CN113791221A - 一种检测急性主动脉夹层的生物标志物及其应用 - Google Patents
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Abstract
本发明公开了一种检测急性主动脉夹层的生物标志物及其应用,属于医药学技术领域。所述生物标志物,包括血清淀粉样蛋白A、富含亮氨酸的α‑2‑糖蛋白和C‑反应蛋白中至少一种;还公开了根据所述的检测急性主动脉夹层的生物标志物在制备区分急性主动脉夹层患者与正常人诊断试剂盒或者诊断试剂中的应用;根据所述的检测急性主动脉夹层的生物标志物在制备区分急性主动脉夹层患者与正常人检测试剂盒或者检测试剂中的应用。该生物标志物通过对急性主动脉夹层患者血清差异表达蛋白筛选获得,可以用于区分急性主动脉夹层患者与正常人,为急性主动脉夹层提供血清标志物,为AAD的临床早期诊断及预后评估提供科学依据。
Description
技术领域
本发明涉及医药学技术领域,特别是涉及一种检测急性主动脉夹层的生物标志物及其应用。
背景技术
急性主动脉夹层(acute aortic dissection,AAD)作为一种严重威胁生命的心血管急症,出现症状后每小时未经治疗的死亡率为1%-2%。现今的临床诊疗过程中,对AAD的诊断主要依靠影像学技术,但其缺乏简便性。虽然目前在AAD的诊断和治疗方面已经取得了进展,但在患者到达医院之前由于不可逆的血管损伤导致的病死率仍然很高,因此缩减从症状出现到接受诊疗的时间对AAD死亡率的减低至关重要。血清学检查作为一种简单、客观、灵敏、经济的检查手段能用以早期准确诊断AAD而成为临床的重要检查手段被广泛应用。
近年来报道的AAD生物标志物主要集中在血管平滑肌细胞损伤、细胞外基质损伤、炎症因子及凝血和纤溶系统激活。如D-二聚体、平滑肌肌球蛋白重链等。然而,由于灵敏度和特异度不高使得这些标志物的广泛应用受到限制,故需寻找可靠的潜在的血清标志物。
研究表明TGF-β/Smad信号转导通路的激活、基质金属蛋白酶(MatrixMetalloproteinases,MMPs)过表达引起细胞外基质的合成与降解失衡、血管平滑肌细胞(Vascular SmoothMuscle Cells,VSMCs)表型转换异常以及系统性炎症反应(TNF-α、IL-6、CRP等)与AAD的发生密切相关。但炎症因子在AAD发生发展过程中的诊断和预测作用尚未完全阐明,需进一步探究。
因此,为了缩减从症状出现到接受诊疗的时间,对AAD发病过程中炎症因子进行分析和测定,对于寻找可靠的血清标志物是非常有必要的。
发明内容
本发明的目的是提供一种检测急性主动脉夹层的生物标志物及其应用,以解决上述现有技术存在的问题,该生物标志物通过对急性主动脉夹层患者血清差异表达蛋白筛选获得,可以用于区分急性主动脉夹层患者与正常人,为急性主动脉夹层提供血清标志物,为AAD的临床早期诊断及预后评估提供科学依据。
为实现上述目的,本发明提供了如下方案:
本发明提供检测急性主动脉夹层的生物标志物,包括血清淀粉样蛋白A、富含亮氨酸的α-2-糖蛋白和C-反应蛋白中至少一种。
本发明还提供根据所述的检测急性主动脉夹层的生物标志物在制备区分急性主动脉夹层患者与正常人诊断试剂盒或者诊断试剂中的应用。
本发明还提供根据所述的检测急性主动脉夹层的生物标志物在制备区分急性主动脉夹层患者与正常人检测试剂盒或者检测试剂中的应用。
优选的是,所述区分急性主动脉夹层患者与正常人的方法包括如下步骤:
(1)收集待测患者的血清样品,并以正常人血清样品作为对照;
(2)检测待测患者和正常人的血清样品中生物标志物的含量,并将所得结果进行比对;
(3)根据比对结果指示待测患者是否为急性主动脉夹层患者;
其中,利用血清淀粉样蛋白A、富含亮氨酸的α-2-糖蛋白和C-反应蛋白三种生物标志物中的至少一种对所述待测对象血清样品进行检测时,若待测对象血清样品中所用的生物标志物的含量高于对照,则指示所述待测对象为急性主动脉夹层。
优选的是,采用ELISA方法检测血清样品中生物标志物的含量。
本发明公开了以下技术效果:
本发明将新型AAD血清标志物结合经典的已证实的血清标志物进行分析组合,筛选其中最佳的标志物组合。通过筛选急性主动脉夹层患者早期诊断的血清标志物,对AAD早期诊断、远期预后及生活质量的改善具有较大的临床价值和实际意义。所筛选的三个蛋白可能参与AAD的发病,并可作为AAD的诊断生物标志物和新的治疗靶点,这为临床急性主动脉夹层早期诊断和预后评估提供了新的方向。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为SAA、LRG1、CRP 3个因子分别作为生物标志物在正常对照组与急性主动脉夹层组中表达水平浓度数值;
