CN108464975A - 辅酶q10在抑制血小板活化和粘附中的应用 - Google Patents
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Abstract
本发明公开了辅酶Q10在抑制血小板活化和粘附中的应用,首次证明了辅酶Q10具有抑制血小板活化以及抑制血小板在纤维蛋白原的粘附铺展功能,而辅酶Q10作为天然存在的营养物质,容易获得,毒副作用小,最大的优势就是可以长期作为膳食补充剂进行心血管疾病的预防/治疗也不会产生毒副作用,通过以辅酶Q10作为膳食营养补充剂发挥抗血小板活化以及粘附功能,可为临床疾病提供有益效果,在心血管疾病治疗中有良好的应用前景。
Description
技术领域
本发明属于生物医学领域,特别涉及辅酶Q10在抑制血小板活化和粘附中的应用。
背景技术
动脉粥样硬化性心血管疾病的发病率和病死率逐年上升,已经成为威胁我国人民健康的主要问题。血小板是血液中最小的细胞,具有保持血管完整性并参与血栓形成防止血管损伤和血液流失的生理防御机制。但是,大量研究报道证实,血小板过度活化以及粘附功能增强会导致机体形成过多的血栓,从而阻碍血流,导致动脉粥样硬化性心血管疾病的发生。因此,血小板在动脉粥样硬化性心血管疾病的发生、发展以及转归中发挥着至关重要的作用,抑制血小板活化以及黏附功能已经成为防治动脉粥样硬化性疾病和血栓性疾病的重要靶标之一。
辅酶Q10的化学名为2,3-二甲氧基-5-甲基-6-聚异戊烯基苯醌,是一种广泛存在于自然界的脂溶性醌类化合物,其结构与维生素K和维生素E相似,在动物脏器(心脏、肝脏、肾脏)、牛肉、豆油、沙丁鱼和花生等食物中含量相对较高。各种属生物体内的辅酶Q异戊二侧链长度有异,人体中主要以辅酶Q10形式存在。辅酶Q10是细胞线粒体电子传递链上重要的中间体,发挥着调节胞浆氧化还原电势及抑制胞内超氧化物形成的作用。大量体内外研究表明,辅酶Q10具有抗衰老,防治心血管疾病,抗肿瘤,调节免疫等重要功能。人体内辅酶Q10的水平会在20岁时候达到巅峰,50岁时减少50%,70岁时减少60%,因此有研究表明人到中年以后,如果能及时补充足够数量的辅酶Q10,便能够延缓主要器官的衰老,并能减缓动脉粥样硬化性心血管疾病的发生发展。
目前研究已经证实,辅酶Q10具有抗氧化,抑制炎症反应,改善血脂水平,抑制动脉斑块的形成等功能,从而发挥抗动脉粥样硬化的发生发展,但是辅酶Q10对血小板活化以及粘附功能的影响未见报道。
发明内容
本发明的首要目的在于提供辅酶Q10在抑制血小板活化的应用。
本发明的再一目的在于提供辅酶Q10在抑制血小板粘附的应用。
本发明所采取的技术方案是:
辅酶Q10在制备抑制激动剂诱导血小板活化的药剂中的应用。
进一步的,所述激动剂为凝血酶、ADP、胶原、花生四烯酸的任意一种。
辅酶Q10在制备抑制凝血酶诱导血小板活化的药剂中的应用。
辅酶Q10在制备抑制血小板粘附或/和铺展功能的药剂中的应用。
辅酶Q10在制备抑制血小板在纤维蛋白原表面粘附或/和铺展的药剂中的应用。
一种抑制血小板活化的试剂盒,该试剂盒中含有辅酶Q10。
一种抑制血小板在纤维蛋白原表面粘附或/和铺展的药剂,该药剂中含有辅酶Q10。
本发明的有益效果是:
本发明首次证明了辅酶Q10具有抑制血小板活化以及粘附的功能,而辅酶Q10作为天然存在的营养物质(大量存在于各种食物中),因此获取的成本低,毒副作用小,最大的优势就是可以长期作为膳食补充剂进行心血管疾病的预防/治疗也不会产生毒副作用,通过以辅酶Q10作为膳食营养补充剂发挥抗血小板活化以及粘附功能,为临床疾病评估提供很好的价值。
当中,可用于增加血小板活化的激动剂有好多种,比如常用的有凝血酶(Thrombin),ADP,胶原,花生四烯酸等,它们各自激活血小板的机制都是不一样的。本发明探讨的是凝血酶诱导的血小板活化与辅酶Q10的关系。
附图说明
图1是辅酶Q10对血小板活化的影响。不同浓度的辅酶Q10(1,10,100μM)或溶剂对照(DMSO)与健康人纯化血小板共孵育50分钟,使用凝血酶激活血小板两分钟,流式细胞术检测血小板表面CD62P的表达,图1左边为不同浓度的辅酶Q10作用的流式图,图1右边为不同浓度的辅酶Q10作用的柱状统计图。*P<0.05,***P<0.001表示与对照组相比,差异具有统计学意义。
图2是辅酶Q10对血小板活化的影响。不同浓度的辅酶Q10(1,10,100μM)或溶剂对照(DMSO)与健康人纯化血小板共孵育50分钟,使用凝血酶激活血小板两分钟,流式细胞术检测血小板表面CD63的表达,图2左边为不同浓度的辅酶Q10作用的流式图,图2右边为不同浓度的辅酶Q10作用的柱状统计图。*P<0.05,***P<0.001表示与对照组相比,差异具有统计学意义。
图3是辅酶Q10对血小板在纤维蛋白原表面粘附铺展的影响。不同浓度的辅酶Q10(1,10,100μM)或溶剂对照(DMSO)与健康人纯化血小板共孵育50分钟,然后在纤维蛋白原表面铺展60分钟,在荧光显微镜下观察并计算粘附铺展后的单个血小板平均表面积。图3左边为不同浓度的辅酶Q10作用的粘附铺展图片,图3右边为不同浓度辅酶Q10作用的平均表面积柱状统计图。*P<0.05,**P<0.01,***P<0.001表示与对照组相比,差异具有统计学意义。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
