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Article(id=1248601466970198847, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1248601466106172220, articleNumber=1001-2494(2024)06-0541-08, orderNo=null, doi=null, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1679155200000, receivedDateStr=2023-03-19, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1775619387619, onlineDateStr=2026-04-08, pubDate=1711036800000, pubDateStr=2024-03-22, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1775619387619, onlineIssueDateStr=2026-04-08, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1775619387619, creator=13701087609, updateTime=1775619387619, updator=13701087609, issue=Issue{id=1248601466106172220, tenantId=1146029695717560320, journalId=1190317699101192196, year='2024', volume='59', issue='6', pageStart='469', pageEnd='558', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1775619387412, creator=13701087609, updateTime=1775619937245, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1248603772348420655, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1248601466106172220, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1248603772348420656, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1248601466106172220, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=541, endPage=548, ext={EN=ArticleExt(id=1248601467272188740, articleId=1248601466970198847, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Analysis of the Inhibitory Effect of Cordycepin on Shear-Induced Platelet Aggregation and Activation by Microfluidic Technique, columnId=1248601467007947584, journalTitle=Chinese Pharmaceutical Journal, columnName=Original article, runingTitle=null, highlight=null, articleAbstract=

OBJECTIVE To observe the inhibitory effect of cordycepin on platelet aggregation and activation induced by different shear rates. METHODS Polydimethylsiloxane (PDMS)-glass microchannel chips were fabricated by soft lithography. The whole blood of normal people anticoagulated with sodium citrate was collected and incubated with different concentrations of cordycepin in vitro, the blood flowed through the straight microchannel or channel with 80% narrow for 150 seconds at the speed of 14.7 μL·min-1 and 50 μL·min-1 respectively. The adhesion and aggregation images of fluorescent labeled platelets on glass surface were photographed with the microscope, and the fluorescent images were analyzed with Image J. The platelet surface coverage percent was used as a quantitative index of platelet aggregation behavior. The effect of cordycepin on platelet calcium mobilization and monocyte-platelet aggregate(MPA) was analyzed by flow cytometry. The risk of cordycepin was assessed through test of blood coagulation. RESULTS Cordycepin inhibits platelet aggregation and the inhibition effect is related to the shear rates. At 14.7 and 50 μL·min-1, platelet aggregation can be inhibited by cordycepin. Cordycepin inhibits platelet calcium mobilization and MPA effectively. It has no effect on exogenous and endogenous coagulation pathways. CONCLUSION Cordycepin can effectively inhibit shear-induced platelet aggregation and is a potential antiplatelet drug.

, correspAuthors=Yuan LI, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Xuemei GAO, Tiancong ZHANG, Xiaojing HUANG, Xuanrong HUAN, Yuan LI), CN=ArticleExt(id=1248601468853441376, articleId=1248601466970198847, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=运用微流控技术分析虫草素对剪切诱导血小板聚集和活化的抑制效果, columnId=1196884516783571586, journalTitle=中国药学杂志, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

目的 分析虫草素对不同非生理性剪切率诱导的血小板聚集和活化的抑制效果。方法 采用软光刻工艺加工聚二甲基硅氧烷-玻璃微通道芯片。收集健康志愿者枸橼酸钠抗凝全血,用钙黄绿素对血小板进行染色标记,并以虫草素体外孵育后,分别以14.7和50 μL·min-1流过带有0.5 mm长的80%狭窄的通道150 s,同时用荧光显微镜实时拍摄血小板在微通道表面的黏附聚集图像;用Image J分析荧光图片,以血小板表面覆盖率作为血小板聚集的量化指标。流式细胞术分析虫草素对血小板钙离子动员,单核细胞-血小板聚集体的抑制效果,并通过凝血检测和溶血实验对虫草素出血风险以及血液相容性做评估。结果 虫草素可抑制剪切诱导的血小板聚集。在14.7和50 μL·min-1条件下,血小板聚集均可被虫草素抑制。虫草素抑制血小板钙离子动员和单核细胞-血小板聚集具有剪切力相关性,并且对外源性凝血途径和内源性凝血途径无影响,具有良好血液相容性。结论 虫草素可有效抑制剪切率诱导的血小板聚集,是一种有潜力的抗血小板药物。

, correspAuthors=李远, authorNote=null, correspAuthorsNote=
*李远,男,硕士,研究员 研究方向:基于微流控芯片技术的临床检验诊断新技术与新方法研究 Tel:18983022607
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高雪梅,女,硕士研究生 研究方向:运用微流控技术分析抗血小板药物活性

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高雪梅,女,硕士研究生 研究方向:运用微流控技术分析抗血小板药物活性

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高雪梅,女,硕士研究生 研究方向:运用微流控技术分析抗血小板药物活性

