CN112305124B - 一种生物标志物及其在疾病诊断中的应用 - Google Patents
一种生物标志物及其在疾病诊断中的应用 Download PDFInfo
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- CN112305124B CN112305124B CN202011195896.5A CN202011195896A CN112305124B CN 112305124 B CN112305124 B CN 112305124B CN 202011195896 A CN202011195896 A CN 202011195896A CN 112305124 B CN112305124 B CN 112305124B
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Abstract
本发明公开了一种生物标志物及其在疾病诊断中的应用,具体的公开了生物标志物PC(20:0/18:1(11Z)),所述PC(20:0/18:1(11Z))在动脉粥样硬化性脑梗死水平降低,以PC(20:0/18:1(11Z))作为检测变量,具有较高的区分动脉粥样硬化性脑梗死和动脉粥样硬化的效能。
Description
技术领域
本发明属于生物医药领域,涉及一种生物标志物及其在疾病诊断中的应用。
背景技术
脑梗死是由多种原因引起的脑部血液供应障碍而导致局部脑组织缺血缺氧性坏死,而出现相应的神经功能缺损的一类疾病,是常见的高致死率、高致残率的神经内科疾病,并且在我国存在其发病率逐渐上升及发病年龄逐渐年轻化趋势。研究表明动脉粥样硬化斑块的不稳定是导致脑梗死的主要原因之一,并且对患者的预后有重要影响。在目前临床实践中,脑梗死的诊断主要依靠临床医师对症状、体征的判断及影像学检查,但是许多疾病如偏头痛、痫性发作等可能与脑梗死有类似的临床表现,影像学客观检查也有一定的局限性,为了使脑梗死患者及时得到有效救治,降低脑梗死的死亡率及病残率临床医生及科学家们致力于找到可以在脑梗死早期辅助诊断的生物标志物。
代谢组学(Metabolomics,Metabonomics)是指生物体系因生物刺激病理生理扰动或遗传信息改变等引起的总体动态的代谢变化。代谢组学作为系统生物学最下游的组学,是整体性研究生命体系功能变化的重要学科分支。代谢组学又分为靶向性代谢组学(Targeted metabolomics)和非靶向性代谢组学(Untargeted metabolomic)靶向性代谢分析主要是定量特定的代谢物,典型的是关注一种或几种相关的代谢通路常用于研究药物代谢的药代动力学,衡量某种疗法或基因修饰的效果靶向性代谢分析的流程是代谢物标准品的液相色谱-质谱联用分析(LC-MS),选择性离子监测,分析方法优化和标准曲线制作,样品前处理,样品提取物LC-MS分析,通过对比样品和标准品进行数据分析,定量特定的代谢物。而非靶向性代谢组学范围宽泛,目标是从生物样本中同时分析尽可能多的代谢物非靶向性代谢组学的步骤是样品前处理,代谢物提取,LC-MS全扫描检测,数据预处理,统计分析及差异代谢物的鉴定。目前代谢组学已经广泛的应用于疾病诊断、药物研发、营养学、毒理学、运动医学等领域。因此,进行脑梗死的代谢组学研究,寻找可实现脑梗死的早期辅助诊断的代谢物,具有重要的临床意义。
发明内容
为了弥补现有技术的不足,本发明的目的在于提供一种与动脉粥样硬化性脑梗死相关的生物标志物,通过检测生物标志物的水平,可以判断患者是否患有动脉粥样硬化性脑梗死,从而为动脉粥样硬化性脑梗死的早期诊断提供一种新的手段。
为了实现上述目的,本发明提供了如下技术方案:
本发明的第一方面提供了一种动脉粥样硬化性脑梗死生物标志物,所述生物标志物为PC(20:0/18:1(11Z))。
本发明的第二方面提供了本发明第一方面所述的生物标志物的筛选方法,包含收集及处理动脉粥样硬化性脑梗死患者和动脉粥样硬化患者的血清样本、色谱分离、质谱数据采集和分析、显著性差异代谢物的筛选、筛选结果的诊断效能验证。
进一步,血清样本处理方法包括:
(1)反相色谱分析血清样本处理方法;
(2)亲水色谱分析血清样本处理方法。
进一步,反相色谱的处理方法包括:
(1)血浆/血清样本在4℃的冰融化为30-60min;
(2)取血清至标记好标签的离心管中,加入的体积比为3:10的甲醇和甲基叔丁基醚;
(3)充分振荡,进行蛋白沉淀,4℃离心,取上层溶液,待测。
进一步,亲水色谱分析血清样本处理方法包括:
(1)血浆/血清样本在4℃的冰融化为30-60min。
(2)取血清至标记好标签的离心管中,加入乙腈;
(3)充分振荡,进行蛋白沉淀;4℃离心,取上层溶液,待测。
进一步,色谱分离采用Thermo Scientific的U3000快速液相色谱使用反相色谱和亲水色谱对血清样本进行分析。
进一步,质谱分析采用装备了热电喷雾离子源的四极杆轨道离子阱质谱仪。
进一步,质谱仪的使用条件为:正负离子离子源电压分别为3.7kv和3.5kV。毛细管加热温度320℃,翘气压力30psi,辅助气压力10psi,容积加热蒸发温度300℃,翘气和辅助气均为氮气,碰撞气为氮气,压力为1.5mTorr,一级全扫描参数为:分辨率70000,自动增益控制目标为1×106,最大隔离时间50ms,质荷比扫描范围50-1500,液质系统由Xcalibur2.2SP1.48软件控制,数据采集和靶向代谢物定量处理均由该软件操作。
本发明的第三方面提供了检测样本中本发明第一方面所述的生物标志物的试剂在制备诊断动脉粥样硬化性脑梗死的产品中的应用。
进一步,所述产品包括用于色谱法、光谱法、质谱法、化学分析法检测所述生物标志物的试剂。
进一步,所述产品包括质谱法联用色谱法检测所述生物标志物的试剂。
进一步,所述样本选自血液、血浆或血清。
本发明提供了一种诊断动脉粥样硬化性脑梗死的试剂盒,所述试剂盒包括检测样本中本发明第一方面所述的生物标志物的试剂;以及使用所述试剂盒评估受试者是否患有或易患动脉粥样硬化性脑梗死的说明书。
进一步,所述试剂盒还包括处理样本的试剂。
本发明的第四方面提供了一种系统,包括:
(1)动脉粥样硬化性脑梗死评估装置,其包括控制单元和存储单元,用于评估受试者是否患有动脉粥样硬化脑梗死;和
(2)彼此通信地连接的信息通信终端装置,其提供关于来自受试者的样本中本发明第一方面所述的生物标志物的存在和/或浓度和/或数量的数据;
其中所述动脉粥样硬化性脑梗死评估装置的控制单元包括:
(1)数据接收单元,其接收从所述信息通信终端设备传输的关于所述样本的所述生物标志物的浓度和/或量的数据;
(2)判别值计算单元,其基于由所述数据接收单元接收的所述样本中所述生物标志物的浓度和/或量值以及具有存储在所述存储单元中的作为解释变量的所述生物标志物的浓度和/或量的判别来计算判别值;
(3)判别值基准评价单元,其基于由所述判别值计算单元计算的判别值,对所述受试者中的动脉粥样硬化性脑梗死的情况进行评价;以及
(4)评估结果发送单元,其将由所述判别值基准评估单元获得的所述受试者的评估结果发送到所述信息通信终端装置。
本发明的第五方面提供一种用于鉴定和评估药剂和/或手术治疗和/或物理治疗对抗动脉粥样硬化性脑梗死的效果的方法,所述方法包括:
(1)收集患有动脉粥样硬化性脑梗死的受试者提供第一样本;
(2)从所述第一样本获得代谢物谱,其中所述第一代谢物谱是使用质谱法获得的;
