CN116699146A - 动脉性肺动脉高压的诊断及危险分层的生物标志物及其应用 - Google Patents
动脉性肺动脉高压的诊断及危险分层的生物标志物及其应用 Download PDFInfo
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Abstract
本申请研究并发现了尿液中白细胞介素‑18结合蛋白和干扰素αβ受体蛋白及其组合可作为动脉性肺动脉高压的诊断生物标志物;本申请还发现了动脉性肺动脉高压中高危患者相较于低危患者,其尿液中白细胞介素‑18结合蛋白和干扰素αβ受体蛋白及其组合表达升高,从而提供了一种动脉性肺动脉高压的无创诊断和危险分层的新方法。
Description
技术领域
本发明属于疾病诊断试剂领域,具体涉及尿液中动脉性肺动脉高压的蛋白标志物及其在诊断及危险分层中的用途。
背景技术
肺高血压是一种高病死率的心血管疾病,其定义为在海平面、静息状态下患者的平均肺动脉压>20mmHg1。2022欧洲心脏病学会(ESC)指南根据病因将肺高血压分为5类,其中第一类为动脉性肺动脉高压,具体病因又包括特发性、遗传性、药物和毒物相关、结缔组织病及先天性心脏病等疾病相关的、对钙通道阻滞剂有反应的、具有明显肺静脉/肺毛细血管受累的以及新生儿持续性肺动脉高压1。据报道,成年人动脉性肺动脉高压的患病率约为每百万人中48-55例1,其病理生理特征为进行性肺血管重构和右室后负荷持续升高,进而出现右心肥厚和右心衰竭,右室功能是动脉性肺动脉高压患者不良预后的主要决定因素,未经治疗的患者中位生存期仅2.8年,目前即使应用相关治疗药物,5年生存率仅为57% 2,3。
动脉性肺动脉高压的常见临床症状,如乏力、头晕、呼吸困难、和下肢水肿,对疾病诊断的特异性不高,部分患者多次就医仍被漏诊和误诊,2021年《中国肺动脉高压患者生存现状白皮书》报道肺动脉高压患者从出现症状至确诊所需年限平均为2.2年,从确诊到起始治疗所需年限平均为2.6年,部分患者确诊时病情已进展为终末期,因此急需一种微创便捷的检测指标能够提示患者患有此类疾病,提高诊断能力、实现早诊早治对于改善患者长期预后至关重要。此外对于高度疑诊动脉性肺动脉高压的患者,需进行有创的右心漂浮导管检查以明确诊断,并每半年复查,收集相关参数明确患者危险分层,以调整治疗方案及评估预后,但是有创的右心导管操作费用较高、有手术并发症风险,也会给患者带来一定程度的不适。
目前临床广泛采用的体液标志物仅有血清NT-proBNP 4,但它对患者的诊断特异性及预后判断价值有限。其他标志物如肌钙蛋白T、CA-125、miR-21和长非编码RNAH19也曾被报道与右心衰竭相关,但它们的临床价值需要进一步验证5-8。
白细胞介素-18 (IL-18)是一种促炎细胞因子,在肺动脉高压患者外周血中表达增加9,但血清IL-18与本申请研究的白细胞介素-18结合蛋白不是同一物质,且本研究的动脉性肺动脉高压按2022 ESC指南的分类为第1类肺高血压,另外血清采样是有创性的。
《血清IL-18水平与慢性左心疾病相关性肺动脉高压患者近期预后的关系》报道了:慢性左心疾病相关性肺动脉高压(PH-LHD)患者血清高IL-18水平与近期预后差有关10,但血清IL-18与白细胞介素-18结合蛋白不是同一物质,慢性左心疾病相关性肺动脉高压按2022 ESC指南的分类为第2类肺高血压,本申请研究的动脉性肺动脉高压为第1类,二者为不同疾病,且血清采样是有创性的。
类似的,在系统性硬化症相关肺动脉高压患者血清中,干扰素-α可能影响促纤维化过程并导致肺动脉高压11,干扰素-α与本申请研究的干扰素αβ受体蛋白不是同一物质,且血清采样是有创性的。
I型干扰素在系统性硬化症相关肺动脉高压患者血清中表达增加,I型干扰素受体在患者肺组织中表达增加,且与疾病严重程度密切相关12,系统性硬化症相关肺动脉高压仅是动脉性肺动脉高压的一小部分,I型干扰素与本申请研究的干扰素αβ受体蛋白不是同一物质,且血清采样是有创性的。I型干扰素受体是在患者肺组织中检验的而不是在体液中。
寻找新的无创采样的生物标志物,且能在右室功能保留时早期诊断动脉性肺动脉高压,并鉴别出不良预后的高危患者至关重要。尿液中白细胞介素-18结合蛋白和干扰素αβ受体蛋白含量是否与动脉性肺动脉高压有相关性、以及是否能对该疾病的危险分层进行预测,目前尚无任何报道和应用,尿液具有无创的采样特点,且能在疾病早期甚至出现临床表现之前即发生分子水平的变化,这两种蛋白如果能成为动脉性肺动脉高压的检测指标,将极大方便该疾病的筛查和早诊早治。
