維生素C后處理對(duì)缺氧復(fù)氧條件下花生四烯酸誘導(dǎo)的血小板活化的影響及其機(jī)制研究
發(fā)布時(shí)間：2018-08-14 10:32
【摘要】：目的:實(shí)驗(yàn)室及臨床試驗(yàn)已經(jīng)證實(shí)血小板活化在缺血再灌注過程中發(fā)揮重要作用,但是其機(jī)制仍不甚明確。有研究表明,缺血再灌注中發(fā)生的血小板活化與活性氧大量產(chǎn)生密切相關(guān)。維生素C(VC)是一種弱酸性維生素,具有強(qiáng)大的抗氧化作用,以往研究發(fā)現(xiàn),維生素C能夠清除活性氧、減少血小板活化、減輕再灌注損傷,但其對(duì)離體缺氧復(fù)氧血小板的作用尚不清楚。本實(shí)驗(yàn)旨在觀察維生素C后處理對(duì)離體缺氧復(fù)氧血小板的作用并闡明其具體作用機(jī)制。方法:研究對(duì)象為新鮮機(jī)采健康人血小板原漿共67人份,血小板濃度為(960~1280)×109/L,由河北省血液中心提供。其納入標(biāo)準(zhǔn)及排除標(biāo)準(zhǔn)均嚴(yán)格按照國(guó)家獻(xiàn)血法的標(biāo)準(zhǔn)。用生理鹽水將血小板原漿稀釋為200×109/L以備用,于血小板采集后4小時(shí)內(nèi)完成實(shí)驗(yàn)。首先進(jìn)行血小板缺氧-復(fù)氧實(shí)驗(yàn)以選取最佳實(shí)驗(yàn)處理時(shí)間,對(duì)血小板進(jìn)行不同缺氧或復(fù)氧時(shí)間處理并用全血電阻抗法檢測(cè)血小板聚集,誘導(dǎo)劑為花生四烯酸(AA0.5mmol/L),建立離體缺氧復(fù)氧血小板模型。選取三種不同抗氧化劑(維生素C、褪黑素、普羅布考)進(jìn)行缺氧后處理,并分別制備成缺氧復(fù)氧組(A/R組)、藥物后處理組(C1-3組),考慮到維生素C的弱酸性同時(shí)設(shè)立鹽酸后處理組(HCl組,p H值與VC組相同,均為3.24)。用全血電阻抗法檢測(cè)血小板聚集率及斜率以觀察不同抗氧化劑對(duì)血小板聚集率的影響。測(cè)定維生素C后處理組中血小板凋亡相關(guān)指標(biāo),用倒置熒光顯微鏡檢測(cè)活性氧(ROS),流式細(xì)胞術(shù)檢測(cè)線粒體膜電位(△ψm),Western Blot檢測(cè)凋亡相關(guān)蛋白Bax、Bcl-2、細(xì)胞色素C(cyto-C)、Caspase-9的表達(dá)。數(shù)據(jù)采用SPSS22.0進(jìn)行統(tǒng)計(jì)分析。計(jì)量資料服從正態(tài)分布時(shí)用均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示,不服從正態(tài)分布時(shí)采用中位數(shù)(四分位數(shù)間距)表示,即M(QR)。正態(tài)分布時(shí)采用單因素方差分析,不服從正態(tài)分布時(shí)組間比較采用多個(gè)相關(guān)樣本的秩和檢驗(yàn)。P0.05認(rèn)為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1不同缺氧或復(fù)氧時(shí)間對(duì)健康人血小板聚集率的影響1.1不同缺氧時(shí)間對(duì)于血小板聚集率的影響與未缺氧組相比,各缺氧組血小板聚集率及聚集斜率均有所升高,且缺氧20min組活化率最大。兩兩比較,發(fā)現(xiàn)缺氧20min組與5min組差異有統(tǒng)計(jì)學(xué)意義(P0.05),而5min、10min、30min組間差異無統(tǒng)計(jì)學(xué)意義(P0.05);1.2不同復(fù)氧時(shí)間對(duì)于血小板聚集率的影響與未復(fù)氧組血小板相比,各復(fù)氧組血小板聚集率及聚集斜率均有所升高,且5min組活化率最大,但各組間差異無統(tǒng)計(jì)學(xué)意義(P0.05);2三種抗氧化劑后處理對(duì)A/R時(shí)血小板聚集率的影響2.1維生素C對(duì)A/R時(shí)血小板聚集率的影響與缺氧復(fù)氧組相比,1000μM維生素C處理組血小板聚集率下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05),而10μM組、100μM組、HCl組與A/R組相比差異無統(tǒng)計(jì)學(xué)意義(P0.05)。