(3)对所述受试者或在所述受试者上施用一种或多种候选药物和/或进行一种或多种物理或手术治疗;
(4)提供来自步骤(3)中的所述受试者的第二样本;
(5)从所述第二样本中获得代谢物谱,其中所述第二代谢物谱是通过质谱法获得的;
(6)将步骤(2)和(5)中获得的所述代谢物谱与参考代谢物谱进行比较;以及
(7)基于步骤(6)中的所述比较,评估所述一种或多种候选药物和/或治疗是否有效对抗动脉粥样硬化性脑梗死;
进一步,步骤(2)和步骤(5)中的代谢物为本发明第一方面所述的生物标志物。
本发明的优点和有益效果:
本发明首次发现了与动脉粥样硬化性脑梗死相关的生物标志物,通过检测生物标志物的水平,可以判断受试者是否患有动脉粥样硬化性脑梗死以及患动脉粥样硬化性脑梗死的风险,以期实现动脉粥样硬化性脑梗死早期的诊断,从而在脑梗早期进行干预治疗,提高患者的生存质量。
附图说明
图1是OPLS-DA统计分析图,其中图A是反向色谱正离子统计分析图;图B是反向色谱负离子统计分析图;图C是亲水色谱正离子统计分析图。
图2是PC(20:0/18:1(11Z))在不同群组中的水平图。
图3是以PC(20:0/18:1(11Z))作为检测变量的诊断效能图。
具体实施方式
本发明为了评估代谢物与动脉粥样硬化性脑梗死之间的相关性,通过收集动脉粥样硬化患者与动脉粥样硬化性脑梗死的样本,综合分析样本的代谢组学,筛选在两个群组中含量呈现显著性差异的代谢物,并进一步分析差异代谢物的诊断效能,从而发现适于动脉粥样硬化性脑梗死诊断和治疗的生物标志物。
在本发明中,术语“生物标志物”意指化合物,优选是代谢物,与来自具有第二表型(例如没有疾病)的受试者或一组受试者的生物样品相比,它在来自具有第一表型(例如患有疾病)的受试者或一组受试者的生物样品中差异地存在(即增加或减少)。术语“生物标志物”和“代谢生物标志物”通常在本发明的上下文中同义使用,并且通常是指一种代谢物的量或两种或更多种代谢物的含量或比率。
生物标志物可以在任何水平上差异地存在,但是一般以如下的水平存在,所述水平增加了至少5%、至少10%、至少15%、至少20%、至少25%、至少30%、至少35%、至少40%、至少45%、至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少100%、至少110%、至少120%、至少130%、至少140%、至少150%、或更多;或一般以如下的水平存在,所述水平减少了至少5%、至少10%、至少15%、至少20%、至少25%、至少30%、至少35%、至少40%、至少45%、至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、或100%(即不存在)。
优选地生物标志物以具有统计显著性(即p值小于0.05和/或q值小于0.10,如使用韦尔奇氏T检验(Welch's T-test)或Wilcoxon秩和检验(Wilcoxon's rank-sum Test)所确定)的水平差异地存在。
术语“样本”与“样品”在本文中可以互换使用,用于本文时指获得自或衍生自受试者(例如感兴趣的个体)的组合物,其包含有待根据例如物理,生化,化学和/或生理特点来表征和/或鉴定的细胞和/或其它分子实体。例如,短语“疾病样本”或其变体指得自感兴趣的受试者的任何样本,预计或已知其包含待表征的细胞和/或分子实体。样本包括但不限于,组织样本(例如肿瘤组织样本),原代或培养的细胞或细胞系,细胞上清,细胞裂解物,血小板,血清,血浆,玻璃体液,淋巴液,滑液,滤泡液(follicular fluid),精液,羊水,乳,全血,血液衍生的细胞,尿液,脑脊髓液,唾液,痰,泪,汗液,粘液,肿瘤裂解物,和组织培养液(tissue culture medium),组织提取物如匀浆化的组织,肿瘤组织,细胞提取物,及其组合。作为优选的实施方式,所述样本选自血液、血清、血浆。