1.选择符合纳入标准的健康志愿者:
①两周内未服用任何抗凝药物以及富含维生素E或者辅酶Q10的膳食补充剂;
②无每天饮用茶、咖啡和红酒的生活习惯;
需同时符合以上两项。
2.收集富血小板血浆(PRP)和纯化血小板
采用一次性采血针进行肘静脉穿刺,抽取健康志愿者外周静脉血10mL,按9:1的体积比加入枸橼酸钠抗凝剂,轻轻混匀。1000rpm离心10min获得富血小板血浆(PRP),将PRP通过凝胶柱子,分离富血小板血浆(PRP)并收集到纯化的血小板,备用。
其中,用于激活血小板的激动剂可为凝血酶(Thrombin)、ADP、胶原、花生四烯酸的任意一种,本实验采用凝血酶激活血小板。
实施例1辅酶Q10对血小板活化功能的抑制:
方法:(1)用浓度为1、10、100μM的辅酶Q10分别与纯化血小板共同孵育50分钟,为辅酶Q10干预后的血小板;
(2)使用检测血小板表面CD62P的抗体(FITC-conjugated anti-huamn CD62P)和CD63的抗体(FITC-conjugated anti-huamn CD63)(购自美国eBioscience公司)与辅酶Q10干预后的血小板共同孵育20分钟;
(3)使用血小板激动剂凝血酶(Thrombin)激活血小板2分钟,使用4%的多聚甲醛固定终止反应;
(4)用流式细胞仪检测血小板表面CD62P和CD63的表达水平,从而判定血小板活化的
情况。同时设立对照组。
结果:如图1~2所示,随着辅酶Q10浓度的增加,发现CD62P的表达量随之下降。辅酶Q10浓度为10μM时,CD62P的表达量与对照组有显著性差异,Q10浓度为100μM时,CD62P的表达量与对照组有极显著性差异。CD63的表达量随着辅酶Q10浓度的增加而下降。当Q10浓度为100μM时,CD63的表达量与对照组有极显著性差异。表明,辅酶Q10有抑制Thrombin诱导的血小板活化的功能。
实施例2辅酶Q10对血小板在纤维蛋白原表面的粘附铺展的影响
方法:(1)用不同浓度的辅酶Q10(1、10、100μM)分别与纯化的血小板共同孵育50分钟后,为辅酶Q10干预后的血小板;
(2)在玻片上铺展纤维蛋白原Fibrinogen(100μg/mL)(购自德国Cayman公司)过夜,用BAS封闭;
(3)然后分别加入经不同浓度的辅酶Q10干预后的血小板,铺展60min之后,加入鬼笔环肽488(1μg/mL);
(4)在荧光显微镜下观察血小板在Fibrinogen表面粘附铺展情况,并计算单个血小板平均表面积(Surface area)。
结果:如图3所示,不同浓度的辅酶Q10(1,10,100μM)或溶剂对照组(DMSO)与健康人纯化血小板共孵育50分钟,然后在纤维蛋白原表面铺展60分钟,在荧光显微镜下观察并计算粘附铺展后的单个血小板平均表面积。发现随着辅酶Q10浓度的增加,单个血小板表面积递减,说明辅酶Q10对血小板的在纤维蛋白原的粘附抑制效果越明显。
综合而言,我们选择了目前热门的膳食营养补充剂辅酶Q10,研究其对血小板活化以及粘附功能的影响。血小板活化、粘附功能增强是引起动脉粥样硬化的重要过程,因此抑制血小板活化以及粘附功能已经成为防治动脉粥样硬化性疾病和血栓性疾病的重要靶标之一。本发明发现,辅酶Q10可以显著抑制凝血酶(Thrombin)诱导的血小板活化,同时可以抑制血小板在纤维蛋白原表面的粘附铺展。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (7)
1.辅酶Q10在制备抑制激动剂诱导血小板活化的药剂中的应用。
2.根据权利要求1所述的应用,其特征在于,所述激动剂为凝血酶、ADP、胶原、花生四烯酸的任意一种。
3.辅酶Q10在制备抑制凝血酶诱导血小板活化的药剂中的应用。
4.辅酶Q10在制备抑制血小板粘附或/和铺展功能的药剂中的应用。
5.辅酶Q10在制备抑制血小板在纤维蛋白原表面粘附或/和铺展的药剂中的应用。
6.一种抑制血小板活化的药剂,其特征在于,该试剂盒中含有辅酶Q10。
7.一种抑制血小板在纤维蛋白原表面粘附或/和铺展的药剂,其特征在于,该试剂盒中含有辅酶Q10。
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NIAZ AHMED CHOUDHURY等: "Plasma serotonin and platelet aggregation during ischemia-reperfusion in dogs: effect of dipyridamole and coenzyme Q10", 《HAEMOSTASIS》 * |
孙姬等: "辅酶Q10对动脉粥样硬化大鼠血脂及炎症因子表达的影响", 《诊断学理论与实践》 * |
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