参考文献
1. Humbert M, Kovacs G, Hoeper MM, et al. 2022 ESC/ERS Guidelines forthe diagnosis and treatment of pulmonary hypertension. Eur Respir J. 2023;61(1):2200879.
2. Vonk Noordegraaf A, Chin KM, Haddad F, Hassoun PM, Hemnes AR,Hopkins SR, et al. Pathophysiology of the right ventricle and of thepulmonary circulation in pulmonary hypertension: an update. Eur Respir J.2019;53(1).
3. 中华医学会呼吸病学分会肺栓塞与肺血管病学组,中国医师协会呼吸医师分会肺栓塞与肺血管病工作委员会,全国肺栓塞与肺血管病防治协作组,等.中国肺动脉高压诊断与治疗指南(2021版)[J].中华医学杂志, 2021, 101(1) : 11-51.
4. Fijalkowska A, Kurzyna M, Torbicki A, Szewczyk G, Florczyk M,Pruszczyk P, et al. Serum N-terminal brain natriuretic peptide as aprognostic parameter in patients with pulmonary hypertension. Chest. 2006;129(5):1313-21.
5. Filusch A, Giannitsis E, Katus HA, Meyer FJ. High-sensitivetroponin T: a novel biomarker for prognosis and disease severity in patientswith pulmonary arterial hypertension. Clin Sci (Lond). 2010;119(5):207-13.
6. Yilmaz MB, Zorlu A, Dogan OT, Karahan O, Tandogan I, Akkurt I.Role of CA-125 in identification of right ventricular failure in chronicobstructive pulmonary disease. Clin Cardiol. 2011;34(4):244-8.
7. Reddy S, Hu DQ, Zhao M, Blay E, Jr., Sandeep N, Ong SG, et al.miR-21 is associated with fibrosis and right ventricular failure. JCIInsight. 2017;2(9).
8. Omura J, Habbout K, Shimauchi T, Wu WH, Breuils-Bonnet S, TremblayE, et al. Identification of Long Noncoding RNA H19 as a New Biomarker andTherapeutic Target in Right Ventricular Failure in Pulmonary ArterialHypertension. Circulation. 2020;142(15):1464-84.
9. Ross DJ, Strieter RM, Fishbein MC, Ardehali A, Belperio JA. Type Iimmune response cytokine-chemokine cascade is associated with pulmonaryarterial hypertension. J Heart Lung Transplant. 2012;31(8):865-73.
10. 石金铮,耿鹤群,李月琴,等.血清IL-18水平与慢性左心疾病相关
性肺动脉高压患者近期预后的关系[J].山东医药, 2020, 60(27):3.DOI:10.3969/j.issn.1002-266X.2020.27.022.