术语“受试者”意指任何动物,还指人类和非人类的动物。术语“非人类的动物”包括所有脊椎动物,例如,哺乳动物,如非人灵长类动物(特别是高等灵长类动物)、绵羊、狗、啮齿类动物(如小鼠或大鼠)、豚鼠、山羊、猪、猫、兔、牛、和任何家畜或宠物;以及非哺乳动物,如鸡,两栖类,爬行动物等。在优选的实施方式中,所述受试者为人。
本发明通过广泛而深入的研究,首次发现了与动脉粥样硬化性脑梗死相关的生物标志物PC(20:0/18:1(11Z))。与动脉粥样硬化患者相比,PC(20:0/18:1(11Z))在动脉粥样硬化性脑梗死患者中含量显著降低。
在本发明中,可以使用任何合适的方法来分析生物样品以确定所述样品中所述生物标志物的水平。合适的方法包括核磁共振光谱法;高效液相色谱法;薄层色谱法;电化学分析法;质谱法;液相色谱-质谱联用;折射率光谱法;紫外光谱法;荧光分析法;放射化学分析法;近红外光谱法;气相色谱法和光散射分析法;酶联免疫吸附测定(ELISA)、抗体连接、其它免疫化学技术、以及其组合。此外,可以间接地测量所述一种或多种生物标志物的水平,例如通过使用测量与期望被测量的生物标志物的水平相关的一种化合物(或多种化合物)的水平的测定。
质谱(MS)分析主要包括液相色谱-质谱联用(LC-MS)和气相色谱-质谱联用(GC-MS),液相色谱又分为超高效液相色谱(UPLC)和高效液相色谱(HPLC)非靶向性代谢组学的分析,色谱可选择与飞行时间(TOF)静电轨道阱(Orbitrap)离子阱飞行时间(IT-TOF)四极杆-飞行时间(Q-TOF)等质谱联用;靶向性代谢组学的分析,色谱可与三重四极杆(QQQ)或四极杆离子阱(Q-Trap)等质谱串联,用多反应监测器(MRM)进行检测。
诊断试剂盒
本发明提供了一种诊断动脉粥样硬化性脑梗死的试剂盒,所述试剂盒包括检测样本中本发明所述的生物标志物的试剂;并且可包括使用所述试剂盒评估受试者是否患有或易患动脉粥样硬化性脑梗死的说明书。
当在实验室环境中处理样本时,可能获得最可靠的结果。例如,可在医生办公室中从受试者获取样本,然后将其发送到医院或商业医学实验室进行进一步测试。然而,在许多情况下,可能希望在临床医生的办公室提供即时结果或允许受试者在家中进行测试。在一些情况下,对于便携式、预包装、一次性的、可由受试者在无协助或指导等的情况下即可使用等等的测试的需求比高度准确度更为重要。在许多情况下,尤其是在有医师随访的情况下,进行初步测试,甚至灵敏度和/或特异度降低的测试也可能就足够了。因此,以试剂盒形式提供的测定可涉及检测和测量相对少量的代谢物,以降低测定的复杂性和成本。
可使用本文所述的能够检测样本代谢物的任何形式的样本测定。通常,所述测定将定量样本中代谢物至一定的程度,例如它们的浓度或量是高于还是低于预定阈值。此类试剂盒可采取测试条、浸杆、盒、药筒、基于芯片或基于珠粒的阵列、多孔板或一系列容器等的形式。提供一种或多种试剂以检测所选样本代谢物的存在和/或浓度和/或量。可将受试者的样本直接分配到测定中,或从存储的或先前获得的样品中间接分配到测定中。高于或低于预定阈值的代谢物的存在或不存在可以例如通过发色、发荧光、电化学发光或其他输出(例如如在酶免疫测定(EIA),诸如酶联免疫测定(ELISA)中)来显示。
