WO2022247047A1 - 一种用于瓣膜性心脏病辅助诊断的新型标志物、辅助诊断的产品 - Google Patents
一种用于瓣膜性心脏病辅助诊断的新型标志物、辅助诊断的产品 Download PDFInfo
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- heart disease
- valvular heart
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
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Definitions
- the invention belongs to the technical field of biomedicine, and in particular refers to a novel marker for auxiliary diagnosis of valvular heart disease and a product for auxiliary diagnosis.
- Valvular Heart Disease refers to abnormal anatomical structure and function of one or more valves caused by congenital developmental malformations or acquired lesions, leading to valve stenosis and/or insufficiency, mainly including aortic valve stenosis ( AS) or regurgitation (AR), mitral stenosis (MS) or regurgitation (MR), etc.
- AS aortic valve stenosis
- AR regurgitation
- MS mitral stenosis
- MR regurgitation
- Valvular heart disease is a common cardiovascular disease that endangers human life and health, and its incidence gradually increases with age. According to research reports, the incidence rate of young patients is low ( ⁇ 2%), the incidence rate of elderly patients is significantly increased, and the incidence rate of VHD over 75 years old rises to 13.2%. With the prolongation of population life and the aggravation of aging, the number of elderly VHD patients in my country is gradually increasing. Due to the poor defense and adaptability of the body of elderly patients, they are often accompanied by various complications. Therefore, the risk of treatment is high and the mortality rate is high. family and social burdens.
- VHD cardiovascular disease
- diagnosis of VHD is mainly based on cardiac color Doppler ultrasound. Although this method is the most intuitive, there may be blind spots, and severe emphysema, thoracic deformity, severe obesity and other factors will also affect the diagnosis, so other indicators are needed. auxiliary.
- BNP B-type natriuretic peptide
- Urokinase-type plasminogen activator receptor (uPAR, also known as PLAUR) is a glycoprotein with a molecular weight of 55-60kD, which consists of 313 amino acid residues and consists of 3 disulfide bonds.
- the three homologous folding regions are named DI, DII, and DIII in sequence from the N-terminus.
- PLAUR does not contain a transmembrane sequence, and its carboxy-terminus (located in the DIII region) is connected to a glycosylated phosphatidylinositol (GPI) anchor, which connects PLAUR to the surface of the phospholipid bilayer of the cell membrane, and its amino-terminus (located in the DI region) provides a binding site for urokinase-type plasminogen activator (uPA), which can bind to uPA with high affinity. Under inflammatory stimuli, PLAUR is shed from the cell membrane under the action of various proteins to form soluble urokinase-type plasminogen activator receptor (suPAR).
- GPI glycosylated phosphatidylinositol
- PLAUR was first known as an inflammatory marker.
- Studies in recent years have found that PLAUR is also closely related to the occurrence and development of cardiovascular diseases such as atherosclerosis and heart failure.
- PLAUR is highly expressed in macrophages, and there is a high concentration of PLAUR in atherosclerotic plaques. Therefore, it is speculated that PLAUR is related to the inflammatory response in atherosclerotic plaques, and the specific mechanism needs to be further studied.
- Another study showed that PLAUR was significantly positively correlated with NT-proBNP levels and the incidence of heart failure. Elevated PLAUR may represent the damage of microvascular circulation, resulting in impaired myocardial function, which is closely related to the occurrence of heart failure. At present, there is no report on the relationship between PLAUR and valvular heart disease in the world.
- the present invention is the first clinical study on serum PLAUR levels in valvular heart disease patients and healthy volunteers, and the results show that PLAUR can be used as a new biomarker for auxiliary diagnosis of valvular heart disease.
- the technical solution of the present invention proposes a novel marker-PLAUR for auxiliary diagnosis of valvular heart disease, and the marker is PLAUR in serum. Then, by using the reagent for detecting the level of PLAUR in serum, a product for assisting the diagnosis of valvular heart disease can be prepared.
- the product is a kit or a chip or a test strip, or some other testing products.
- Fig. 1 is a technical flow chart of the present invention
- Figure 2 is a graph showing the detection results of NT-ProBNP in serum of healthy volunteers and patients with valvular heart disease;
- Figure 3 is a graph showing the detection results of hsCRP levels in the serum of healthy volunteers and patients with valvular heart disease
- Fig. 4 is a graph showing the detection results of PLAUR in serum of healthy volunteers and patients with valvular heart disease.