11. Eloranta ML, Franck-Larsson K, Lövgren T, Kalamajski S, RönnblomA, Rubin K, et al. Type I interferon system activation and association withdisease manifestations in systemic sclerosis. Ann Rheum Dis. 2010;69(7):1396-402.
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发明内容
发明所要解决的问题:
鉴于目前临床诊断上尚没有特异和无创的动脉性肺动脉高压诊断用生物标志物,本研究基于质谱的定量蛋白组学和ELISA方法,在野百合碱致肺动脉高压大鼠模型和动脉性肺动脉高压患者的尿液中发现了一组全新的、能较好区分健康对照和动脉性肺动脉高压患者、以及患者不同危险分层的蛋白标志物,包括白细胞介素-18结合蛋白和干扰素αβ受体蛋白。本申请研究了尿液中白细胞介素-18结合蛋白和干扰素αβ受体蛋白及其组合作为动脉性肺动脉高压的诊断和危险分层生物标志物及其应用,提供了一种动脉性肺动脉高压的无创诊断和危险分层的新方法。
一、动脉性肺动脉高压的诊断和危险分层
在具体的实施方案中,诊断是指动脉性肺动脉高压的诊断,危险分层是指动脉性肺动脉高压低危与中高危患者的鉴别及动脉性肺动脉高压大鼠右心功能失代偿期与代偿期的鉴别。
人动脉性肺动脉高压的诊断标准包括海平面、静息状态下平均肺动脉压≥25mmHg且肺毛细血管楔压≤15mmHg。
大鼠的动脉性肺动脉高压的诊断标准:大鼠右心导管检查右室收缩压及平均肺动脉压水平升高,超声心动图检查示三尖瓣收缩期位移及心排量显著下降。
人动脉性肺动脉高压的危险分层标准见表1
表1 2015年ESC/ERS指南的简化的人动脉性肺动脉高压的危险分层标准
预后因素 | 低危 | 中危 | 高危 |
I:WHO分级 | I,II | III | IV |
II:6分钟步行试验 | >440m | 165~440m | <165m |
III:NT-proBNP/BNP或右心房压(RAP) | BNP <50ng/L,NT-proBNP <300ng/L or RAP < 8mmHg | BNP 50~300ng/L,NT-proBNP 300~1400ng/LorRAP 8~14mmHg | BNP >300ng/L,NT-proBNP>1400ng/L or RAP >14mmHg |
IV:心指数(CI)或静脉血氧饱和度(SvO2) | CI ≥2.5L/min/m2 orSvO2>65% | CI 2.0~2.4L/min/m2orSvO2 60%~65% | CI < 2.0 L/min/m2orSvO2<60% |
根据表1,动脉性肺动脉高压低危患者是指至少满足3个低危标准且无高危标准,其他归为中高危患者。
二、动脉性肺动脉高压模型大鼠的尿液中相关蛋白的筛选
用野百合碱造动脉性肺动脉高压大鼠模型,收集模型大鼠的尿液样品及富集尿蛋白,进行蛋白酶解,构建文库,样品分别用1D-LC-MS/MS分析,数据非依赖检测结果显示白细胞介素-18结合蛋白及干扰素αβ受体蛋白为显著差异蛋白。
质谱定量检测尿液中白细胞介素-18结合蛋白,干扰素αβ受体蛋白,结果显示:
两种蛋白组合,动脉性肺动脉高压模型大鼠与对照组AUC分析结果为0.954,动脉性肺动脉高压右心失代偿期大鼠与右心代偿期大鼠AUC分析结果为0.817。