在一个实施方案中,试剂盒可包含固体基片诸如芯片、载玻片、阵列等,其具有能够检测和/或定量固定在基片上的预定位置处的一种或多种样本代谢物的试剂。作为说明性实例,可向芯片提供固定在离散的预定位置的试剂,以用于检测和定量样本中生物标志物的存在和/或浓度和/或量。如上所述,在患有动脉粥样硬化性脑梗死的受试者的样本中发现所述生物标志物的水平降低。芯片可被配置成使得仅当这些代谢物中的一种或多种的浓度超过阈值时才提供可检测的输出(例如颜色变化),所述阈值被选择或区分指示对照受试者的生物标志物的浓度和/或量与指示患有或易患动脉粥样硬化性脑梗死的患者的生物标志物的浓度和/或量。因此,可检测到的输出(诸如颜色变化)的存在立即表明样本中包含显著降低水平的生物标志物,表明受试者患有或易患动脉粥样硬化性脑梗死。
当比较两个不同群体(例如,一个患病而另一个不患病)的测试结果时,很少观察到两组之间的完美分离。实际上,测试结果的分布会重叠,因此,当选择并应用区分两个群体的截断点或标准值时,在一些情况下该疾病会正确分类为阳性(真阳性分数),但一些疾病病例将归类为阴性(假阴性分数)。另一方面,一些没有疾病的病例将被正确地分类为阴性(真阴性分数),而一些没有疾病的病例将被分类为阳性(假阳性分数)。
可使用诸如ROC曲线分析之类的工具来评估这样的测试的性能,或测试将疾病组与健康组区分开的准确度。ROC曲线是使用灵敏度作为y轴,将1-特异性作为x轴,使用各种截断值生成的灵敏度和特异度谱图的图形表示。在ROC曲线中,针对不同的截断点绘制了真阳性率(灵敏度)与FP率(100-特异性)的函数关系。ROC曲线上的每个点代表对应于特定决策阈值的灵敏度/特异性对。具有完美判别力的测试(两个分布中没有重叠)的ROC曲线穿过左上角(灵敏度为100%,特异度为100%)。因此,从质量上讲,曲线图越靠近左上角,测试的总体准确度越高。ROC曲线(AUC)下面积反映了测试的准确度,并显示在曲线图的左下角。
药剂和/或物理治疗和/或手术治疗的疗效评估
代谢组学分析是鉴定和评估潜在药剂和/或新的物理和/或手术治疗对动脉粥样硬化性脑梗死的效果的理想选择。通过本文之前所述的方法,可在治疗之前和之后从受试者采集样本一次或多次。治疗可包括以一种或多种剂量向或对受试者施用一种或多种药剂,和/或向或对受试者进行一种或多种物理和/或手术治疗。可以以多种不同方式进行药剂施用,包括但不限于注射、口服施用、贴剂或软膏剂敷用。
可以将从样本获得的代谢物谱相互比较和/或与动脉粥样硬化的受试者的代谢物谱进行比较。所述比较可以通过受试者样本中代谢物谱的变化来指示药剂和/或物理治疗和/或手术治疗的功效。
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法,通常按照常规条件。
实施例动脉粥样硬化脑梗死相关代谢物的筛选及效能判断
1、样本收集
收集21例动脉粥样硬化脑梗死患者及21例动脉粥样硬化患者的血液样本。
动脉粥样硬化脑梗死组纳入标准:
1)受试者已签署知情同意书
2)符合《中国急性缺血性脑卒中诊疗指南(2014版)》急性脑梗死诊断标准。
3)病因分型为动脉粥样硬化型脑梗死。
4)年龄18-65周岁。
5)BMI 18.5-23.9kg/m2。
6)血常规:红细胞计数、MCHC、血红蛋白、白细胞计数、淋巴细胞计数、中性粒细胞计数、单核细胞计数在正常范围。
7)TG、TC、HDL-C、LDL-C、血糖、糖化血红蛋白在正常范围。
排除标准:
1)合并其他疾病:神经系统疾病(既往脑梗死、脑出血、多发性硬化等);各种慢性消化系统疾病,3个月内患有急性消化系统疾病;循环系统疾病(冠心病、心力衰竭、房颤);呼吸系统疾病(慢性阻塞性肺疾病、慢性支气管炎、哮喘);代谢性疾病(肥胖、高脂血症、糖尿病、代谢综合征、骨质疏松症);泌尿系统疾病(慢性肾脏病、肾衰竭、肾结石);血液系统疾病(贫血);其他(痛风、抑郁、精神疾病、慢性疲劳综合征、纤维肌痛症、食物过敏、肿瘤)。