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DOI: 10.3390/biom10101448., articleTitle=Assessment of fibrinogen macromolecules interaction with red blood cells membrane by means of laser aggregometry, flow cytometry, and optical tweezers combined with microfluidics, refAbstract=null), Reference(id=1248712180661666157, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, doi=null, pmid=null, pmcid=null, year=2020, volume=8, issue=36, pageStart=8305, pageEnd=8514, url=null, language=null, rfNumber=[39], rfOrder=38, authorNames=KIM S, YE S H, ADAMO A, journalName=J Mater Chem B, refType=null, unstructuredReference=KIM S, YE S H, ADAMO A, et al. A biostable, anti-fouling zwitterionic polyurethane-urea based on PDMS for use in blood-contacting medical devices[J]. J Mater Chem B, 2020, 8(36):8305-8514., articleTitle=A biostable, anti-fouling zwitterionic polyurethane-urea based on PDMS for use in blood-contacting medical devices, refAbstract=null), Reference(id=1248712180741357934, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, doi=null, pmid=null, pmcid=null, year=2008, volume=85, issue=3, pageStart=829, pageEnd=839, url=null, language=null, rfNumber=[40], rfOrder=39, authorNames=WU Y, ZHANG M, HAUCH K D, journalName=J Biomed Mater Res A, refType=null, unstructuredReference=WU Y, ZHANG M, HAUCH K D, et al. Effect of adsorbed von Willebrand factor and fibrinogen on platelet interactions with synthetic materials under flow conditions[J]. J Biomed Mater Res A, 2008, 85(3):829-839., articleTitle=Effect of adsorbed von Willebrand factor and fibrinogen on platelet interactions with synthetic materials under flow conditions, refAbstract=null), Reference(id=1248712180804272495, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, doi=null, pmid=null, pmcid=null, year=2018, volume=106, issue=10, pageStart=2777, pageEnd=2788, url=null, language=null, rfNumber=[41], rfOrder=40, authorNames=HORBETT T A, journalName=J Biomed Mater Res A, refType=null, unstructuredReference=HORBETT T A. Fibrinogen adsorption to biomaterials[J]. J Biomed Mater Res A, 2018, 106(10):2777-2788., articleTitle=Fibrinogen adsorption to biomaterials, refAbstract=null)], funds=[Fund(id=1248712175938879772, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, awardId=2023GDRC008, language=CN, fundingSource=重庆市科卫联合医学科研项目中青年高端人才项目资助(2023GDRC008), fundOrder=null, country=null), Fund(id=1248712175997600030, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, awardId=YJSCX202204, language=CN, fundingSource=重庆医科大学附属永川医院研究生创新基金项目资助(YJSCX202204), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1248712170922492028, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, xref=null, ext=[AuthorCompanyExt(id=1248712170930880637, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, companyId=1248712170922492028, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Central Laboratory, Yongchuan Hospital of Chongqing Medical University, Chongqing 402160, China), AuthorCompanyExt(id=1248712170943463550, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, companyId=1248712170922492028, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=重庆医科大学附属永川医院中心实验室, 重庆 402160)])], figs=[ArticleFig(id=1248712174672199921, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=EN, label=Fig.1, caption=Microfluidic technology platform

A-the schematic diagram of the microfluidic chip; B-the schematic diagram of the microfluidic chip used in the experiment; C-the physical image of the chip; D-the data analysis process; The white clusters in the diagram are platelet bodies.

, figureFileSmall=ZadertBwYwQYDIx9TM8O5Q==, figureFileBig=U2veNqFETEG41y/1LhsvZQ==, tableContent=null), ArticleFig(id=1248712174764474613, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=CN, label=图1, caption=微流控技术平台

A-微流控芯片技术平台示意图;B-实验所用微流控芯片示意图; C-芯片实物图;D-数据分析流程;白色团簇为血小板体。

, figureFileSmall=ZadertBwYwQYDIx9TM8O5Q==, figureFileBig=U2veNqFETEG41y/1LhsvZQ==, tableContent=null), ArticleFig(id=1248712174869332216, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=EN, label=Fig.2, caption=Fluid dynamics simulation of microfluidic chip

A-Simulation heat map of shear rate along the wall at the bottom of the chip; B-Distribution curve of shear rate corresponding to the volume flow rate at each outlet.

, figureFileSmall=MvbwlvrjwI0zJrFZCYaI6A==, figureFileBig=UF8fk8dt2xsRnFLmAYxBWw==, tableContent=null), ArticleFig(id=1248712174940635387, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=CN, label=图2, caption=微流控芯片流体力学仿真

A-芯片底部沿壁的剪切率仿真热图;B-各出口体积流量所对应的剪切率大小分布曲线图。

, figureFileSmall=MvbwlvrjwI0zJrFZCYaI6A==, figureFileBig=UF8fk8dt2xsRnFLmAYxBWw==, tableContent=null), ArticleFig(id=1248712175028715775, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=EN, label=Fig.3, caption=Fluorescence image and Wright's staining smear of shear induced platelet aggregation. n=5,$\stackrel{-}{x}$±s

A-the fluorescence imaging image at the 150th second under microfluidic control, with white dots representing platelet aggregates: a-the positive control group at 14.7 μL·min-1; b-cordycepin group at 14.7 μL·min-1; c-the positive control group at 50 μL·min-1; d-cordycepin group at 50 μL·min-1; B-the statistical histogram of platelet surface coverage in each group in Figure 3A. Compared with the positive control group at 14.7 μL·min-1, 1)P<0.01; Compared with the positive control group at 50 μL·min-1, 2)P<0.001; C-the aggregation of platelets after shearing by Wright's staining(×100): a-untreated platelets, as a negative control; b-positive control group at 14.7 μL·min-1; c-cordycepin group at 14.7 μL·min-1; d-positive control group at 50 μL·min-1; e-cordycepin group at 50 μL·min-1; SIPA-shear-induced platelet aggregation.