- Example 1 Serum collection from healthy volunteers or patients with valvular heart disease
- Embodiment 2 Serum NT-ProBNP level detection
- Sample preparation Freshly separated serum can be tested directly; if it is a frozen sample, it needs to be thawed at room temperature and mixed thoroughly by low-speed vortex or inversion.
- NT-ProBNP level was detected according to the instructions of Brain Natural Peptide N-terminal Precursor Protein Assay Kit (chemiluminescent method) (New Industry, 130206004M).
- Sample preparation Freshly separated serum can be tested directly; if it is a frozen sample, it needs to be thawed at room temperature and mixed thoroughly by low-speed vortex or inversion.
- hsCRP level was detected according to the instructions of the C-reactive protein assay kit (chemiluminescent method) (New Industry, 130216002M).
- Embodiment 4 Detection of serum PLAUR level
- Sample preparation Freshly separated serum can be tested directly; if it is a frozen sample, it needs to be thawed and centrifuged again, and then the supernatant is taken for testing.
- PLAUR level detection The PLAUR level was detected according to the instructions of the human urokinase-type plasminogen activator receptor (PLAUR/UPAR) enzyme-linked immunosorbent assay kit (CUSABIO, CSB-E04752h).
- PLAUR/UPAR human urokinase-type plasminogen activator receptor
- MI mitral regurgitation
- AI aortic regurgitation
- TI tricuspid regurgitation
- MS mitral stenosis
- RHD rheumatic heart disease
- AF atrial fibrillation
- AS aortic valve narrow.
- the present invention discovers for the first time a new biomarker PLAUR that can be used as an auxiliary diagnosis of valvular heart disease. It has the advantage of fast detection speed, and the result can be obtained in only one working day. At the same time, the sensitivity/accuracy of detection is much higher Based on NT-ProBNP and hsCRP, and can achieve high-throughput operation.
- the detection of PLAUR level in serum can be used alone as a new diagnostic basis for valvular heart disease, and its accuracy is higher than that of existing markers; of course, it can also be combined with original biomarkers such as B-type sodium Urinary peptides can be used to comprehensively diagnose valvular heart disease based on multiple detection results, thereby improving the diagnostic accuracy of valvular heart disease.