2)既往有输血史、消化系统疾病手术史及外伤史。
3)心电图异常的患者。
4)3个月内服用以下药物:抗生素、泻药、氯硝西泮、性激素类药物、口服避孕药、美沙拉嗪、TNF-α抑制剂、免疫抑制剂、抗抑郁药、PPI、卢帕他定、阿片类、钙剂、维生素D、二甲双胍、叶酸、β-交感神经吸入剂、中药。
5)3个月内服用益生菌制剂。
6)本次发病前应用抗血小板及他汀类药物。
7)进行静脉溶栓和血管内介入治疗的患者。
8)妊娠期或哺乳期妇女。
9)本研究期间,患者已入选或计划入选另一项临床药物或装置/干预性研究。
动脉粥样硬化组纳入标准:
1)受试者已签署知情同意书。
2)颈部血管超声和/或颈部血管影像学表现为颅内外血管动脉粥样硬化。
3)年龄18-65周岁。
4)BMI 18.5-23.9kg/m2。
5)血常规:红细胞计数、MCHC、血红蛋白、白细胞计数、淋巴细胞计数、中性粒细胞计数、单核细胞计数在正常范围。
6)TG、TC、HDL-C、LDL-C、血糖、糖化血红蛋白在正常范围。
排除标准:
1)存在其他疾病:神经系统疾病(脑梗死、脑出血、多发性硬化等);各种慢性消化系统疾病,3个月内患有急性消化系统疾病;循环系统疾病(冠心病、心力衰竭、房颤);呼吸系统疾病(慢性阻塞性肺疾病、慢性支气管炎、哮喘);代谢性疾病(肥胖、高脂血症、糖尿病、代谢综合征、骨质疏松症);泌尿系统疾病(慢性肾脏病、肾衰竭、肾结石);血液系统疾病(贫血);其他(痛风、抑郁、精神疾病、慢性疲劳综合征、纤维肌痛症、食物过敏、肿瘤)。
2)既往有输血史、消化系统疾病手术史及外伤史。
3)心电图异常。
4)3个月内服用以下药物:抗生素、泻药、氯硝西泮、性激素类药物、口服避孕药、美沙拉嗪、TNF-α抑制剂、免疫抑制剂、抗抑郁药、PPI、卢帕他定、阿片类、钙剂、维生素D、二甲双胍、叶酸、β-交感神经吸入剂、中药、抗血小板药物及他汀类药物。
5)3个月内服用益生菌制剂。
6)妊娠或哺乳期妇女。
7)本研究期间,受试者已入选或计划入选另一项临床药物或装置/干预性研究。
2、非靶向代谢组学检测
2.1血清样本制备
2.1.1反相色谱分析血清样本处理方法
1)血浆/血清样本在4℃的冰融化为30-60min。
2)取40μl血清至标记好标签的1.5ml离心管中,加入300μl的甲醇和1ml甲基叔丁基醚。
3)充分振荡15s,进行蛋白沉淀。12000rpm,4℃,离心10min,取上层溶液100μl,置于200μl内衬管中,待测。
2.1.2亲水色谱分析血清样本处理方法:
1)血浆/血清样本在4℃的冰融化为30-60min。
2)取50μl血清至标记好标签的1.5ml离心管中,加入150μl的乙腈。
3)充分振荡15s,进行蛋白沉淀。12000rpm,4℃,离心10min,取上层溶液100μl,置于200μl内衬管中,待测。
2.2色谱条件
色谱分离采用Thermo Scientific的U3000快速液相色谱使用反相色谱和亲水色谱对血清样本进行分析。
2.2.1反相色谱分离条件
色谱柱:waters UPLC HSS T3(1.8μm 2.1mm*100mm);
流动相:A(乙腈/水4:6,0.1%甲酸,10mM乙酸铵)和B(乙腈/异丙醇9:1,0.1%甲酸,10mM乙酸铵);
洗脱程序:见表1;
流速:0.3ml/min;
进样量为1.0μL;
柱温:50℃。
表1 C18反相色谱测定洗脱程序
2.2.1亲水色谱分离条件
色谱柱:waters UPLC BEH Amide(1.7μm 2.1mm*100mm);
流动相:A(乙腈,0.1%甲酸,10mM乙酸铵)和B(水,0.1%甲酸,10mM乙酸铵);
洗脱程序:见表2;