, figureFileSmall=c6RHYvlzkjf58WWotVMyyg==, figureFileBig=ohlXZPsjRvqBt5gctEhHog==, tableContent=null), ArticleFig(id=1248712175112601857, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=CN, label=图3, caption=剪切力诱导血小板聚集的荧光图像和瑞氏染色涂片. n=5,$\stackrel{-}{x}$±s

A-微流控下第150秒时的荧光成像图,其中白色点状为血小板聚集体: a-14.7 μL·min-1下的阳性对照组;b-14.7 μL·min-1下虫草素实验组;c-50 μL·min-1下的阳性对照组;d-50 μL·min-1下的虫草素实验组;B-图3A中各组血小板表面覆盖率的统计直方图。与14.7 μL·min-1下阳性对照组相比,1)P<0.01;与50 μL·min-1下的阳性对照组相比,2)P<0.001;C-剪切后血小板聚集瑞氏染色(×100):a-静息状态的血小板,阴性对照;b-14.7 μL·min-1下的阳性对照组; c-14.7 μL·min-1下虫草素实验组;d-50 μL·min-1下的阳性对照组;e-50 μL·min-1下的虫草素实验组;SIPA-剪切力诱导的血小板聚集体。

, figureFileSmall=c6RHYvlzkjf58WWotVMyyg==, figureFileBig=ohlXZPsjRvqBt5gctEhHog==, tableContent=null), ArticleFig(id=1248712175204876551, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=EN, label=Fig.4, caption=Shear-induced platelet calcium mobilization time-fluorescence flow scatter image and analysis statistical diagram. n=5,$\stackrel{-}{x}$±s

A-calcium ion flow detection loop gate strategy: a-for all events of CD61-FSC on flow cytometry, circle the target cell population platelets in the P1 gate; b-to remove adhesions, P2 gate circles the population of individual platelets; c-the time-Fluo-4AM average fluorescence intensity plot of platelets; B-analysis of the average fluorescence intensity of calcium ions: a-a plot of the average fluorescence intensity and time of Fluo-4AM, in which 10 time windows were extracted to record the changes in the average fluorescence intensity of Fluo-4; b-a normalized graph representing the fluorescence evolution of Fluo-4, represented as the ratio of fluorescence intensity to baseline fluorescence at each time point after shearing; C-the comparison histogram of the ratios of each group corresponding to Fig b in Figure 4B. Compared with the positive control group at 14.7 μL·min-1, 1)P<0.05; Compared with the positive control group at 50 μL·min-1, 2)P<0.001.

, figureFileSmall=oAq7vyKgTnfwyDNptQQ4oA==, figureFileBig=vKbmomAclVCgjPXMmjejfw==, tableContent=null), ArticleFig(id=1248712175309734154, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=CN, label=图4, caption=剪切激活的血小板钙离子时间-荧光流式散点图像与分析统计图. n=5,$\stackrel{-}{x}$±s

A-钙离子流式检测圈门策略代表图:a-流式细胞仪上样CD61-FSC所有事件,P1门圈出目标细胞群体血小板;b-去黏体,P2门圈出单个血小板的群体;c-血小板的时间-Fluo-4AM平均荧光强度图;B-钙离子平均荧光强度分析图:a-Fluo-4AM平均荧光强度与时间的点图,其中提取了10个时间窗口,以记录Fluo-4的平均荧光强度变化;b-Fluo-4荧光演变的归一化图,表示为剪切后每个时间点的荧光强度与基线荧光的比率;C-图4B中b图所对应的各组比率的比较直方图。与14.7 μL·min-1下阳性对照组相比,1)P<0.05; 与50 μL·min-1下的阳性对照组相比,2)P<0.001。

, figureFileSmall=oAq7vyKgTnfwyDNptQQ4oA==, figureFileBig=vKbmomAclVCgjPXMmjejfw==, tableContent=null), ArticleFig(id=1248712175393620238, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=EN, label=Fig.5, caption=Flow scatter plot of shear induced platelet-monocyte polymer. n=5,$\stackrel{-}{x}$±s

A-gate strategy for flow cytometry detection of monocyte platelet binding: a-CD14-SSA all cell events, P2 gate circled monocyte population; b-circle the proportion of monocytes bound to platelets in the P3 gate; B-representative flow charts for each group: a-represents untreated platelets, as a negative control; b-positive control group at 14.7 μL·min-1; c-cordycepin group at 14.7 μL·min-1; d-positive control group at 50 μL·min-1; e-cordycepin group at 50 μL·min-1; C-statistical histograms of the percentage results of each group of aggregates in Figure; Compared with the positive control group at 14.7 μL·min-1, 1)P<0.05; Compared with the positive control group at 50 μL·min-1, 2)P<0.001.

