CAJ | 학술논문

目的：在血站标准存储血小板条件下，探讨花生四烯乙醇胺（N-Arachidonoylethanolamine， ANA）对血小板细胞相关凋亡的影响。方法利用 Amicus 血细胞分离机从无偿献血志愿者采集血小板，向其中一组加入终浓度为0.5μmol/L的ANA为ANA组，未加入ANA的另一组作为对照组，置于（22±2）℃水平振荡的振荡仪保存7 d。采用流式细胞仪检测血小板磷脂酰丝氨酸（PS）膜外表达；ELISA检测可溶性P-选择素的释放，Western blot检测血小板caspase-3和caspase-9的表达以及免疫共沉淀分析 BCL-XL 和 Bak 蛋白相互作用。结果 ANA 组 PS 表达阳性率显著低于对照组[（8.29±1.44）% vs.（14.24±2.47）%，P＜0.05]；ANA 组可溶性 P-选择素含量显著低于对照组[（75.08±6.35）ng/ml vs.（90.37±8.91）ng/ml，P＜0.05]；ANA 组BCL-XL和Bak蛋白的结合量显著高于对照组，约为对照组的2.6倍（P＜0.05）；ANA 组caspase-9活性形式的量占总量（包括caspase-9酶原形式和活性形式）的百分比显著低于对照组[（9.63±1.47）%vs.（23.24±2.47）%]；ANA 组caspase-3活性形式的量占总量（包括caspase-3酶原形式和活性形式）的百分比显著低于对照组[（6.3±1.4）%vs.（13.2±2.5）%]，两组相比差异均具有统计学意义。结论 ANA促进BCL-XL和Bak蛋白的结合，抑制了caspase-3和caspase-9的活化，在一定程度上抑制血小板凋亡。
목적：재혈참표준존저혈소판조건하，탐토화생사희을순알（N-Arachidonoylethanolamine， ANA）대혈소판세포상관조망적영향。방법이용 Amicus 혈세포분리궤종무상헌혈지원자채집혈소판，향기중일조가입종농도위0.5μmol/L적ANA위ANA조，미가입ANA적령일조작위대조조，치우（22±2）℃수평진탕적진탕의보존7 d。채용류식세포의검측혈소판린지선사안산（PS）막외표체；ELISA검측가용성P-선택소적석방，Western blot검측혈소판caspase-3화caspase-9적표체이급면역공침정분석 BCL-XL 화 Bak 단백상호작용。결과 ANA 조 PS 표체양성솔현저저우대조조[（8.29±1.44）% vs.（14.24±2.47）%，P＜0.05]；ANA 조가용성 P-선택소함량현저저우대조조[（75.08±6.35）ng/ml vs.（90.37±8.91）ng/ml，P＜0.05]；ANA 조BCL-XL화Bak단백적결합량현저고우대조조，약위대조조적2.6배（P＜0.05）；ANA 조caspase-9활성형식적량점총량（포괄caspase-9매원형식화활성형식）적백분비현저저우대조조[（9.63±1.47）%vs.（23.24±2.47）%]；ANA 조caspase-3활성형식적량점총량（포괄caspase-3매원형식화활성형식）적백분비현저저우대조조[（6.3±1.4）%vs.（13.2±2.5）%]，량조상비차이균구유통계학의의。결론 ANA촉진BCL-XL화Bak단백적결합，억제료caspase-3화caspase-9적활화，재일정정도상억제혈소판조망。
Objective To investigate the effect of N-Arachidonoylethanolamine(ANA) on apoptotic proteins of platelets(PLTs) stored under standard blood bank storage conditions. Methods Samples taken from collected apheresis PLTs by the Amicus instrument were split into two parts. An aliquot of 0.5 μmol/L ANA was added to one part of storage PLTs as the ANA group, and another part without ANA as the control group. These samples were stored on a flat-bed shaker at (22±2)℃for up to 7 days. The expression of phosphatidyl serine (PS) positive was determined by flow cytometer. Soluble P-selectin were measured by ELISA, the expressions of caspase-3 and caspase-9 were detected by Western blotting. BCL-XL interaction with Bak was detected by immunoprecipitation experiments. Results The rate of PLT PS positive decreased significantly in ANA group than that in control group [(8.29±1.44)%vs. (14.24±2.47)%, P<0.05]. There was a significant difference in soluble P-selectin contents between the experimental group [(75.08±6.35)ng/ml] and the control group[(90.37±8.91)ng/ml, P<0.05]. Higher amounts of Bak protein could be co-precipitated with BCL-XL in ANA group than that in control group (about 2.6 fold, P<0.05). The expression of cleaved caspase-9 decreased significantly in ANA group than that in control group [(9.63±1.47)%vs. (23.24±2.47)%, P<0.05]. The expression of cleaved caspase-3 decreased significantly in ANA group than that in control group[(6.3±1.4)% vs. (13.2±2.5)%, P<0.05]. Conclusion ANA promoted the interaction of BCL-XL with Bak, and inhibited activations of caspase-9 and caspase-3, which suggested that ANA could inhibit PLT apoptosis to some extend.