1000μM維生素C較10μM、100μM來說,血小板聚集抑制率最大且差異有統(tǒng)計(jì)學(xué)意義(P0.05);2.2褪黑素對(duì)A/R時(shí)血小板聚集率的影響與缺氧復(fù)氧組相比,100μM褪黑素處理組血小板聚集率下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05),而0.01μM組、1μM組與A/R組相比差異無統(tǒng)計(jì)學(xué)意義(P0.05)。1000μM褪黑素較0.01μM、1μM來說,血小板聚集抑制率最大且差異有統(tǒng)計(jì)學(xué)意義(P0.05),而0.01μM組、1μM組不僅未表現(xiàn)出抑制作用反而呈現(xiàn)活化血小板作用;2.3普羅布考對(duì)A/R時(shí)血小板聚集率的影響與缺氧復(fù)氧組相比,100μM普羅布考處理組血小板聚集率下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05),而1μM組、10μM組與A/R組相比差異無統(tǒng)計(jì)學(xué)意義(P0.05)。100μM普羅布考較1μM、10μM來說,血小板聚集抑制率最大且差異有統(tǒng)計(jì)學(xué)意義(P0.05);3維生素C對(duì)血小板凋亡相關(guān)指標(biāo)變化影響3.1活性氧(ROS)與Control組(指血小板置于空氣中,未進(jìn)行缺氧復(fù)氧)相比,A/R組、VC組、HCl組中ROS產(chǎn)生明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05);與A/R組相比,VC組ROS減少,差異有統(tǒng)計(jì)學(xué)意義(P0.05),而HCl組無類似變化,提示維生素C具有強(qiáng)大的清除活性氧的作用,且該作用與VC本身的弱酸性無關(guān);3.2線粒體膜電位(△ψm)與Control組相比,A/R組、VC組、HCl組中低△ψm血小板百分比明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05);VC組與A/R組相比,低△ψm血小板百分比相對(duì)減少,差異有統(tǒng)計(jì)學(xué)意義(P0.05),而HCl組與A/R組相比,差異無統(tǒng)計(jì)學(xué)意義(P0.05),提示維生素C減輕線粒體損傷與其抗氧化作用相關(guān),而與其酸性無關(guān);3.3凋亡相關(guān)蛋白Bax、Bcl-2、cyto-C、Caspase-9的表達(dá)與Control組相比,A/R組、VC組、HCl組中cyto-C、Bax、Bcl-2、Caspase-9表達(dá)明顯增多,差異有統(tǒng)計(jì)學(xué)意義(P0.05),提示缺氧后線粒體損傷,凋亡途徑激活。與A/R組相比,VC組cyto-C、Bax、Caspase-9表達(dá)減少,Bcl-2表達(dá)增多,凋亡程度減輕,差異有統(tǒng)計(jì)學(xué)意義(P0.05),而HCl組無此變化。提示維生素C可通過抑制血小板凋亡來降低血小板活化。結(jié)論:1不同抗氧化劑均可濃度依賴性地抑制血小板活化聚集。2維生素C后處理通過清除ROS,下調(diào)cyto-C、Bax、Caspase-9的表達(dá),上調(diào)Bcl-2的表達(dá),減輕血小板凋亡,進(jìn)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96
本文編號(hào)：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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 王秀娟;張平平;趙麗麗;涂_g;戴克勝;;活性氧參與血小板凋亡調(diào)控的研究[J];中華血液學(xué)雜志;2014年06期
2 趙麗麗;阮長(zhǎng)耿;戴克勝;;血小板凋亡的最新研究進(jìn)展[J];中華血液學(xué)雜志;2012年08期
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