上述结果表明:模型大鼠尿液中白细胞介素-18结合蛋白,干扰素αβ受体蛋白两种组合可用于动脉性肺动脉高压的诊断和危险分层,可制成诊断和危险分层的试剂或芯片。
说明:上述质谱定量检测是基于蛋白质特征性标签肽段的质谱检测。标签肽是指能够代表某一个蛋白的肽段,其特征在于存在且特异性仅存在于某蛋白质氨基酸序列中。
三、动脉性肺动脉高压患者尿液中相关蛋白的筛选
用酶联免疫吸附试验(如采用双抗体夹心酶联免疫吸附试验)在人尿液中筛选动脉性肺动脉高压相关蛋白质,结果显示:
白细胞介素-18结合蛋白单独用于动脉性肺动脉高压患者的诊断和危险分层的AUC分别为0.872,0.764;干扰素αβ受体蛋白单独用于动脉性肺动脉高压患者的诊断和危险分层的AUC分别为0.876,0.801;两种蛋白组合,动脉性肺动脉高压患者与对照组AUC分析结果为0.935,动脉性肺动脉高压患者中高危组与低危组AUC分析结果为0.861。
上述结果表明:
相较于健康对照,人尿液中白细胞介素-18结合蛋白、干扰素αβ受体蛋白任意一种的表达升高或其组合的表达升高指示受试者患有动脉性肺动脉高压,可制成诊断试剂或芯片,两种蛋白组合显著优于单用任意一种蛋白。
相较于低危患者,人尿液中白细胞介素-18结合蛋白、干扰素αβ受体蛋白任意一种的表达升高或其组合的表达升高指示患者为中高危患者,可制成危险分层试剂或芯片,两种蛋白组合显著优于单用任意一种蛋白。
说明:健康对照是指未患有动脉性肺动脉高压的个体。
基于上述技术方案,本发明具有以下有益效果:采用基于蛋白质特征性标签肽段的质谱检测或酶联免疫吸附试验定量检测方法检测尿液中白细胞介素-18结合蛋白和干扰素αβ受体蛋白,可以结合标准品建立人尿液中这两种蛋白的基线,通过与正常对照组的含量范围比较,对动脉性肺动脉高压患者进行诊断,通过不同危险分层组的含量范围比较,对动脉性肺动脉高压患者进行危险分层,从而提供了一种动脉性肺动脉高压的无创诊断及危险分层新方法。
附图说明
图1、数据非依赖采集模式下尿液蛋白组区分动脉性肺动脉高压大鼠和正常对照组PLS-DA图;
图2、数据非依赖采集模式下,质谱检测大鼠尿液白细胞介素-18结合蛋白(左)、干扰素αβ受体蛋白(右)的含量变化;
图3、平行反应监测模式下大鼠尿液白细胞介素-18结合蛋白标签肽段SSKDPC[Carbamidomethyl (C)]SSWSPAVPTK(左)、干扰素αβ受体蛋白标签肽段ETYVPIIVVHR(右)的含量变化,白细胞介素-18结合蛋白标签肽段序列如序列SEQ ID NO .1所示,干扰素αβ受体蛋白标签肽段序列如序列SEQ ID NO .2所示 ;
图4、两种蛋白质组合预测大鼠动脉性肺动脉高压的ROC曲线,其中,
A、两种蛋白质组合预测大鼠动脉性肺动脉高压的ROC曲线;
B、两种蛋白质组合预测大鼠右心失代偿期动脉性肺动脉高压的ROC曲线;
图5、ELISA检测人尿液白细胞介素-18结合蛋白、干扰素αβ受体蛋白的含量变化;
图6、人白介素-18结合蛋白预测动脉性肺动脉高压及危险分层的ROC曲线,其中,
A、人白介素-18结合蛋白预测动脉性肺动脉高压的ROC曲线;
B、人白介素-18结合蛋白预测动脉性肺动脉高压危险分层的ROC曲线;
图7、人干扰素αβ受体蛋白预测动脉性肺动脉高压及危险分层的ROC曲线,其中,
A、人干扰素αβ受体蛋白预测动脉性肺动脉高压的ROC曲线
B、人干扰素αβ受体蛋白预测动脉性肺动脉高压危险分层的ROC曲线;
图8、两种尿液蛋白质组合预测人动脉性肺动脉高压及危险分层的ROC曲线,其中,
A、两种蛋白质组合预测动脉性肺动脉高压的ROC曲线;
