembrane potential was calculated with the Nernst equation. Results There was a notable change in the transmembrane potential of platelet during storage in vitro. The mean transmembrane po- tential of platelets from 20 healthy volunteers was （ - 56 ＋ 4） mV, the CV value was 5.5% , which was similar to the results obtained by patch clamp （ - 50 - 60） inV. The transmembrane potential of platelet rose during storage in vitro. During the storage period, platelet transmembrane potential of 1 d, 3 d, 5 d were -58 ±4mV, -54 ± 8mV, and -46 ± 3mV, respectively. There was a significant difference compared with ld and 5d. Condtlsion The method for the determination of plate- let transmembrane potential with flow cytometry is relatively accurate and reliable.