图2为SAA、LRG1、CRP3个因子分别作为生物标志物在正常对照组与急性主动脉夹层组进行对比分析的ROC曲线;
图3为SAA+LRG1在正常对照组与急性主动脉夹层组进行对比分析的ROC联合曲线;
图4为SAA+CRP在正常对照组与急性主动脉夹层组进行对比分析的ROC联合曲线;
图5为LRG1+CRP在正常对照组与急性主动脉夹层组进行对比分析的ROC联合曲线;
图6为SAA+LRG1+CRP在正常对照组与急性主动脉夹层组进行对比分析的ROC联合曲线。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1急性主动脉夹层患者血清差异表达蛋白筛选
根据性别和年龄随机分组,分别选取正常对照者及急性主动脉夹层患者各10例,随后采用非标记定量蛋白质组学技术(Label-free quantification,LFQ)分析两组患者升主动脉组织标本的蛋白质谱的变化,筛选差异表达蛋白并对其生物学功能进行分析。具体方法如下:
(1)蛋白提取
样品从-80℃取出,4℃,12000g离心10min,去除细胞碎片,上清液转移至新的离心管,用Thermo公司生产的试剂盒参照PierceTMTop 12Abundant Protein Depletion SpinColumns Kit说明书去除高丰度蛋白。利用BCA试剂盒进行蛋白浓度测定。
(2)胰蛋白酶酶解
蛋白溶液中加入二硫苏糖醇使其终浓度为5mM,56℃还原30min。之后加入碘代乙酰胺使其终浓度为11mM,室温避光孵育15min。以1:50的质量比例(胰酶:蛋白)加入胰酶,37℃酶解过夜。再以1:100的质量比例(胰酶:蛋白)加入胰酶,继续酶解4h。
(3)HPLC分级
肽段用高pH反向HPLC分级,色谱柱为Agilent 300Extend C18(5μm粒径,4.6mm内径,250mm长)。操作如下:肽段分级梯度为8%-32%乙腈、pH 9,60min时间分离60个组分,随后肽段合并为若干个组分,合并后的组分经真空冷冻干燥后进行后续操作。
(4)液相色谱-质谱联用(LC-MS/MS)分析
肽段用液相色谱流动相A相溶解后使用NanoElute超高效液相系统进行分离。随后肽段被注入Capillary离子源中进行电离然后进tims-TOF Pro质谱进行分析。肽段母离子及其二级碎片都使用TOF进行检测和分析。二级质谱扫描范围设置为100-1700m/z。数据采集模式使用平行累积串行碎裂(PASEF)模式。
(5)数据库搜索
二级质谱数据使用Maxquant(v1.6.6.0)进行检索。检索参数设置:数据库为Homo_sapiens_9606_SP_20191115(20380条序列),添加了反库以计算随机匹配造成的假阳性率(FDR),并且在数据库中加入了常见的污染库,用于消除鉴定结果中污染蛋白的影响;该数据库具有严格的胰蛋白酶特异性,允许最多2个缺失的切割。所需的最小肽段长度设定为7个氨基酸。
(6)生物信息学分析
在GO富集分析过程中,通过GO注释将蛋白分为生物进程、细胞组成、分子功能3大类。Kyoto Encyclopedia of Genes and Genomes(KEGG)数据库用于通路的富集分析,最后根据KEGG网站(http://www.genome.jp/kegg/)通路层级分类方法将这些通路进行分类。InterPro(http://www.ebi.ac.uk/interpro/)数据库用于分析差异表达蛋白的功能结构域的富集情况。在三种功能富集分析(GO,KEGG途径和蛋白质结构域)中,采用Fisher’s精确双端检验比较差异表达的蛋白质与所有蛋白质的富集,使用标准FDR对照方法对多种假设检验进行了校正,校正后的P<0.05被认为显著富集。
(7)通过分析采用4D-LFQ技术筛选出86个差异蛋白的生物进程、细胞组成和分子功能及通路富集,选取与AAD发生机制密切相关的3个炎症蛋白进一步扩大样本验证。
(8)统计结果
上述三种炎症蛋白在正常对照者及急性主动脉夹层患者中存在显著差异,差异具有统计学意义(见表1)
表1采用4D-LFQ技术筛选的血清中炎症反应相关蛋白
实施例2急性主动脉夹层患者血清标志物扩大样本验证
根据性别和年龄随机分组,选取79个健康人作为对照,急性主动脉夹层患者145例,进行ELISA实验验证上述3种蛋白因子能否判断患者患有主动脉夹层。实验步骤如下:
(1)检测前准备工作
①提前1h从冰箱中取出试剂盒、样本,平衡至室温。