B、两种蛋白质组合预测中高危动脉性肺动脉高压的ROC曲线。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
下述实施例所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所有的材料、试剂等,如无特殊说明,均可从商业途径获得。
实施例1尿液中动脉性肺动脉高压疾病相关蛋白检测
材料与试剂
1)仪器:Orbitrap Exploris 480质谱仪(Thermo Scientific公司)。
2)主要试剂:胰蛋白酶(Promega公司);C18固相萃取小柱(3CC, 60 mg, Waters公司);C18反相色谱柱(4.6 mm×250 mm, C18, 3 μm, Waters 公司)。
3)样本:13例野百合碱诱导的动脉性肺动脉高压大鼠(8例右心功能代偿期,5例右心功能失代偿期)的尿液和8例正常对照大鼠的尿液,为雄性Sprague–Dawley大鼠。
我们用数据非依赖性采集方法(Data independent acquisition, DIA)在尿液中筛选动脉性肺动脉高压相关蛋白质。
1、大鼠尿液样品的收集及尿蛋白的富集
代谢笼过夜收集大鼠尿液,5000g的转速离心30min,去除沉淀。上清用乙醇沉淀,4度过夜以析出蛋白质。将蛋白沉淀用裂解液复溶。采用Bradford方法测量收集到的尿蛋白的浓度。蛋白样品用SDS-PAGE分析。
2、蛋白酶解
用膜上酶切方法进行蛋白酶解。蛋白样品先用20mM DTT还原95℃ 5min,再用50mMIAA对暴露的巯基进行烷基化,室温避光反应45min。将还原烷基化蛋白液转移至30KD超滤管中,离心弃掉下层废液。膜上蛋白样品用UA溶液(含8M尿素)清洗两次,并用25mM碳酸氢铵溶液清洗两次。膜上蛋白样品用1:50胰酶37℃过夜酶解,离心收集酶解后多肽。酶切后多肽溶液用C18萃取柱纯化,真空抽干纯化产物。复溶后多肽用BCA试剂盒进行多肽浓度测定。
3、构建文库
为了构建谱图库,将所有尿液样品(疾病组及对照组)等量混合。并进行离线高pH高效液相色谱分离,收集的洗脱液置于旋转真空干燥仪中,真空抽干后复溶于1‰甲酸中进行LC-MS/MS分析。每一个组分的样品用数据依赖性采集方法(DDA, Data dependentacquisition)方式采集,采集到的原始数据用Proteome Discoverer(Thermo Scientific,Germany)软件检索,检索条件如下:酶切方式:胰蛋白酶;容许最多两个漏切位点;固定修饰:半胱氨酸烷基化;可变修饰:天冬氨酸和谷氨酰胺脱氨化,甲硫氨酸氧化;赖氨酸和多肽N端氨甲酰化;母离子质量误差:10ppm;子离子质量误差:0.02Da。蛋白水平FDR<1%,每个蛋白至少含有一个unique肽。检索结果导入Spectronaut Pulsar (Biognosys,Switzerland)软件产生数据库。
4、DIA分析实验数据:
21例样品分别用1D-LC-MS/MS分析。每个样品DIA方式进行数据采集。DIA采集的数据用Spectronaut软件处理。检索上述构建的谱图库,搜索参数同上。导出数据结果。以1.5倍以上变化和p<0.05作为筛选条件,白细胞介素-18结合蛋白及干扰素αβ受体蛋白为显著差异蛋白(图2)。
实施例2尿液中白细胞介素-18结合蛋白,干扰素αβ受体蛋白的质谱检测
材料与试剂
1)仪器:Orbitrap Fusion Lumos Tribrid质谱仪(Thermo Scientific公司)。