, figureFileSmall=wQ8c20ZPaJY66u137Rverg==, figureFileBig=3ZolQc4WHoMKe2qa/MpOpg==, tableContent=null), ArticleFig(id=1248712175477506318, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=CN, label=图5, caption=剪切诱导的血小板-单核细胞聚合体流式散点结果图. n=5,$\stackrel{-}{x}$±s

A-单核细胞-血小板结合的流式检测圈门策略:a-CD14-SSA所有细胞事件,P2门圈出单核细胞群体,b-P3门圈出单核细胞中与血小板结合的占比; B-各组流式图代表图:a-静息状态的血小板,阴性对照; b-14.7 μL·min-1下的阳性对照组; c-14.7 μL·min-1下虫草素实验组; d-50 μL·min-1下的阳性对照组; e-50 μL·min-1下的虫草素实验组;C-图B中各组聚集体百分比结果值的统计直方图;与14.7 μL·min-1下阳性对照组相比,1)P<0.05;与50 μL·min-1下的阳性对照组相比,2)P<0.001。

, figureFileSmall=wQ8c20ZPaJY66u137Rverg==, figureFileBig=3ZolQc4WHoMKe2qa/MpOpg==, tableContent=null), ArticleFig(id=1248712175565586704, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=EN, label=Tab.1, caption=

Results of hemolysis value of cordycepin on erythrocyte destruction. n=5,$\stackrel{-}{x}$±s

, figureFileSmall=null, figureFileBig=null, tableContent=
Cordycepin/μmol·L-1 Hemolysis rate/%
50 0.12±0.02
100 0.19±0.07
200 0.49±0.01
400 0.79±0.13
), ArticleFig(id=1248712175641084178, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=CN, label=表1, caption=

虫草素对红细胞破坏的溶血值检测结果. n=5,$\stackrel{-}{x}$±s

, figureFileSmall=null, figureFileBig=null, tableContent=
Cordycepin/μmol·L-1 Hemolysis rate/%
50 0.12±0.02
100 0.19±0.07
200 0.49±0.01
400 0.79±0.13
), ArticleFig(id=1248712175716581653, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=EN, label=Tab.2, caption=

Results of the effect of cordycepin on coagulation. n=3,$\stackrel{-}{x}$±s

, figureFileSmall=null, figureFileBig=null, tableContent=
Group c/μmol·L-1 PT/s APTT/s
Control 0 11.55±0.78 30.20±0.28
Cordycepin 400 11.40±0.85ns 30.10±0.42 ns
t value - 0.18 0.28
P value - 0.87 0.99
), ArticleFig(id=1248712175792079128, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601466970198847, language=CN, label=表2, caption=

虫草素对凝血项的影响结果分析. n=3,$\stackrel{-}{x}$±s

, figureFileSmall=null, figureFileBig=null, tableContent=
Group c/μmol·L-1 PT/s APTT/s
Control 0 11.55±0.78 30.20±0.28
Cordycepin 400 11.40±0.85ns 30.10±0.42 ns
t value - 0.18 0.28
P value - 0.87 0.99
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运用微流控技术分析虫草素对剪切诱导血小板聚集和活化的抑制效果
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高雪梅 , 张天聪 , 黄小静 , 宦宣容 , 李远 *
中国药学杂志 | 研究论文 2024,59(6): 541-548
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中国药学杂志 | 研究论文 2024, 59(6): 541-548
运用微流控技术分析虫草素对剪切诱导血小板聚集和活化的抑制效果
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高雪梅, 张天聪, 黄小静, 宦宣容, 李远*
作者信息
  • 重庆医科大学附属永川医院中心实验室, 重庆 402160

    • 高雪梅,女,硕士研究生 研究方向:运用微流控技术分析抗血小板药物活性

通讯作者:

