CAJ | 학술논문

目的探讨血站标准存储条件下花生四烯乙醇胺(ANA)对血小板凋亡的抑制作用及相关机制.方法取志愿者血小板,分别单独加入0.5 μmol/L ANA或联合加入0.5 μmol/L ANA和l μmol/L大麻素1型受体抑制剂利莫那班(Rimonabant,SR141716),在(22±2)℃水平振荡仪中振荡保存7d,同时设不加药物的血小板作为对照组.流式细胞术检测血小板膜磷脂酰丝氨酸(PS)表达水平;Western blot法检测凋亡相关蛋白表达;用细胞色素C(Cyt-C)检测试剂盒检测其从线粒体释放到胞质水平;免疫共沉淀法检测BCL-XL和Bak蛋白相互作用.结果 ANA处理组PS表达阳性率(早期细胞凋亡率)显著低于对照组[(8.29±1.44)％对(14.24±2.47)％];ANA处理组Cyt-C从线粒体释放到胞质的百分比显著低于对照组[(3.29±1.44)％对(15.24±3.40)％];ANA处理组血小板凋亡相关蛋白磷酸化(p)-Akt和p-Bad表达水平均高于对照组[(71.33±10.26)％对(35.00±6.00)％; (39.00±9.64)％对(10.33±1.53)％,P值均＜ 0.05];ANA处理组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);ANA处理组活化的caspase-9和caspase-3水平均显著低于对照组[(9.63±1.47)％对(23.24±2.47)％;(6.30±1.40)％对(13.20±2.50)％].ANA+ SR141716组各项检测指标与对照组结果相似,两组相比差异无统计学意义.结论ANA通过影响凋亡蛋白的表达水平,抑制Cyt-C释放到胞质使血小板免于凋亡而起到保护作用.
목적 탐토혈참표준존저조건하화생사희을순알(ANA)대혈소판조망적억제작용급상관궤제.방법 취지원자혈소판,분별단독가입0.5 μmol/L ANA혹연합가입0.5 μmol/L ANA화l μmol/L대마소1형수체억제제리막나반(Rimonabant,SR141716),재(22±2)℃수평진탕의중진탕보존7d,동시설불가약물적혈소판작위대조조.류식세포술검측혈소판막린지선사안산(PS)표체수평;Western blot법검측조망상관단백표체;용세포색소C(Cyt-C)검측시제합검측기종선립체석방도포질수평;면역공침정법검측BCL-XL화Bak단백상호작용.결과 ANA처리조PS표체양성솔(조기세포조망솔)현저저우대조조[(8.29±1.44)％대(14.24±2.47)％];ANA처리조Cyt-C종선립체석방도포질적백분비현저저우대조조[(3.29±1.44)％대(15.24±3.40)％];ANA처리조혈소판조망상관단백린산화(p)-Akt화p-Bad표체수평균고우대조조[(71.33±10.26)％대(35.00±6.00)％; (39.00±9.64)％대(10.33±1.53)％,P치균＜ 0.05];ANA처리조BCL-XL화Bak단백적결합량현저고우대조조(약위대조조적2.6배);ANA처리조활화적caspase-9화caspase-3수평균현저저우대조조[(9.63±1.47)％대(23.24±2.47)％;(6.30±1.40)％대(13.20±2.50)％].ANA+ SR141716조각항검측지표여대조조결과상사,량조상비차이무통계학의의.결론ANA통과영향조망단백적표체수평,억제Cyt-C석방도포질사혈소판면우조망이기도보호작용.
Objective To investigate the mechanism of N-Arachidonoylethanolamine (ANA) on inhibiting platelets (PLT) apoptosis under standard blood bank storage conditions.Methods Samples taken from collected apheresis PLT by the Amicus instrument were split into three parts.An aliquot of 0.5 μmol/L ANA were added to one part of storage PLT as the ANA group; an aliquot of 0.5 μmol/L ANA and 1 μmol/L SR141716 was added to the another part as the ANA + SR141716 group; and the third part without ANA and SR141716 as the control group.These samples were stored on a flat-bed shaker at (22± 2) ℃ for 7 days.The expression of phosphatidyl serine (PS) positive,phospho (p)-Akt,Akt,p-Bad,Bad,caspase-3,caspase-9,cytochrome C (Cyt-C) and BCL-XL interaction with Bak were detected.Results The rate of PLT PS positive in ANA group decreased significantly than that in control group [(8.29 ±1.44)％ vs (14.24 ± 2.47)％,P＜0.05].The release of Cyt-C from mitochondria to cytosol in ANA group decreased significantly compared with control group [(3.29±1.44) ％ vs (15.24±3.40) ％,P＜0.05].Also the expressions of p-Akt and p-Bad in ANA group increased significantly than those in control group [(71.33±10.26)％ vs (35.00±6.00)％,P＜0.05; (39.00±9.64)％ vs (10.33±1.53)％,P＜0.05,respectively].Higher amounts of Bak protein were co-precipitated with BCL-XL in ANA group than that in control group (about 2.6 fold,P＜0.05).The expressions of cleaved caspase-9 and caspase-3 in ANA group decreased significantly than those in control group [(9.63± 1.47)％ vs (23.24±2.47)％,P＜0.05; (6.30± 1.40)％ vs (13.20±2.50) ％,P＜0.05,respectively].There were no significantly changes between ANA+ SR141716 and control groups (P＞0.05).Conclusion ANA protected PLTs from apoptosis as a result of inhibiting the release of Cvt-C from mitochondria to cvtosol by modifying the expressions of apoptosis-relative proteins.