维生素C后处理对缺氧复氧条件下花生四烯酸诱导的血小板活化的影响及其机制研究
发布时间：2018-08-14 10:32
【摘要】：目的:实验室及临床试验已经证实血小板活化在缺血再灌注过程中发挥重要作用,但是其机制仍不甚明确。有研究表明,缺血再灌注中发生的血小板活化与活性氧大量产生密切相关。维生素C(VC)是一种弱酸性维生素,具有强大的抗氧化作用,以往研究发现,维生素C能够清除活性氧、减少血小板活化、减轻再灌注损伤,但其对离体缺氧复氧血小板的作用尚不清楚。本实验旨在观察维生素C后处理对离体缺氧复氧血小板的作用并阐明其具体作用机制。方法:研究对象为新鲜机采健康人血小板原浆共67人份,血小板浓度为(960~1280)×109/L,由河北省血液中心提供。其纳入标准及排除标准均严格按照国家献血法的标准。用生理盐水将血小板原浆稀释为200×109/L以备用,于血小板采集后4小时内完成实验。首先进行血小板缺氧-复氧实验以选取最佳实验处理时间,对血小板进行不同缺氧或复氧时间处理并用全血电阻抗法检测血小板聚集,诱导剂为花生四烯酸(AA0.5mmol/L),建立离体缺氧复氧血小板模型。选取三种不同抗氧化剂(维生素C、褪黑素、普罗布考)进行缺氧后处理,并分别制备成缺氧复氧组(A/R组)、药物后处理组(C1-3组),考虑到维生素C的弱酸性同时设立盐酸后处理组(HCl组,p H值与VC组相同,均为3.24)。用全血电阻抗法检测血小板聚集率及斜率以观察不同抗氧化剂对血小板聚集率的影响。测定维生素C后处理组中血小板凋亡相关指标,用倒置荧光显微镜检测活性氧(ROS),流式细胞术检测线粒体膜电位(△ψm),Western Blot检测凋亡相关蛋白Bax、Bcl-2、细胞色素C(cyto-C)、Caspase-9的表达。数据采用SPSS22.0进行统计分析。计量资料服从正态分布时用均数±标准差((?)±s)表示,不服从正态分布时采用中位数(四分位数间距)表示,即M(QR)。正态分布时采用单因素方差分析,不服从正态分布时组间比较采用多个相关样本的秩和检验。P0.05认为差异有统计学意义。结果:1不同缺氧或复氧时间对健康人血小板聚集率的影响1.1不同缺氧时间对于血小板聚集率的影响与未缺氧组相比,各缺氧组血小板聚集率及聚集斜率均有所升高,且缺氧20min组活化率最大。两两比较,发现缺氧20min组与5min组差异有统计学意义(P0.05),而5min、10min、30min组间差异无统计学意义(P0.05);1.2不同复氧时间对于血小板聚集率的影响与未复氧组血小板相比,各复氧组血小板聚集率及聚集斜率均有所升高,且5min组活化率最大,但各组间差异无统计学意义(P0.05);2三种抗氧化剂后处理对A/R时血小板聚集率的影响2.1维生素C对A/R时血小板聚集率的影响与缺氧复氧组相比,1000μM维生素C处理组血小板聚集率下降,差异有统计学意义(P0.05),而10μM组、100μM组、HCl组与A/R组相比差异无统计学意义(P0.05)。1000μM维生素C较10μM、100μM来说,血小板聚集抑制率最大且差异有统计学意义(P0.05);2.2褪黑素对A/R时血小板聚集率的影响与缺氧复氧组相比,100μM褪黑素处理组血小板聚集率下降,差异有统计学意义(P0.05),而0.01μM组、1μM组与A/R组相比差异无统计学意义(P0.05)。1000μM褪黑素较0.01μM、1μM来说,血小板聚集抑制率最大且差异有统计学意义(P0.05),而0.01μM组、1μM组不仅未表现出抑制作用反而呈现活化血小板作用;2.3普罗布考对A/R时血小板聚集率的影响与缺氧复氧组相比,100μM普罗布考处理组血小板聚集率下降,差异有统计学意义(P0.05),而1μM组、10μM组与A/R组相比差异无统计学意义(P0.05)。100μM普罗布考较1μM、10μM来说,血小板聚集抑制率最大且差异有统计学意义(P0.05);3维生素C对血小板凋亡相关指标变化影响3.1活性氧(ROS)与Control组(指血小板置于空气中,未进行缺氧复氧)相比,A/R组、VC组、HCl组中ROS产生明显增加,差异有统计学意义(P0.05);与A/R组相比,VC组ROS减少,差异有统计学意义(P0.05),而HCl组无类似变化,提示维生素C具有强大的清除活性氧的作用,且该作用与VC本身的弱酸性无关;3.2线粒体膜电位(△ψm)与Control组相比,A/R组、VC组、HCl组中低△ψm血小板百分比明显增加,差异有统计学意义(P0.05);VC组与A/R组相比,低△ψm血小板百分比相对减少,差异有统计学意义(P0.05),而HCl组与A/R组相比,差异无统计学意义(P0.05),提示维生素C减轻线粒体损伤与其抗氧化作用相关,而与其酸性无关;3.3凋亡相关蛋白Bax、Bcl-2、cyto-C、Caspase-9的表达与Control组相比,A/R组、VC组、HCl组中cyto-C、Bax、Bcl-2、Caspase-9表达明显增多,差异有统计学意义(P0.05),提示缺氧后线粒体损伤,凋亡途径激活。与A/R组相比,VC组cyto-C、Bax、Caspase-9表达减少,Bcl-2表达增多,凋亡程度减轻,差异有统计学意义(P0.05),而HCl组无此变化。提示维生素C可通过抑制血小板凋亡来降低血小板活化。结论:1不同抗氧化剂均可浓度依赖性地抑制血小板活化聚集。2维生素C后处理通过清除ROS,下调cyto-C、Bax、Caspase-9的表达,上调Bcl-2的表达,减轻血小板凋亡,进而抑制血小板聚集。
[Abstract]:OBJECTIVE: Laboratory and clinical trials have confirmed that platelet activation plays an important role in the process of ischemia-reperfusion, but its mechanism is still unclear. Studies have shown that platelet activation during ischemia-reperfusion is closely related to the production of reactive oxygen species (ROS). Previous studies have shown that vitamin C scavenges reactive oxygen species, reduces platelet activation and reduces reperfusion injury, but its effect on isolated hypoxic-reoxygenated platelets is still unclear. The platelet plasma was diluted to 200 Platelet hypoxia-reoxygenation test was used to select the best treatment time. Platelets were treated with different hypoxia or reoxygenation time and platelet aggregation was detected by whole blood electrical impedance spectroscopy. Arachidonic acid (AA0.5mmol/L) was used as inducer