CN113667740B - 分子标志物组合在诊断心血管疾病中的应用 - Google Patents
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Abstract
本发明涉及生物技术、生物医药领域,具体涉及分子标志物组合在诊断心血管疾病中的应用。本发明提供了一种诊断心血管疾病的标志物组合,所述标志物组合包括APBA2、MBD5和RFC2;同时,本发明还提供了检测所述标志物组合的试剂在制备诊断受试者是否患有心血管疾病的产品中的应用。优选地,所述心血管疾病是冠状动脉疾病。
Description
技术领域
本发明涉及生物技术、生物医药领域,具体涉及分子标志物组合在诊断心血管疾病中的应用。
背景技术
冠状动脉疾病是一种包含稳定型心绞痛、非稳定型心绞痛、心肌梗塞和猝死的疾病。当冠状动脉较大的分支完全或几乎完全堵塞时,相应的心肌因得不到血液的供应而坏死,就会发生心肌梗塞。心肌梗塞时有胸痛,性质与心绞痛相似,但更加剧烈。与心绞痛不同的是没有一定的诱发因素,疼痛持续时间较长,往往达几小时甚至1~2天,范围也较广,可波及前胸与中上腹部。严重的可有休克、心力衰竭、心律不齐、恶心、呕吐、发热等症状。心肌梗塞发生时有一系列特殊的心电图变化,同时血清谷氨酸-草酰乙酸转氨酶与肌酸磷酸激酶等含量增高,这两项检查对诊断急性心肌梗塞很有价值。冠状动脉硬化时,心肌缺血,心功能减退,甚至心力衰竭。此外,也常引起各种心律失常。如果患了冠状动脉疾病而不予治疗,动脉阻塞情况会日益严重。接着,供应心脏的血液会大量减少,会有心脏病发作的危险,而且可能因此死亡。不过,即使是在心脏病大发作之后,心脏还是可能恢复到相当健康的程度。但有时候由于心脏受到了严重的损伤,使得它的泵血功能减弱了,这会造成心力衰竭。
冠状动脉疾病好发于家族有冠状动脉病史、高血脂症、代谢症候群、高血压、糖尿病、抽烟、肥胖、缺乏运动、过度紧张及精神压力的人群。
目前常用的治疗手段包括:经皮冠状动脉扩张术及支架植入术,利用心导管,将气球充气于血管狭窄处;若狭窄依旧严重可植入支架,增加血管内径以及冠状动脉血流量;冠状动脉绕道术,以外科手术方式在狭窄血管位置附近,利用自体血管或人工血管移植,以建立一个新的血流路线,供应血流量到狭窄处以下的血管,改善心肌缺氧的状况,达到治疗的效果;药物治疗,主要是避免冠状动脉粥状硬化更趋恶化。
目前冠状动脉疾病趋于年轻化,有必要提早了解自身情况,加强锻炼,定期检查。所有筛选可临床应用的标志物具有较高的实用意义。
发明内容
发明人通过对数据库信息进行生物信息学分析,筛选到了可以在患者和健康对照体内差异表达的标志物,进一步对标志物进行组合得到了高效的诊断标志物组合,所述标志物组合在临床应用中有重要的意义。
标志物组合
一方面,本发明提供了一种诊断心血管疾病的标志物组合,所述标志物组合包括APBA2、MBD5和RFC2。
优选地,所述标志物组合选自以下任意一组:
(1)APBA2+MBD5+RFC2;
(2)APBA2+MBD5+RFC2+FKBP1A;
(3)APBA2+MBD5+LIN7A+RFC2。
本发明中,所述“+”代表联合使用。
优选地,所述心血管疾病包括动脉粥样硬化、冠状动脉疾病、外周动脉疾病、动脉瘤、血栓症、栓塞、心肌梗塞、局部缺血。
优选地,所述心血管疾病是冠状动脉疾病。
优选地,所述APBA2在患者中低表达。
优选地,所述MBD5在患者中高表达。
优选地,所述RFC2在患者中高表达。
优选地,所述FKBP1A在患者中高表达。
优选地,所述LIN7A在患者中高表达。
诊断疾病的应用
另一方面,本发明提供了检测前述标志物组合或前述标志物组合中任意一个标志物的表达量的试剂在制备诊断受试者是否患有心血管疾病的产品中的应用。
优选地,所述受试者为哺乳动物。例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物。
优选地,所述受试者为人。
优选地,所述检测表达量的试剂包括检测mRNA表达量和/或蛋白表达量的试剂。
优选地,所述检测包括定量或定性的检测。
优选地,所述检测mRNA表达量的试剂包括以下方法中使用到的试剂:基于PCR原理的检测方法、Southern杂交方法、Northern杂交方法、点杂交方法、荧光原位杂交方法、DNA微阵列方法、ASO法、高通量测序平台方法。
优选地,所述基于PCR原理的检测方法包括以下至少任意一种:将提取RNA的步骤、mRNA逆转录为cDNA的步骤、测量cDNA的含量的步骤。
优选地,所述测量cDNA的含量的方法包括但不限于PCR、NASBA、RPA、SDA、LAMP、HAD、NEAR、MDA、RCA、LCR、RAM。
优选地,所述检测mRNA表达量的试剂包括特异性引物和/或探针。
优选地,所述探针包括杂交型探针和水解型探针(Taqman探针)。