*李远,男,硕士,研究员 研究方向:基于微流控芯片技术的临床检验诊断新技术与新方法研究 Tel:18983022607
Analysis of the Inhibitory Effect of Cordycepin on Shear-Induced Platelet Aggregation and Activation by Microfluidic Technique
Xuemei GAO, Tiancong ZHANG, Xiaojing HUANG, Xuanrong HUAN, Yuan LI*
Affiliations
  • Central Laboratory, Yongchuan Hospital of Chongqing Medical University, Chongqing 402160, China
出版时间: 2024-03-22
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摘要
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目的 分析虫草素对不同非生理性剪切率诱导的血小板聚集和活化的抑制效果。方法 采用软光刻工艺加工聚二甲基硅氧烷-玻璃微通道芯片。收集健康志愿者枸橼酸钠抗凝全血,用钙黄绿素对血小板进行染色标记,并以虫草素体外孵育后,分别以14.7和50 μL·min-1流过带有0.5 mm长的80%狭窄的通道150 s,同时用荧光显微镜实时拍摄血小板在微通道表面的黏附聚集图像;用Image J分析荧光图片,以血小板表面覆盖率作为血小板聚集的量化指标。流式细胞术分析虫草素对血小板钙离子动员,单核细胞-血小板聚集体的抑制效果,并通过凝血检测和溶血实验对虫草素出血风险以及血液相容性做评估。结果 虫草素可抑制剪切诱导的血小板聚集。在14.7和50 μL·min-1条件下,血小板聚集均可被虫草素抑制。虫草素抑制血小板钙离子动员和单核细胞-血小板聚集具有剪切力相关性,并且对外源性凝血途径和内源性凝血途径无影响,具有良好血液相容性。结论 虫草素可有效抑制剪切率诱导的血小板聚集,是一种有潜力的抗血小板药物。

血小板聚集  /  微流控技术  /  壁剪切率  /  虫草素

OBJECTIVE To observe the inhibitory effect of cordycepin on platelet aggregation and activation induced by different shear rates. METHODS Polydimethylsiloxane (PDMS)-glass microchannel chips were fabricated by soft lithography. The whole blood of normal people anticoagulated with sodium citrate was collected and incubated with different concentrations of cordycepin in vitro, the blood flowed through the straight microchannel or channel with 80% narrow for 150 seconds at the speed of 14.7 μL·min-1 and 50 μL·min-1 respectively. The adhesion and aggregation images of fluorescent labeled platelets on glass surface were photographed with the microscope, and the fluorescent images were analyzed with Image J. The platelet surface coverage percent was used as a quantitative index of platelet aggregation behavior. The effect of cordycepin on platelet calcium mobilization and monocyte-platelet aggregate(MPA) was analyzed by flow cytometry. The risk of cordycepin was assessed through test of blood coagulation. RESULTS Cordycepin inhibits platelet aggregation and the inhibition effect is related to the shear rates. At 14.7 and 50 μL·min-1, platelet aggregation can be inhibited by cordycepin. Cordycepin inhibits platelet calcium mobilization and MPA effectively. It has no effect on exogenous and endogenous coagulation pathways. CONCLUSION Cordycepin can effectively inhibit shear-induced platelet aggregation and is a potential antiplatelet drug.