to establish in vitro hypoxia-reoxygenation platelet model. Three different antioxidants (vitamin C, melatonin, probucol) were selected. Hypoxia-reoxygenation group (A/R group) and drug-treatment group (C1-3 group) were prepared after hypoxia-reoxygenation, and HCl group (HCl group, with the same p H value as VC group, all 3.24) were set up considering the weak acidity of vitamin C. Platelet aggregation rate and slope were measured by whole blood electrical impedance to observe the platelet aggregation of different antioxidants. The expression of apoptosis-related proteins Bax, Bcl-2, cytochrome C and Caspase-9 were detected by flow cytometry, reverse fluorescence microscopy and flow cytometry. The data were analyzed by SPSS22.0. The mean (?) + standard deviation ((?) + s) was used when the data obeyed the normal distribution, and the median (quartile spacing) was used when the data did not obey the normal distribution, i.e. M (QR). 1 The effect of different hypoxia or reoxygenation time on platelet aggregation rate in healthy people 1.1 The effect of different hypoxia time on platelet aggregation rate was higher than that of the NON-HYPOXIA group, and the platelet aggregation rate and aggregation slope were higher in each hypoxia group, and the activation rate was the highest in the 20-minute hypoxia group. The difference between the 20-minute hypoxia group and the 5-minute hypoxia group was statistically significant. Significance (P 0.05), but there was no significant difference between 5 min, 10 min, 30 min groups (P 0.05); 1.2 Different reoxygenation time on platelet aggregation rate compared with non-reoxygenation group, platelet aggregation rate and aggregation slope were increased in each reoxygenation group, and 5 min group activation rate was the largest, but there was no significant difference between each group (P 0.05); Effect of oxidant post-treatment on platelet aggregation rate in A/R group 2.1 Effect of vitamin C on platelet aggregation rate in A/R group was significantly lower than that in hypoxia-reoxygenation group (P 0.05). There was no significant difference in platelet aggregation rate between 10 mu M group, 100 mu M group and HCl group (P 0.05). 1000 mu mu mu mu vitamin C group and A/R group (P 0.05). The platelet aggregation inhibitory rate of melatonin C was the highest and the difference was statistically significant (P 0.05) compared with 10 and 100 mu M. The platelet aggregation rate of melatonin 2.2 in A/R group was significantly lower than that of hypoxia-reoxygenation group (P 0.05), but there was no significant difference between 0.01 mu M group and 1 mu M group (P 0.05). Significance (P The platelet aggregation rate decreased significantly (P 0.05), but there was no significant difference between 1_ M group, 10_ M group and A / R group (P 0.05). 