2)主要试剂:胰蛋白酶(Promega公司);C18固相萃取小柱(3CC, 60 mg, Waters公司)。
3)样本:10例野百合碱诱导的动脉性肺动脉高压大鼠(5例右心功能代偿期,5例右心功能失代偿期)的尿液和8例正常对照大鼠的尿液,为雄性Sprague–Dawley大鼠。
为后面更好地对这些差异蛋白质组学分析结果进行应用检测,我们对于鉴定到的差异蛋白质,采用靶向质谱分析方法对差异蛋白质进行定量分析。平行反应监测是一种基于二级质谱信号的靶标质谱定量分析技术,相较于传统的选择反应监测技术,不需要预先设计靶向蛋白质的母离子/子离子的配对信息,节约实验设计和操作时间;且选择性更高,灵敏度更佳,重现性更好,在复杂背景中的抗干扰能力更强。其相较于免疫方法,不再受制于商业化抗体,克服了基于免疫方法中抗体特异性和滴度的限制。
1、样品制备
10例动脉性肺动脉高压大鼠的尿液和8例正常对照组的尿液,分别取4ml尿液进行胰蛋白酶酶解。酶解后样本用BCA法进行多肽浓度测定。每个样品取10.5μg(0.5μg/μL)加入1.5μL iRT标准肽。并取等量样本制备混合样。
2、PRM多肽筛选
选取待验证的差异蛋白进行PRM验证。用Skyline 3.6软件进行PRM多肽筛选。先用混合多肽进行筛选。每个差异蛋白有约3个标签肽段作为候选,筛选在尿液蛋白质组数据库中有较好谱图的,且在混合样品中能够鉴定到的或具有较高信噪比峰的多肽进行后续定量分析。
3、PRM检测
对2中筛选到的可用于PRM验证的标签多肽进行PRM分析,包括白细胞介素-18结合蛋白标签肽段SSKDPC[Carbamidomethyl (C)]SSWSPAVPTK(SEQ ID NO .1)及干扰素αβ受体蛋白标签肽段ETYVPIIVVHR(SEQ ID NO .2)。对上述18例样本分别验证,每个样本用schedule模式对待验证的多肽进行分析。为保证数据质量,在所有样品上样之前和上样之后,以及每5-6个样品之间,各进行一次混合样品的分析作为质量控制,观察整个分析过程中,仪器信号的稳定性。为保证数据质量,每个样品中加入iRT标准肽分析,观察分析过程中,色谱保留时间的稳定性。每个样品进行两次技术重复分析。不同组样品打乱顺序穿插进行质谱分析,以减少系统误差。
4、PRM数据分析
用Skyline 3.6软件进行PRM数据分析。所有结果导入到Skyline软件中,人工挑选正确的峰,将所有样品的所有多肽结果导出。用Progenesis软件提取每个样品的+2~+5电荷的总离子流强度(TIC)。将每个样品的每个多肽的质谱结果用该样品的总离子流强度均一化,校正上样量和质谱信号强度的误差。对每个多肽结果进行定量分析,筛选不同组之间的差异蛋白,并与数据非依赖检测结果进行比较。用Metaboanalyst进行受试者操作特征曲线(receiver operating characteristic,ROC)分析,筛选出能够具有高灵敏度和特异性的生物标志物或标志物组合。
结果表明,两种蛋白的定量结果与数据非依赖检测结果一致,且具有较高的灵敏度和特异性(图2,3)。我们将这两种蛋白进行ROC分析,整合结果显示两种蛋白组合动脉性肺动脉高压大鼠与对照组AUC分析结果为0.954,动脉性肺动脉高压失代偿期大鼠与代偿期大鼠AUC分析结果为0.817(图4)。
实施例3尿液中白细胞介素-18结合蛋白,干扰素αβ受体蛋白的ELISA检测
材料与试剂
1)主要试剂:人白细胞介素-18结合蛋白ELISA试剂盒(睿信生物,泉州,中国),人干扰素αβ受体蛋白ELISA试剂盒(睿信生物,泉州,中国)。
2)样本:58例动脉性肺动脉高压患者(31例低危患者,27例中高危患者)的尿液和22例正常对照受试者的尿液,来自北京协和医院。