platelet aggregation  /  microfluidic technology  /  shear rate  /  cordycepin
高雪梅, 张天聪, 黄小静, 宦宣容, 李远. 运用微流控技术分析虫草素对剪切诱导血小板聚集和活化的抑制效果. 中国药学杂志, 2024 , 59 (6) : 541 -548 .
Xuemei GAO, Tiancong ZHANG, Xiaojing HUANG, Xuanrong HUAN, Yuan LI. Analysis of the Inhibitory Effect of Cordycepin on Shear-Induced Platelet Aggregation and Activation by Microfluidic Technique[J]. Chinese Pharmaceutical Journal, 2024 , 59 (6) : 541 -548 .
正文
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血小板在人体中参与凝血的生理性反应调控[1-3]。在正常生理条件下,凝血反应启动时,凝血级联途径激活凝血酶,凝血酶不仅可以使纤维蛋白原变成纤维蛋白单体,还可与血小板表面凝血酶受体结合,激活血小板。纤维蛋白单体形成纤维蛋白网,网络附近的血小板等血细胞进一步形成血栓。血小板被激活后,血小板内钙池可释放出大量钙离子[4-5],参与血小板的活化过程,激活的血小板还可分泌二磷酸腺苷(adenosine diphosphate,ADP)和血栓素A2(thromboxane A2,TXA2)等凝血因子激活附近其他血小板,最终通过血小板的GPⅡb/Ⅲa受体和纤维蛋白原结合发生最终的聚集[6-8]。活化的血小板表面表达的P-选择素可与单核细胞上受体P-选择素糖蛋白配体-1结合,形成单核细胞-血小板聚合体(MPA),促使炎症的发生[9]。
剪切是体内激活血小板的重要因素。在正常生理条件下,静脉中壁剪切率低于500 ·s-1,大动脉剪切率约1 000 ·s-1[10]。在病理性条件下,如动脉粥样硬化造成血栓或血管内植入医疗器械时,血管内出现非生理性狭窄,血流状态出现紊乱,血流剪切率会出现急剧升高接着再急剧降低的情况,狭窄处会形成瞬时升高的病理性高剪切率。此时的非生理性剪切会诱导血小板活化,使血管内出现病理性血小板聚集[11-13]。预防非生理性剪切诱导的血小板聚集对预防血栓的形成具有重要意义。
虫草素是蛹虫草的主要成分[14]。研究表明,蛹虫草中含有虫草素、虫草多糖、虫草酸、麦角固醇,还有丰富的人体必需氨基酸、微量元素等多种生物活性物质,其中虫草素,又名3'-脱氧腺苷,是蛹虫草中特有的活性成分[15]。虫草素在抗肿瘤、抗白血病和免疫调节方面有较多研究,它还具有抗炎和降血脂等心血管保护作用。虫草素可降低胶原和ADP诱导的人类血小板聚集[16-18],但现有的研究进展仅在静态条件下以血小板激动剂激活血小板,缺乏对流动状态影响血小板活性的考虑[19],即目前还没有虫草素对剪切率诱导血小板聚集的抑制效果研究。明确虫草素对剪切力诱导血小板聚集的抑制作用对临床上治疗心血管疾病研发新型抗血小板药物及探寻新的治疗方案具有一定价值。
微流控技术是一项可以体外模拟血液流变的技术,不仅可以还原血液流动环境,还可以提供实验过程中所需的不同剪切率条件[20-22]。在微流控通道中,血小板可通过表面受体与相应蛋白配体结合黏附到通道底部,而其他血液内活细胞不会黏附到底部,所以可通过荧光标记全血活细胞以分析血小板黏附行为[23]。本课题组前期建立的微流控技术芯片模型和其他组的微流控实验中使用的芯片相比,通道底面为洁净裸玻璃,玻璃上无胶原等黏附蛋白的修饰;玻璃对于血液而言是一种外源性植入材料,可直接吸附血浆中的蛋白(纤维蛋白原、血管性血友病因子等)以介导血小板的黏附聚集,减小由于蛋白包被不均等带来的结果误差,并且由于不同剪切下介导血小板聚集的主要蛋白不同,未包被的通道底部体现了不同剪切下相应蛋白黏附而介导的血小板聚集。本实验在不同的剪切率条件下分析了虫草素对血小板聚集和活化的抑制效果研究,通过观察流动条件下虫草素对血小板黏附聚集的抑制效果,为临床中虫草素的相关用量提供更多参考依据。
1 材料与方法
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1.1 试剂与仪器
虫草素(≥98%,上海源叶生物科技有限公司);Calcein-A M荧光染料、anti-hu CD61、anti-hu CD41a、anti-hu CD14 (美国Intivrogen公司); 磷酸盐缓冲液(PBS)、红细胞裂解液(美国BD pharmingen公司);Sylgard 184聚二甲基硅氧烷(美国Dow Corning公司);HQ-6100感光干膜(长兴化工材料有限公司);3.2%枸橼酸钠静脉血液真空采集管(山东威高集团有限公司)。
紫外曝光灯(实验室自制);FM-360覆膜机(杭州新彩科技有限公司);PDG-32G-2等离子清洗机(德国Harrick公司);1390喷墨打印机[爱普生(中国)有限公司];RSP01-CS双向推拉型精密注射泵(嘉善瑞创电子科技有限公司);Ⅸ71倒置荧光显微镜(日本奥林巴公司斯);单色制冷相机(加拿大Qicam公司);Streampix视频录制软件(加拿大Norpix公司)。
1.2 方法
1.2.1 微流控芯片模型制备