100_ M probucol had the highest platelet aggregation inhibition rate compared with 1_ M, 10_ M, and the difference was statistically significant (P 0.05); 3_ vitamin C had the greatest effect on platelet apoptosis related indicators (R_ 1). ROS production in A/R, VC and HCl groups was significantly higher than that in C ontrol group (P The effect was not related to the weak acidity of VC itself; 3.2 Mitochondrial membrane potential (m) was significantly higher in A/R group, VC group and HCl group than in Control group (P 0.05); the percentage of low M platelets in VC group was significantly lower than that in A/R group (P 0.05), while that in HCl group was significantly lower than that in A/R group (P 0.05). The expression of apoptosis-related proteins Bax, Bcl-2, cyto-C and Caspase-9 was significantly higher in A/R group, VC group and HCl group than in C ontrol group (P 0.05). Compared with A/R group, the expression of cyto-C, Bax and Caspase-9 decreased, the expression of Bcl-2 increased, and the degree of apoptosis decreased in VC group (P 0.05). There was no significant difference in HCl group. Degree-dependent inhibition of platelet activation and aggregation.2 Vitamin C postconditioning inhibits platelet aggregation by scavenging ROS, down-regulating the expression of cyto-C, Bax and Caspase-9, up-regulating the expression of Bcl-2 and reducing platelet apoptosis.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
本文编号：2182578
[Abstract]:OBJECTIVE: Laboratory and clinical trials have confirmed that platelet activation plays an important role in the process of ischemia-reperfusion, but its mechanism is still unclear. Studies have shown that platelet activation during ischemia-reperfusion is closely related to the production of reactive oxygen species (ROS). Previous studies have shown that vitamin C scavenges reactive oxygen species, reduces platelet activation and reduces reperfusion injury, but its effect on isolated hypoxic-reoxygenated platelets is still unclear. The platelet plasma was diluted to 200 Platelet hypoxia-reoxygenation test was used to select the best treatment time. Platelets were treated with different hypoxia or reoxygenation time and platelet aggregation was detected by whole blood electrical impedance spectroscopy. Arachidonic acid (AA0.5mmol/L) was used as inducer to establish in vitro hypoxia-reoxygenation platelet model. Three different antioxidants (vitamin C, melatonin, probucol) were selected. Hypoxia-reoxygenation group (A/R group) and drug-treatment group (C1-3 group) were prepared after hypoxia-reoxygenation, and HCl group (HCl group, with the same p H value as VC group, all 3.24) were set up considering the weak acidity of vitamin C. Platelet aggregation rate and slope were measured by whole blood electrical impedance to observe the platelet aggregation of different antioxidants. The expression of apoptosis-related proteins Bax, Bcl-2, cytochrome C and Caspase-9 were detected by flow cytometry, reverse fluorescence microscopy and flow cytometry. The data were analyzed by SPSS22.0. The mean (?) + standard deviation ((?) + s) was used when the data obeyed the normal distribution, and the median (quartile spacing) was used when the data did not obey the normal distribution, i.e. M (QR). 