优选地,所述探针的两端连接有淬灭基团和/或荧光基团。
优选地,所述荧光基团包括但不限于FAM(羧基荧光素,Carboxy fluorescein,绿色荧光)、FITC(异硫氰酸荧光素,Fluorescein isothiocyanate)、TET(四氯-6-羧基荧光素,Tetrachloro fluorescein)、HEX(六氯-6-甲基荧光素,Hexachloro fluorescein)、JOE(2,7-二甲基-4,5-二氯-6-羧基荧光素)、罗丹明类(Rhodamine染料如R110,TAMRA、TexasRed等)、ROX、AlexaFluor染料、ATTO染料、DyLight染料、cyanine染料(如Cy2,Cy3,Cy3.5,Cy3b,Cy5,Cy5.5,Cy7,Cy7.5)、FluoProbes染料、SulfoCy染料、Seta染料、IRIS染料、SeTau染料、SRfluor染料、Square染料。
优选地,本文所述淬灭基团包括但不限于3'-BBQ-650(Black Berry Quencher650)、BHQ-0(Black Hole Quencher 0,3')、BHQ-1(Black Hole Quencher 1,3')、BHQ-2(Black Hole Quencher 2,3')、BHQ-3(Black Hole Quencher 3,3')、BHQ-1(Black HoleQuencher-1,5')、BHQ-2(Black Hole Quencher-2,5')、BHQ-3(Black Hole Quencher-3,5')、Atto 540Q、Atto 575Q、Atto 612Q。
优选地,所述检测蛋白表达量的试剂包括以下方法中使用的试剂:蛋白质印迹(Western Blot法)、酶联免疫吸附测定(ELISA)、放射性免疫测定(RIA)、夹心测定、免疫组织化学染色、质谱法、免疫沉淀分析法、补体结合分析法、流式细胞荧光分边技术和蛋白质芯片法。
优选地,所述检测蛋白表达量的试剂包括特异性抗体或其片段,所述特异性抗体或其片段能特异性的与蛋白结合。
优选地,所述检测蛋白表达量的试剂还包括二抗,所述二抗能与前述的特异性抗体或其片段结合并且显色。
优选地,所述显色通过显色剂体现,所述显色剂包括但不限于荧光染料(包括荧光分子)、化学发光标记物、荧光素酶、金属离子、生物素、放射性同位素、在UV光谱中进行吸收的分子、在近红外辐射中能够进行吸收的分子或远红外辐射中能够进行吸收的分子。
优选地,所述荧光染料包括但不限于罗丹明、对甲氨基酚、荧光素、硫代荧光素、氨基荧光素、羧基荧光素、氯代荧光素、甲基荧光素、磺基荧光素、氨基对甲氨基酚、羧基对甲氨基酚、氯代对甲氨基酚、甲基对甲氨基酚、磺基对甲氨基酚、氨基罗丹明、羧基罗丹明、氯代罗丹明、甲基罗丹明、磺基罗丹明、以及硫代罗丹明、菁、吲哚碳菁、氧杂碳菁、噻碳菁、部花青、菁染料、噁二唑衍生物、吡啶基噁唑、硝基苯噁二唑、苯并硝基苯、芘衍生物、瀑布蓝、噁嗪衍生物、尼罗红、尼罗蓝、甲酚紫、噁嗪170、吖淀衍生物、前黄素、吖啶橙、吖啶黄、芳基甲川衍生物、金胺、噻吨染料、磺化噻吨染料、亚历克萨荧光(AlexaFluor)、结晶紫、孔雀绿、四吡咯衍生物、卟啉、酞菁、胆红素、Cy5.5、吲哚菁绿(ICG)、DyLight750或IRdye800。
优选地,所述化学发光标记物包括但不限于过氧化物酶、碱性磷酸酶、荧光素酶、水母发光蛋白、官能化的铁-卟啉衍生物、鲁米那、鲁米诺、异鲁米诺、吖啶酯、磺酰胺等。
优选地,所述荧光素酶包括但不限于长腹水蚤(Gaussia)荧光素酶、海肾(Renilla)荧光素酶、腰鞭毛虫荧光素酶、萤火虫荧光素酶、真菌荧光素酶、细菌荧光素酶和弯喉萤(vargula)荧光素酶。
优选地,所述的抗体或其片段还可以直接与显色剂结合,检测到所述显色剂显示就表示蛋白有表达。
产品
另一方面本发明提供了一种诊断受试者是否患有心血管疾病试剂盒,所述试剂盒包括检测前述标志物组合或前述标志物组合中任意一个标志物的试剂。
优选地,所述试剂盒还包括检测基因表达量所使用的仪器。
优选地,所述试剂盒还包括检测其他疾病标志物的试剂和/或仪器。
另一方面本发明还提供了前述试剂盒在制备诊断心血管疾病的产品中的应用。
方法
另一方面本发明提供诊断受试者是否患有心血管疾病的方法,所述方法包括根据前述标志物组合或前述标志物组合中任意一个标志物的表达量的检测,得出诊断结果。
优选地,所述方法还包括检测受试者的前述标志物组合或前述标志物组合中任意一个标志物的表达量。
优选地,所述检测需要采集来自患者的样本。
进一步,所述方法包括:
(1)收集受试者样本;
(2)提取受试者样本中的RNA或蛋白,检测前述标志物组合或前述标志物组合中任意一个标志物的表达量;
(3)根据表达量的检测,得出诊断结果。