分析血小板黏附聚集的微流控芯片由微通道和两侧的样品池及出口组成,其中通道高70 μm,宽1 mm,长10 mm,样品池直径7 mm,出口直径1.5 mm,80%狭窄模型的狭窄位于通道中央,长度为0.5 mm,狭窄处宽200 μm。将聚二甲基硅氧烷(polydimethylsiloxane, PDMS)基质和固化剂混合而成的预聚物浇铸在芯片阳模上,抽真空除气泡后进行加热固化。利用平头打孔器打孔形成样品池和出口,氧等离子清洗机处理后与清洗干净的载玻片进行不可逆键合,形成PDMS-玻璃微流控芯片。该简易微流控装置中剪切率及所对应的出口体积流量由solidworks软件进行流体仿真得到。
1.2.2 血液样品采集及处理
采用12名健康人全血,健康志愿者由重庆市血液中心永川分中心招募。纳入标准为志愿者自述1个月内无服药史、手术史和酗酒史,血小板数量、红细胞比容、凝血功能均在正常参考值区间。本研究经重庆医科大学附属永川医院伦理委员会批准,采血前志愿者和患者均签署知情同意。采集的血液样本用质量分数3.2%枸橼酸钠抗凝管保存。
1.2.3 微流控技术观察动态血小板聚集
检测设备及分析过程见图1~2。以14.7和50 μL·min-1抽吸狭窄通道中荧光标记血样(即模拟非生理性狭窄血管剪切率1 000·s-1和5 000·s-1),仿真结果见图2。同时用高速摄像机拍照记录血小板的黏附聚集情况。用Image J分析血小板表面覆盖率。
1.2.4 剪切诱导血小板聚集观察
将枸橼酸钠抗凝全血与钙黄绿素以500∶1的体积比混合(钙黄绿素终浓度为4 μmol·L-1)。随后将血液样本均分为2管,每管200 μL。1管作为实验组(虫草素浓度为400 μmol·L-1),另一管作为阳性对照组(加虫草素等体积生理盐水)。实验组和阳性对照组同时进行37 ℃孵育30 min。将通道用3%牛血清白蛋白(BSA)进行封闭处理后用PBS润洗以用于剪切收集血样。剪切收集的血样做瑞氏涂片染色。阴性组不做任何处理直接进行瑞氏涂片染色。
1.2.5 钙离子释放检测
向枸橼酸钠抗凝全血加入终浓度为5 μmol·L-1的Fluo-4AM和CD61荧光抗体避光孵育15 min后将其分为阴性对照组(加等体积生理盐水),阳性对照组(加等体积生理盐水)和实验组(虫草素浓度为400 μmol·L-1),各组在37 ℃下孵育30 min。将通道用3%BSA进行封闭处理后用牛血清白蛋白PBS润洗以用于剪切收集血样。阴性对照组直接流式采集分析Fluo-4AM荧光强度,阳性对照组和实验组上机采集数据1 min后,经剪切收集后流式上机采集9 min。用CytExpert软件记录时长共10 min,并进行血小板的Fluo-4AM荧光强度分析。分析方法参照Mar等[24]实验。
1.2.6 单核细胞-血小板聚合体检测
单核细胞-血小板聚合体流式检测方法参考Gremmel等[25]实验:枸橼酸钠抗凝全血分装为2管,各200 μL。1管作为实验组(虫草素浓度为400 μmol·L-1),另一管作为实验阳性对照组(加虫草素等体积生理盐水)。将通道用3%BSA进行封闭处理后用PBS润洗以用于剪切收集血样。向每管中加入CD41和CD14荧光抗体常温避光孵育15 min后剪切收集,用1 mL 1%多聚甲醛固定20 min后再用1×红细胞裂解液裂解红细胞10 min,450 g离心5 min后去上清,用200 μL生理盐水重悬底部沉底。流式细胞仪上机记录10 000个细胞,分析单核细胞-血小板聚合体的百分比。
1.2.7 虫草素溶血实验
枸橼酸钠抗凝全血200 g离心5 min后吸取底部红细胞200 μL,生理盐水洗涤1次后用生理盐水重悬。将1 mL药物与20 μL红细胞混匀震荡孵育后200 g离心5 min,吸取上清液200 μL加入96孔板中进行吸光度检测,每个样本做3个复孔取均值计算。阳性对照组用1 mL双蒸水与20 μL红细胞混匀,阴性对照组用1 mL生理盐水与20 μL红细胞混匀。溶血率的计算见公式1。其中ODt代表实验组光密度,ODn代表阳性对照组光密度,ODP代表阴性对照组光密度。
溶血率(%)=$\frac{(\mathrm{O}{\mathrm{D}}_{\mathrm{t}}-\mathrm{O}{\mathrm{D}}_{\mathrm{n}})}{(\mathrm{O}{\mathrm{D}}_{\mathrm{p}}-\mathrm{O}{\mathrm{D}}_{\mathrm{n}})}$×100%
1.2.8 凝血项目检测
将枸橼酸钠抗凝全血3 000 r·min-1离心10 min,收集上清,分为实验组(虫草素浓度为400 μmol·L-1)和对照组(加等体积生理盐水),在37 ℃下孵育30 min后用血凝仪进行上机检测。检测指标为凝血酶原时间(prothrombin time,PT)和活化部分凝血活酶时间(activated partial thromboplatin time,APTT)。
1.3 统计学分析
所有计量资料用平均值±标准差表示,统计分析采用 Graph pad软件,多组间均值比较采用单因素重测方差分析(ANOVA),采用 Tukey 检验进行两两均值比较; 两组均值比较采用配对t检验,P<0.05表示差异有统计学意义。
2 结果
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2.1 血小板聚集情况和虫草素抑制血小板聚集效果
以血小板表面覆盖率参数分析血小板在不同剪切率条件下的黏附聚集情况。在1 000 ·s-1低剪切率时(14.7 μL·min-1),血小板以分散点状聚集;在5 000 ·s-1高剪切率下(50 μL·min-1),血小板聚集沿流线方向呈头大尾小的条索状,并且条索状面积随着剪切率增加而增大,聚集体个数随剪切率增加而减少。瑞氏染色结果可见随剪切率增大,血小板聚集体增大。通过药物组与对照组的比较,发现虫草素对剪切诱导血小板聚集有明显抑制作用(图3)。
2.2 钙离子释放检测结果