②20×洗涤缓冲液的稀释:蒸馏水按1:20稀释,即1份20×洗涤缓冲液加19份蒸馏水。
③实验所需试剂及耗材:
a.ELISA试剂盒,如表2:
表2ELISA试剂盒组成
试剂盒组成 | 96孔配置 | 48孔配置 | 备注 |
微孔酶标板 | 8孔×12条 | 8孔×6条 | 无 |
标准品 | 0.3mL×6管 | 0.3mL×6管 | 无 |
样本稀释液 | 6mL | 3mL | 无 |
检测抗体-HRP | 10mL | 5mL | 无 |
20×洗涤缓冲液 | 25mL | 15mL | 按说明书进行稀释 |
底物A | 6mL | 3mL | 无 |
底物B | 6mL | 3mL | 无 |
终止液 | 6mL | 3mL | 无 |
封板膜 | 2张 | 2张 | 无 |
说明书 | 1份 | 1份 | 无 |
自封袋 | 1个 | 1个 | 无 |
b.所需自备实验器材:酶标仪(450nm);高精度加样器及枪头:0.5-10μL、2-20μL、20-200μL、200-1000μL;37℃恒温箱;蒸馏水或去离子水。
c.操作步骤
a)从室温平衡20min后的铝箔袋中取出所需板条,剩余板条用自封袋密封放回4℃。
b)设置标准品孔和样本孔,标准品孔各加不同浓度的标准品50μL;
c)样本孔中加入待测样本50μL;空白孔不加。
d)除空白孔外,标准品孔和样本孔中每孔加入辣根过氧化物酶(HRP)标记的检测抗体100μL,用封板膜封住反应孔,37℃水浴锅或恒温箱温育60min。
e)弃去液体,吸水纸上拍干,每孔加满洗涤液(350μL),静置1min,甩去洗涤液,吸水纸上拍干,如此重复洗板5次(也可用洗板机洗板)。
f)每孔加入底物A、B各50μL,37℃避光孵育15min。
g)每孔加入终止液50μL,15min内,在450nm波长处测定各孔的OD值。
d.通过上述ELISA方法,以SAA、LRG1、CRP、SAA+LRG1、SAA+CRP、LRG1+CRP、SAA+LRG1+CRP分别作为生物标志物对急性主动脉夹层组和正常组进行分析检测,所得数据采用SPSS26.0统计学软件进行分析并绘制受试者工作特征(ROC)曲线,明确新型标志物能否成为新的AAD诊断标志物应用于临床,同时探索联合指标对AAD诊断价值。
(2)结果分析
如图1所示,该图为SAA、LRG1、CRP 3个因子作为生物标志物在正常对照组和急性主动脉夹层组中表达水平浓度数值,结果显示:3个因子均能够很好的区分健康人与急性主动脉夹层患者。
如图2和表3所示,SAA、LRG1、CRP 3个因子作为生物标志物在区分正常对照组和急性主动脉夹层组时的ROC曲线,结果显示:3个因子的曲线下面积均大于0.7,对急性主动脉夹层具有很好的诊断价值。
表3 3个因子用于区分健康人和急性主动脉夹层患者时的ROC统计结果
图3-图6为不同蛋白因子组合后确定最佳诊断组合蛋白因子,结果显示通过各个因子的联合作用的ROC统计分析,确定区分急性主动脉夹层和正常对照组的诊断组合。经过进一步分析,确定最佳的区分急性主动脉夹层和健康人的诊断因子组合为SAA+LRG1+CRP(见表4)。
表4最佳因子组合用于区分健康人和急性主动脉夹层患者时的ROC统计结果
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (5)
1.检测急性主动脉夹层的生物标志物,其特征在于,包括血清淀粉样蛋白A、富含亮氨酸的α-2-糖蛋白和C-反应蛋白中至少一种。
2.根据权利要求1所述的检测急性主动脉夹层的生物标志物在制备区分急性主动脉夹层患者与正常人诊断试剂盒或者诊断试剂中的应用。
3.根据权利要求1所述的检测急性主动脉夹层的生物标志物在制备区分急性主动脉夹层患者与正常人检测试剂盒或者检测试剂中的应用。
4.根据权利要求2或者3所述的应用,其特征在于,所述区分急性主动脉夹层患者与正常人的方法包括如下步骤:
(1)收集待测患者的血清样品,并以正常人血清样品作为对照;
(2)检测待测患者和正常人的血清样品中生物标志物的含量,并将所得结果进行比对;
(3)根据比对结果指示待测患者是否为急性主动脉夹层患者;
其中,利用血清淀粉样蛋白A、富含亮氨酸的α-2-糖蛋白和C-反应蛋白三种生物标志物中的至少一种对所述待测对象血清样品进行检测时,若待测对象血清样品中所用的生物标志物的含量高于对照,则指示所述待测对象为急性主动脉夹层。
5.根据权利要求4所述的应用,其特征在于,采用ELISA方法检测血清样品中生物标志物的含量。
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