优选地,所述样本包括:血液、鼻上皮细胞、组织、尿液、唾液、精液、乳汁、脑脊髓液、泪液、痰、粘液、淋巴、胞液、腹水、胸膜积液、羊水、膀胱冲洗液和支气管肺泡灌洗液。
优选地,所述样本是血液。
附图说明
图1为在数据库GSE12288中差异基因的差异箱线图,A是FKBP1A、B是FN3KRP、C是LEF1、D是RFC2、E是LIN7A、F是TUT1、G是APBA2、H是MBD5。
图2为在数据库GSE20680中差异基因的差异箱线图,A是FKBP1A、B是FN3KRP、C是LEF1、D是RFC2、E是LIN7A、F是TUT1、G是APBA2、H是MBD5。
图3是APBA2_RFC2在数据库GSE12288中诊断的ROC曲线;纵坐标是敏感度,横坐标是特异性。
图4是MBD5+TUT1+FKBP1A在数据库GSE12288中诊断的ROC曲线;纵坐标是敏感度,横坐标是特异性。
图5是APBA2+MBD5+RFC2在数据库GSE12288中诊断的ROC曲线;纵坐标是敏感度,横坐标是特异性。
图6是APBA2+MBD5+RFC2+FKBP1A在数据库GSE12288中诊断的ROC曲线;纵坐标是敏感度,横坐标是特异性。
图7是APBA2+MBD5+LIN7A+RFC2在数据库GSE12288中诊断的ROC曲线;纵坐标是敏感度,横坐标是特异性。
图8是APBA2+LIN7A+FN3KRP+LEF1在数据库GSE12288中诊断的ROC曲线;纵坐标是敏感度,横坐标是特异性。
具体实施方式
下面结合实施例对本发明做进一步的说明,以下所述,仅是对本发明的较佳实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容,依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化,均落在本发明的保护范围内。
实施例1、筛选并验证标志物组合的诊断效果
下载数据库GSE12288(110患者+112对照)、GSE20680(143患者+52对照)的数据,使用R包进行差异表达分析,筛选在患者和对照中差异表达(p<0.05)的基因。对比对两个数据库的筛选结果,筛选在两个数据库中表达量变化一致的标志物,分析结果表明FKBP1A、FN3KRP、LEF1、RFC2、LIN7A、TUT1、APBA2、MBD5在两个数据库中的变化一致,差异分析如图1所示。
为提高诊断的准确性,对FKBP1A、FN3KRP、LEF1、RFC2、LIN7A、TUT1、APBA2、MBD5进行组合,各组合的ROC值及附图如下表1所示:
表1、标志物组合及其AUC值
标志物、标志物组合 | AUC | 附图 | 最佳阈值(特异度,灵敏度) |
FKBP1A | 0.576948052 | ||
FN3KRP | 0.581737013 | ||
LEF1 | 0.585957792 | ||
RFC2 | 0.592288961 | ||
LIN7A | 0.605519481 | ||
TUT1 | 0.610146104 | ||
APBA2 | 0.618506494 | ||
MBD5 | 0.63538961 | ||
APBA2+RFC2 | 0.654626623 | 3 | 0.532(0.718,0.571) |
MBD5+TUT1+FKBP1A | 0.62987013 | 4 | 0.547(0.727,0.509) |
APBA2+MBD5+RFC2 | 0.701298701 | 5 | 0.474(0.627,0.723) |
APBA2+MBD5+RFC2+FKBP1A | 0.710470779 | 6 | 0.471(0.600,0.723) |
APBA2+MBD5+LIN7A+RFC2 | 0.712987013 | 7 | 0.476(0.655,0.723) |
APBA2+LIN7A+FN3KRP+LEF1 | 0.640827922 | 8 | 0.510(0.636,0.598) |
根据表1数据分析可知APBA2+MBD5+RFC2三个标志物组合在一起的时候诊断ROC大于0.7,向以上三标志物组合中再加入其他标志物,可以继续提高AUC值。
Claims (2)
1.检测以下任一所述标志物组合的表达水平的试剂在制备诊断受试者是否患有冠状动脉疾病的产品中的应用:
(1)APBA2+MBD5+RFC2;
(2)APBA2+MBD5+RFC2+FKBP1A;
(3)APBA2+MBD5+LIN7A+RFC2;
所述表达水平为mRNA表达水平。
2.如权利要求1所述的应用,其特征在于,所述检测需要采集来自受试者的样品,所述样品是血液。
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