选择记录了10个时间窗口的血小板钙离子平均荧光强度。以ADP激活组作为阳性对照组,可见剪切力激活血小板钙池内钙离子的释放,而这也是血小板后续发生活化的基础(图4A)。实验组分别代表在14.7和50 μL·min-1的入口体积流量下的钙离子平均荧光强度。在各剪切力条件下,血小板钙池的钙离子释放随剪切力的增加而增加,虫草素有效地抑制剪切诱导的血小板钙释放(图4B)。
2.3 单核细胞-血小板聚合体
通过在单核细胞群中找到结合血小板的群体可直观地分析单核细胞-血小板聚合体的情况。剪切可有效激活血小板,形成单核细胞-血小板聚合体。剪切率越大,单核细胞-血小板聚合体越多,即血小板的活化程度越大,并且虫草素可有效抑制单核细胞-血小板聚合体的形成,见图5。
2.4 虫草素溶血分析检测
药物使用过程中,应避免对人体产生损伤。通过对药物是否会造成溶血进行分析,可判断药物的血液相容性,良好的血液相容性是药物投入临床使用的基础。通过观察浓度梯度的虫草素溶血实验结果可以发现虫草素不会导致严重的溶血反应,这保证了虫草素作为药物使用时的安全,见表1。溶血率低于5%即符合医用要求,不会导致严重的溶血反应。
2.5 凝血项检测结果
PT和APTT评估虫草素对凝血的抑制作用,这分别反映了外部和内部途径的凝血功能。虫草素显著抑制了剪切应力诱导的血小板聚集但不抑制PT和APTT(表2),表明虫草素抑制剪切应力导致的血小板聚集,但不影响凝血系统功能。
3 讨论
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心血管疾病是人类主要死亡原因之一,血小板功能紊乱可诱发凝血系统障碍并进一步诱导心血管疾病的发生[26]。有效防止血小板的非生理性活化聚集将有利于预防心血管疾病的发生。目前临床上常使用阿司匹林、氯吡格雷等抗血小板药物治疗凝血,预防血栓形成[27-29],但仍会存在有出血的风险,抗血小板治疗方案需要进一步完善。对于现有药物的改进、新药的开发,以及新靶点的探索都是发展的方向。
针对剪切诱导血小板聚集通路的抑制是一探讨的热点。非生理性剪切可促使血小板发生活化聚集,例如在植入医疗器械的血管内,会形成非生理性狭窄,血小板在局部会经历一段剪切力瞬时升高又瞬时降低的过程[30-31]。在该剪切过程中,血管性血友病因子(von willebrand factor, vWF)会被拉伸延长并暴露出与血小板结合的位点A2,A2可与血小板膜蛋白GPⅠb结合促使血小板发生活化黏附,并最终激活血小板膜蛋白GPⅡb/Ⅲa,活化的GPⅡb/Ⅲa可与纤维蛋白原(fibrinogen, Fg)结合,介导血小板的不可逆聚集,最后形成血栓[32-34]。针对非生理性剪切诱导的血小板聚集通路进行抗血小板药物的研发,可以有效预防体内血栓的形成。
随着微流控技术的发展,使得体外模拟血管流场环境成为一种可行的方法。在微流控芯片中,不仅可以通过控制出口体积流量以达到实验所需的高剪切条件,还可模拟还原血细胞流经血管时的动态过程,如红细胞碰撞使得血小板沿壁流动[35-37]。除此之外,本实验所使用的微流控芯片底部为洁净的裸玻璃,不仅减少了由于蛋白包被不均所带来的实验误差,而且还原了医源植入性材料对血小板黏附聚集的吸附效应[38-41]。在玻璃表面,血液中的蛋白如Fg、vWF等会吸附到玻璃上,提供血小板黏附聚集的结合位点。血小板在玻璃表面的聚集与流体剪切力相关,血小板沿流线形成聚集体,剪切力越大,血小板聚集程度越强;血小板单个聚集体体积随剪切力增加而增大。
虫草属真菌是一种用于治疗炎症和癌症的药物。据研究报道,虫草素是蛹虫草的一种主要成分,可以降低胶原诱导的人血小板中[Ca2+]i水平和TXA2的产生,有体外抗血栓形成的作用。在此基础上可继续进一步验证虫草素的抗血小板活性以及对其抑制效果的影响因素探究。本研究发现,虫草素对非生理性剪切诱导的血小板聚集和活化有明显的抑制作用,实验检测了PT和APTT指标,发现高浓度的虫草素并不会对凝血途径造成影响,虫草素的使用不会带来出血风险,并且不会造成溶血。表明虫草素值得在临床上进一步探究,是一种有潜力的抗血小板药物。
基金
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  • 重庆市科卫联合医学科研项目中青年高端人才项目资助(2023GDRC008)
  • 重庆医科大学附属永川医院研究生创新基金项目资助(YJSCX202204)
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  • 接收时间：2023-03-19
  • 首发时间：2026-04-08
  • 出版时间：2024-03-22
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  • 收稿日期：2023-03-19
基金
重庆市科卫联合医学科研项目中青年高端人才项目资助(2023GDRC008)
重庆医科大学附属永川医院研究生创新基金项目资助(YJSCX202204)
作者信息
    重庆医科大学附属永川医院中心实验室, 重庆 402160

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*李远,男,硕士,研究员 研究方向:基于微流控芯片技术的临床检验诊断新技术与新方法研究 Tel:18983022607
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2种不同金属材料的力学参数
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Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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