流速:0.3ml/min;
进样量:1.0μL;
柱温:40℃。
表2 HILIC测定极性小分子洗脱程序
2.3质谱条件
质谱分析采用装备了热电喷雾离子源的四极杆轨道离子阱质谱仪。正负离子离子源电压分别为3.7kv和3.5kV。毛细管加热温度320℃。翘气压力30psi,辅助气压力10psi。容积加热蒸发温度300℃。翘气和辅助气均为氮气。碰撞气为氮气,压力为1.5mTorr。一级全扫描参数为:分辨率70000,自动增益控制目标为1×106,最大隔离时间50ms,质荷比扫描范围50-1500。液质系统由Xcalibur 2.2SP1.48软件控制,数据采集和靶向代谢物定量处理均由该软件操作。
3、靶向代谢组学检测
3.1血清样本处理方法
1)血浆样本于4℃放置30min解冻。
2)取50μl血浆样本至1.5ml离心管中,加入150μl的甲醇(含有吲哚乙酸-D2500ppb,吲哚丙酸-D2 50ppb组成),旋涡震荡30min。
3)12000rpm离心5min,取上清液100μl,置于200μl内衬管中,待测。
3.2色谱条件
色谱分离采用Waters ACQUITY UPLC I-CLASS超高压液相色谱系统,色谱分离条件如下:
色谱柱:Waters UPLC BEH C8(1.7μm 2.1mm*100mm);
流动相:A(水,0.5Mm NH4F)和B(甲醇);
洗脱梯度:见表3;
流速:0.3ml/min;
进样量:1.0μL;
柱温:45℃。
表3洗脱程序
3.3质谱条件
质谱分析仪器为Waters公司XEVO TQ-XS型串联四级杆质谱仪。正离子离子源电压为3kv,锥孔电压为20V。去溶剂温度550℃,源温度150℃。去溶剂气流速1000L/Hr,锥孔气流速7L/h。
3.4靶向代谢组数据处理
靶向代谢组数据峰面积计算采用masslynx定量软件,保留时间允许误差15s。浓度计算采用单点同位素内标法获取定量结果。
4、数据处理
4.1数据质控
为了评价样品采集过程中系统的稳定性和重复性,使用了质控样本。质控样本是所有样本均移取固定体积混合均匀后得到的。指控样本的前处理方法和其他样品一样。为了得到可信赖的且可重复性的代谢物,三个因素需要考虑:1)保留时间,2)信号强度,3)质量准确度。本次实验首先采用5个空白样本平衡色谱柱,再采用3个质控样本平衡柱条件。然后每间隔6-8个样本插入1个质控样本用于监测整个液质系统的稳定性和重复性。同时计算质控样本中提取的代谢特征的变异系数值,变异系数超过15%的代谢特征被删除。
4.2 PCA分析
所有采集好的数据,无论是何种分离模式或是正负离子模式,均采用ProgenesisQI软件处理,包括的步骤依次为导入原始数据、峰对齐、峰提取、归一化处理,最终形成保留时间、质荷比和峰强度的表格。反相色谱和亲水色谱提取峰的时间依次为1至16和1至12min。各种添加剂离子如加氢和加钠等均去卷积到每一个离子特征。代谢物鉴定采用人类代谢组数据库和脂质数据库进行一级分子量匹配。
4.3 OPLS-DA分析
为了获得在动脉粥样硬化性脑梗死组(BL)和在动脉粥样硬化组(AS)组呈现显著差异的代谢物信息,进一步采用监督性的多维统计方法即偏最小二乘方判别分析(OPLS-DA)对两组样本进行统计分析。
采用OPLS-DA模型的VIP(Variable Importance in the Projection)值(阈值>1),并结合t-test的p值(p<0.05)来寻找差异性表达代谢物。差异性代谢物的定性方法为:搜索在线数据库(HMDB)(比较质谱的质荷比m/z或者精确分子质量mass,误差限制0.01Da)。
4.4 ROC分析