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Abstract
一种用于瓣膜性心脏病辅助诊断的新型标志物、辅助诊断的产品，其中可作为辅助诊断瓣膜性心脏病的新型生物标志物为PLAUR，具有检测速度快的优点，仅需一个工作日即可拿到结果，同时检测的敏感性、准确率远高于NT-ProBNP和hsCRP，并可实现高通量操作。
Description
本发明创造属于生物医药技术领域，特别是指一种用于瓣膜性心脏病辅助诊断的新型标志物、及辅助诊断的产品。
瓣膜性心脏病(Valvular Heart Disease，VHD)是指先天发育畸形或后天病变引起的一个或多个瓣膜解剖结构和功能异常，导致瓣膜口狭窄和(或)关闭不全，主要包括主动脉瓣狭窄(AS)或关闭不全(AR)、二尖瓣狭窄(MS)或关闭不全(MR)等。
心脏瓣膜的狭窄或不全一旦发生，会直接影响到心脏内正常的血液流动，增加心脏负担，造成心脏功能的损害，最终导致患者心力衰竭而死亡。瓣膜性心脏病作为一类常见的危害人类生命和健康的心血管病，其发病率随着年龄增长逐渐增加。根据研究报道，年轻患者发病率低(<2％)，老年患者发病率显著加，75岁以上VHD发病率升至13.2％。随着人口寿命的延长和老龄化的加剧，我国老年VHD患者逐渐增加，又由于老年患者机体防御和适应能力差，常伴有各种并发症，因此治疗风险大、死亡率高，已成为重大的家庭和社会负担。
及时、准确、快速的诊断，尽早治疗，是控制VHD病程发展的最有效方式。目前，对于VHD的诊断主要以心脏彩超为主，虽然这种方式最为直观，但是有可能存在盲区，并且严重肺气肿、胸廓畸形、严重肥胖等因素也会影响诊断，因此需要其它指标来进行辅助。目前，被大家公认的VHD诊断的生物标志物只有B型钠尿肽(B-type Matriuretic Peptide，BNP)，其主要在心脏中合成，并受到心肌压力的调控，是目前使用最广泛的心肌病变标志物。欧洲心脏病协会(the European Society of Cardiology，ESC)和欧洲心胸外科协会(the European Association for Cardio-Thoracic Surgery，EACTS)发布的2017瓣膜性心脏病管理指南中，将血清BNP作为主动脉瓣狭窄(AS)和二尖瓣返流(MR)的严重程度和预后标志。但目前为止，对其阈值的定义尚不明确，也没有明确的使用建议。此外，其它的一些情况(如气道疾病、肥胖、房颤、肝硬化、年龄差异等)也会造成BNP水平的变化，因此限制了其在评估个体患者方面的应用。新型生物标志物的研发和应用将会给VHD的诊断带 来新的突破。
尿激酶型纤溶酶原激活物受体(urokinase-type plasminogen activator receptor，uPAR，亦称PLAUR)是一个分子质量为55-60kD的糖蛋白，包含313个氨基酸残基，通过二硫键组成3个同源折叠区域，从N端开始依次被命名为DI、DⅡ、DⅢ。PLAUR不含跨膜序列，其羧基末端(位于DⅢ区域中)连接一个糖基化磷脂酰肌醇(GPI)锚，GPI锚将PLAUR连接于细胞膜的磷脂双分子层表面，其氨基末端(位于DI区域中)则提供尿激酶型纤溶酶原激活物(uPA)的结合位点，可与uPA高亲和力结合。在炎症刺激下，PLAUR在多种蛋白作用下从细胞膜上脱落，形成可溶性尿激酶型纤溶酶原激活物受体(suPAR)。在不同的生理病理状态下，血清PLAUR浓度的高低可反映机体的炎症反应状态和免疫系统的激活程度。因此，PLAUR最早作为一种炎症标志物被熟知。近些年的研究发现，PLAUR还与动脉粥样硬化、心力衰竭等心血管疾病发生、发展密切相关。比如，PLAUR在巨噬细胞中高表达，在粥样斑块中存在高浓度PLAUR，因此推测PLAUR与动脉粥样斑块中的炎症反应有关，具体机制有待进一步研究。另有研究结果表明，PLAUR与NT-proBNP水平和心力衰竭发病率呈明显正相关。PLAUR升高可能代表了微血管循环的受损，从而导致心肌功能受损，这与心力衰竭的发生是密切相关的。目前世界上并没有PLAUR与瓣膜性心脏病相关的报道。
发明内容
本发明首次在瓣膜性心脏病病人和健康志愿者中进行的血清PLAUR水平的临床研究，结果表明，PLAUR可以作为一个新型生物标志物用于瓣膜性心脏病的辅助诊断。
研究过程如下：
1、用常规的，公认的方法采集健康志愿者或瓣膜性心脏病病人的血清/血浆。
2、用脑自然肽N端前体蛋白测定试剂盒(化学发光法)测定健康志愿者或瓣膜性心脏病病人血清NT-ProBNP水平。
3、用ELISA试剂盒检测健康志愿者或瓣膜性心脏病病人血清/血浆中PLAUR水平。
因此，本发明的技术方案提出一种用于瓣膜性心脏病辅助诊断的新型标志物-PLAUR，且该标志物为血清中的PLAUR。那么，利用检测血清中PLAUR水平的试剂，可制得一种辅助诊断瓣膜性心脏病的产品。所述产品为试剂盒或者芯片或者试纸，又或者其他的一些检测产品。