我们用酶联免疫吸附试验在人尿液中筛选动脉性肺动脉高压相关蛋白质。
1、人尿液样品的收集及尿蛋白的富集
收集空腹晨尿,1000g的转速离心20min,去除沉淀。取上清进行后续检测。
2、酶联免疫吸附试验
设置标准品孔、0值孔、空白孔和样本孔,标准品孔各加不同浓度的标准品50μL,0值孔加样本稀释液50μL,空白孔不加,样本孔加待测样本50μL。除空白孔外,标准品孔、0值孔和样本孔,加入辣根过氧化物酶(HRP)标记的检测抗体100μL。用封板膜盖住反应板,37℃水浴锅或恒温箱避光孵育60min。使用自动洗板机洗板5次,充分拍干反应板。将底物A和B按1:1体积充分混合,所有孔中加入底物混合液100μL。用封板膜盖住反应板,37℃水浴锅或恒温箱避光孵育15min。所有孔加入终止液50μL,在酶标仪上读取各孔吸光度(OD值)。
3、数据处理
以标准品浓度做为横坐标(6个标准品孔,加1个0值孔,共7个浓度点),对应的吸光度(OD值)作为纵坐标,利用计算机软件,采用四参数Logistic曲线拟合,创建标准曲线方程,通过样本的吸光度(OD值),利用方程计算样品的浓度值。
进而,我们对其进行进一步ROC分析,从而判断这两种蛋白质区分动脉性肺动脉高压患者和对照组,以及中高危患者与低危患者的能力。结果显示,这两种蛋白单独使用的AUC均大于0.75,白细胞介素-18结合蛋白单独用于动脉性肺动脉高压患者的诊断和危险分层的AUC分别为0.872,0.764(图6);干扰素αβ受体蛋白单独用于动脉性肺动脉高压患者的诊断和危险分层的AUC分别为0.876,0.801(图7)。
进而,我们将这两种蛋白的结果进行整合分析。我们整合结果显示两种蛋白组合用于动脉性肺动脉高压患者与对照组AUC分析结果为0.935,动脉性肺动脉高压患者中高危组与低危组AUC分析结果为0.861(图8)。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (8)
1.一组尿液中的蛋白质在制备动脉性肺动脉高压诊断和危险分层的试剂及芯片中的应用,其特征在于,所述尿液中的蛋白质包括白细胞介素-18结合蛋白、干扰素αβ受体蛋白中的任意一种或两种组合。
2.根据权利要求1所述的应用,其特征在于,所述危险分层是指大鼠右心功能失代偿期和代偿期的鉴别,以及动脉性肺动脉高压中高危和低危患者之间的鉴别。
3.根据权利要求1所述的应用,其特征在于,相较于健康对照,所述白细胞介素-18结合蛋白、干扰素αβ受体蛋白中的任意一种或两种组合在动脉性肺动脉高压患者尿液中表达升高。
4.根据权利要求1所述的应用,其特征在于,相较于低危患者,所述尿液中白细胞介素-18结合蛋白、干扰素αβ受体蛋白中的任意一种或两种组合表达升高,指示受试者动脉性肺动脉高压危险分层为中高危。
5.根据权利要求1-4任意一项所述的应用,其特征在于,鉴定和定量检测所述的白细胞介素-18结合蛋白、干扰素αβ受体蛋白的试剂为质谱鉴定标签肽段或酶联免疫吸附法试剂盒。
6.根据权利要求5所述的应用,其特征在于,所述的质谱鉴定标签肽段是指在数据非依赖质谱采集和平行反应监控质谱采集模式中使用。
7.根据权利要求5所述的应用,其特征在于,所述酶联免疫吸附法试剂盒采用双抗体夹心酶联免疫吸附试验进行蛋白鉴定。
8.根据权利要求1所述的应用,其特征在于,所述白细胞介素-18结合蛋白、干扰素αβ受体蛋白来源于大鼠或人的尿液样本。
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