根据代谢物的水平,绘制受试者工作特征曲线(ROC),计算二项精确置信空间,分析差异代谢物的诊断效能。
5、结果
质控结果显示,质控样本相对聚集在一起,系统重复性较好,所采集的数据可以进行进一步的研究。
反向色谱正离子、反向色谱负离子、亲水色谱正离子的结果分别如表4和图1所示。
表4 OPLS-DA分析模型参数
生物信息学分析结果显示,与动脉粥样硬化组相比,PC(20:0/18:1(11Z))在动脉粥样硬化脑梗死组的水平显著降低(图2)。
以PC(20:0/18:1(11Z))的含量作为检测变量判断诊断效能,结果显示,曲线下面积为0.900,截断值为2747218.938,敏感性为0.810,特异性为1.000(图3),具有较高的敏感性和特异性以及准确性。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
Claims (7)
1.检测样本中生物标志物PC(20:0/18:1(11Z))的试剂在制备诊断动脉粥样硬化性脑梗死的产品中的应用,所述样本选自血液、血浆或血清。
2.根据权利要求1所述的应用,其特征在于,所述产品包括用于色谱法、光谱法、质谱法、化学分析法检测所述生物标志物的试剂。
3.根据权利要求2所述的应用,其特征在于,所述产品包括质谱法联用色谱法检测所述生物标志物的试剂。
4.根据权利要求1所述的应用,其特征在于,所述产品包括使用所述产品评估受试者是否患有或易患动脉粥样硬化性脑梗死的说明书。
5.根据权利要求1所述的应用,其特征在于,所述产品还包括处理样本的试剂。
6.一种系统,其特征在于,包括:
(1)动脉粥样硬化性脑梗死评估装置,其包括控制单元和存储单元,用于评估受试者是否患有动脉粥样硬化脑梗死;和
(2)彼此通信地连接的信息通信终端装置,其提供关于来自受试者的样本中的生物标志物PC(20:0/18:1(11Z))的存在和/或浓度和/或数量的数据;
其中所述动脉粥样硬化性脑梗死评估装置的控制单元包括:
(1)数据接收单元,其接收从所述信息通信终端设备传输的关于所述样本的所述生物标志物的浓度和/或量的数据;
(2)判别值计算单元,其基于由所述数据接收单元接收的所述样本中所述生物标志物的浓度和/或数量以及具有存储在所述存储单元中的作为解释变量的所述生物标志物的浓度和/或量的判别来计算判别值;
(3)判别值基准评价单元,其基于由所述判别值计算单元计算的判别值,对所述受试者中的动脉粥样硬化性脑梗死的情况进行评价;以及
(4)评估结果发送单元,其将由所述判别值基准评估单元获得的所述受试者的评估结果发送到所述信息通信终端装置;
其中,所述样本选自血液、血浆或血清。
7.生物标志物PC(20:0/18:1(11Z))在制备鉴定和评估药剂和/或手术治疗和/或物理治疗对抗动脉粥样硬化性脑梗死的效果的产品中的应用,其特征在于,鉴定和评估步骤包括:
(1)收集患有动脉粥样硬化性脑梗死的受试者提供第一样本;
(2)从所述第一样本获得生物标志物PC(20:0/18:1(11Z))的第一代谢物谱,其中所述第一代谢物谱是使用质谱法获得的;
(3)对所述受试者或在所述受试者上施用一种或多种候选药物和/或进行一种或多种物理或手术治疗;
(4)提供来自步骤(3)中的所述受试者的第二样本;
(5)从所述第二样本中获得第二代谢物谱,其中所述第二代谢物谱是通过质谱法获得的;
(6)将步骤(2)和(5)中获得的所述代谢物谱与参考代谢物谱进行比较;以及
(7)基于步骤(6)中的所述比较,评估所述一种或多种候选药物和/或治疗是否有效对抗动脉粥样硬化性脑梗死;
其中,所述第一样本和第二样本选自血液、血浆或血清。
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