图1为本发明的技术流程图；
图2为健康志愿者和瓣膜性心脏病病人血清中NT-ProBNP检测结果图；
图3为健康志愿者和瓣膜性心脏病病人血清中hsCRP水平检测结果图；
图4为健康志愿者和瓣膜性心脏病病人血清中PLAUR检测结果图。
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合具体实施例进行详细描述。
本发明的技术流程如图1所示。
实施例1：健康志愿者或瓣膜性心脏病病人血清采集
1.1健康志愿者样招募：健康志愿者通过自愿的方式招募，并签订知情同意书。本次纳入研究的健康志愿者人数为27人，其中男性14人，女性13人。年龄分布为，21～30岁8人，31～40岁11人，41～50岁3人，51～60岁5人。
1.2瓣膜性心脏病病人收集:本次纳入研究的瓣膜性心脏病病人由广东省人民医院南海医院收治，并完成采血工作。本研究在患者知情并自愿签订知情同意书的情况下开展。此次研究的患者中男性23人，女性25人。年龄分布为，<20岁1人，31～40岁5人，41～50岁9人，51～60岁15人，61～70岁30人，>70岁3人。
1.3分离血清:将采血管于室温下1300g离心10min，收集上层血清至新的离心管中。收集的血清可以直接进行NT-ProBNP或PLAUR水平的检测，或者进行分装后于-80℃储存备用。
实施例2：血清NT-ProBNP水平检测
2.1：样品准备：新鲜分离的血清可以直接进行检测；若是冷冻保存的样本，需在室温环境下解冻后，通过低速涡旋或颠倒使其充分混匀。
2.2：NT-ProBNP水平检测：按照脑自然肽N端前体蛋白测定试剂盒(化学发光法)(新产业，130206004M)说明书对NT-ProBNP水平进行检测。
结果分析：如图2、表1所示，瓣膜性心脏病病人血清NT-ProBNP水平显著高于健康志愿者组，但仍有34.5％(20/58)的已确诊患者NT-ProBNP水平在正常范围，说明以NT-ProBNP水平作为瓣膜性心脏病生物标志物的敏感性/准确性不理想。因此，临床上仅用 NT-ProBNP这一个生物标志物来评价是不全面的，需要结合多个指标，才能做出准确的判断。
实施例3：血清hsCRP水平检测
3.1：样品准备：新鲜分离的血清可以直接进行检测；若是冷冻保存的样本，需在室温环境下解冻后，通过低速涡旋或颠倒使其充分混匀。
3.2：hsCRP水平检测：按照C反应蛋白测定试剂盒(化学发光法)(新产业，130216002M)说明书对hsCRP水平进行检测。
结果分析：如图3、表1所示，血清hsCRP水平在已确诊的瓣膜性心脏病病人中有31.0％(18/58)显示在正常范围，说明以hsCRP水平作为瓣膜性心脏病生物标志物的敏感性/准确性不理想。
实施例4：血清PLAUR水平检测
4.1：样品准备：新鲜分离的血清可以直接进行检测；若是冷冻保存的样本，需解冻后再次离心，然后取上清进行检测。
4.2：PLAUR水平检测：按照人尿激酶型纤溶酶原激活物受体(PLAUR/UPAR)酶联免疫试剂盒(CUSABIO，CSB-E04752h)说明书对PLAUR水平进行检测。
结果分析：如图4、表1所示，在健康志愿者中，健康志愿者血清PLAUR平均水平(1.05ng/mL)显著低于瓣膜性心脏病病人组，并且约44％的健康人中检测不到PLAUR。若以健康人群PLAUR平均值作为评价标准，在瓣膜性心脏病患者中仅有3.4％(2/58)的患者低于此标准。因此，PLAUR可以作为瓣膜性心脏病辅助诊断的生物标志物，并且其敏感性/准确率远高于NT-ProBNP和hsCRP。
表1瓣膜性心脏病病人血清PLAUR、NT-proBNP和hsCRP水平
注释：MI，二尖瓣关闭不全；AI，主动脉瓣关闭不全；TI，三尖瓣关闭不全；MS，二尖瓣狭窄；RHD，风湿性心脏病；AF，心房颤动；AS，主动脉瓣狭窄。
本发明首次发现可作为辅助诊断瓣膜性心脏病的新型生物标志物PLAUR，其具有检测速度快的优点，仅需1个工作日即可拿到结果，同时，检测的敏感性/准确率远高于NT-ProBNP和hsCRP，并可实现高通量操作。因此，血清中PLAUR水平检测可以单独作为一个新的瓣膜性心脏病的诊断依据，相对于现有的标志物，其准确度更高；当然，也可以结合原有的生物标志物如B型钠尿肽，根据检测的多个结果对瓣膜性心脏病进行综合诊断，从而提高瓣膜性心脏病的诊断准确率。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (5)
- 一种用于瓣膜性心脏病辅助诊断的新型标志物，其特征在于，该标志物为PLAUR。
- 根据权利要求1所述的一种用于瓣膜性心脏病辅助诊断的新型标志物，其特征在于，所述标志物为血清或血浆中的PLAUR。
- 一种辅助诊断瓣膜性心脏病的产品，其特征在于，包括用于检测PLAUR水平的试剂。
- 根据权利要求3所述的一种诊断瓣膜性心脏病的产品，其特征在于，检测对象为血清或血浆。
- 根据权利要求3所述的一种诊断瓣膜性心脏病的产品，其特征在于，所述产品为试剂盒或者芯片或者试纸。
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