1 The effect of different hypoxia or reoxygenation time on platelet aggregation rate in healthy people 1.1 The effect of different hypoxia time on platelet aggregation rate was higher than that of the NON-HYPOXIA group, and the platelet aggregation rate and aggregation slope were higher in each hypoxia group, and the activation rate was the highest in the 20-minute hypoxia group. The difference between the 20-minute hypoxia group and the 5-minute hypoxia group was statistically significant. Significance (P 0.05), but there was no significant difference between 5 min, 10 min, 30 min groups (P 0.05); 1.2 Different reoxygenation time on platelet aggregation rate compared with non-reoxygenation group, platelet aggregation rate and aggregation slope were increased in each reoxygenation group, and 5 min group activation rate was the largest, but there was no significant difference between each group (P 0.05); Effect of oxidant post-treatment on platelet aggregation rate in A/R group 2.1 Effect of vitamin C on platelet aggregation rate in A/R group was significantly lower than that in hypoxia-reoxygenation group (P 0.05). There was no significant difference in platelet aggregation rate between 10 mu M group, 100 mu M group and HCl group (P 0.05). 1000 mu mu mu mu vitamin C group and A/R group (P 0.05). The platelet aggregation inhibitory rate of melatonin C was the highest and the difference was statistically significant (P 0.05) compared with 10 and 100 mu M. The platelet aggregation rate of melatonin 2.2 in A/R group was significantly lower than that of hypoxia-reoxygenation group (P 0.05), but there was no significant difference between 0.01 mu M group and 1 mu M group (P 0.05). Significance (P The platelet aggregation rate decreased significantly (P 0.05), but there was no significant difference between 1_ M group, 10_ M group and A / R group (P 0.05). 100_ M probucol had the highest platelet aggregation inhibition rate compared with 1_ M, 10_ M, and the difference was statistically significant (P 0.05); 3_ vitamin C had the greatest effect on platelet apoptosis related indicators (R_ 1). ROS production in A/R, VC and HCl groups was significantly higher than that in C ontrol group (P The effect was not related to the weak acidity of VC itself; 3.2 Mitochondrial membrane potential (m) was significantly higher in A/R group, VC group and HCl group than in Control group (P 0.05); the percentage of low M platelets in VC group was significantly lower than that in A/R group (P 0.05), while that in HCl group was significantly lower than that in A/R group (P 0.05). The expression of apoptosis-related proteins Bax, Bcl-2, cyto-C and Caspase-9 was significantly higher in A/R group, VC group and HCl group than in C ontrol group (P 0.05). Compared with A/R group, the expression of cyto-C, Bax and Caspase-9 decreased, the expression of Bcl-2 increased, and the degree of apoptosis decreased in VC group (P 0.05). There was no significant difference in HCl group. Degree-dependent inhibition of platelet activation and aggregation.2 Vitamin C postconditioning inhibits platelet aggregation by scavenging ROS, down-regulating the expression of cyto-C, Bax and Caspase-9, up-regulating the expression of Bcl-2 and reducing platelet apoptosis.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
【参考文献】
相关期刊论文 前2条
1 王秀娟;张平平;赵丽丽;涂_g;戴克胜;;活性氧参与血小板凋亡调控的研究[J];中华血液学杂志;2014年06期
2 赵丽丽;阮长耿;戴克胜;;血小板凋亡的最新研究进展[J];中华血液学杂志;2012年08期
，本文编号：2182578
本文链接：https://www.wllwen.com/yixuelunwen/yiyaoxuelunwen/2182578.html
最近更新
教材专著
