阅读说明：本技术 动脉硬化及动脉硬化相关疾病标志物 (Arteriosclerosis and arteriosclerosis-related disease marker ) 是由 南野直人 八木宽阳 锦织充広 植田初江 松田均 村上裕辅 于 2019-03-29 设计创作，主要内容包括：本发明的目的是提供用于检测动脉硬化相关疾病或用于评价动脉硬化进展程度的新型生物标志物。具体地,本发明涉及一种用于检测动脉硬化相关疾病或用于评价动脉硬化进展程度的标志物,其包含NPC2(Niemann-Pick disease type C2,尼曼-匹克病C2型)蛋白和/或IGFBP7(Insulin-like growth factor-binding protein7,胰岛素样生长因子结合蛋白7)蛋白。(It is an object of the present invention to provide novel biomarkers for detecting arteriosclerosis-associated diseases or for evaluating the degree of progression of arteriosclerosis. In particular, the present invention relates to a marker for detecting an arteriosclerosis-associated disease or for evaluating the degree of progression of arteriosclerosis, comprising an NPC2(Niemann-Pick disease type C2 ) protein and/or an IGFBP7(Insulin-like growth factor-binding protein7, Insulin-like growth factor-binding protein 7) protein.)

1. A method for detecting an arteriosclerosis-associated disorder, the method comprising: determining, in a sample obtained from a subject, the steps of 1) and/or 2) below:

1) NPC2(niemann-pick disease type C2) protein;

2) IGFBP7(insulin-like growth factor binding protein 7) protein.

2. A method for detecting or screening for recurrence of an arteriosclerosis-associated disease, the method comprising: determining the following steps 1) and/or 2) in a sample obtained from a subject at risk of recurrence of an arteriosclerosis-associated disease:

1) NPC2(niemann-pick disease type C2) protein;

2) IGFBP7(insulin-like growth factor binding protein 7) protein.

3. A method for detecting or screening a subject at risk of developing an arteriosclerosis-associated disease in the future, the method comprising: determining, in a sample obtained from a subject, the steps of 1) and/or 2) below:

1) NPC2(niemann-pick disease type C2) protein;

2) IGFBP7(insulin-like growth factor binding protein 7) protein.

4. A method for evaluating the effect of a prophylactic or therapeutic agent for arteriosclerosis-associated diseases or arteriosclerosis, which comprises:

a step of measuring the following 1) and/or 2) in a sample obtained from a subject in need of prevention or treatment of arteriosclerosis-associated diseases or arteriosclerosis, before administration of a preventive or therapeutic agent for arteriosclerosis-associated diseases or arteriosclerosis;

a step of determining the following 1) and/or 2) in a sample obtained from the subject after administration of an arteriosclerosis-associated disease or a preventive or therapeutic agent for arteriosclerosis;

alternatively, the method comprises: the step of determining the following 1) and/or 2) from samples obtained at two or more time points in chronological order from a subject in need of prevention or treatment of arteriosclerosis-associated diseases or arteriosclerosis after administration of a preventive or therapeutic agent for arteriosclerosis-associated diseases or arteriosclerosis:

1) NPC2(niemann-pick disease type C2) protein;

2) IGFBP7(insulin-like growth factor binding protein 7) protein.

5. The method of any one of claims 1-4, wherein the arteriosclerosis-associated disorder is selected from the group consisting of: myocardial infarction, peripheral arterial disease, aortic aneurysm, aortic dissection, atherosclerotic cerebral infarction, transient ischemic attack, renal artery stenosis, internal carotid artery stenosis, angina pectoris.

6. A method for assessing the degree of progression of arteriosclerosis, the method comprising: determining, in a sample obtained from a subject, the steps of 1) and/or 2) below:

1) NPC2(niemann-pick disease type C2) protein;

2) IGFBP7(insulin-like growth factor binding protein 7) protein.

7. The method of any one of claims 1 to 6, wherein the sample is selected from the group consisting of: serum, plasma, blood, urine and saliva.

8. The method according to any one of claims 1 to 7, wherein the subject is an animal in need of prevention or treatment of arteriosclerosis-associated diseases or arteriosclerosis, and the method evaluates or determines the effect of a prophylactic or therapeutic agent for arteriosclerosis-associated diseases or arteriosclerosis using the NPC2 protein and/or IGFBP7 protein as an index.

9. The method of any one of claims 1-8, wherein the subject is a human.

10. The method of any one of claims 1 to 9, wherein the assay is an immunoassay or mass spectrometry.

11. A marker for detecting an arteriosclerosis-associated disease, comprising the following 1) and/or 2):

1) NPC2 protein;

2) IGFBP7 protein.

12. The marker of claim 11, wherein the atherosclerosis-related disease is selected from the group consisting of: myocardial infarction, peripheral arterial disease, aortic aneurysm, aortic dissection, atherosclerotic cerebral infarction, transient ischemic attack, renal artery stenosis, internal carotid artery stenosis, angina pectoris.

13. A marker for evaluating the degree of progression of arteriosclerosis, comprising the following 1) and/or 2):

1) NPC2 protein;

2) IGFBP7 protein.

14. An assay reagent for detecting an arteriosclerosis-associated disease or for evaluating the degree of progression of arteriosclerosis, comprising an antibody or an aptamer for use in assaying the marker of any one of claims 11 to 13.

15. The assay reagent of claim 14, wherein the antibody is a monoclonal antibody and/or a polyclonal antibody.

16. A kit for detecting an arteriosclerosis-associated disease or for evaluating the degree of progression of arteriosclerosis, which comprises the measuring reagent according to claim 14 or 15.

17. The marker, assay reagent or kit according to any one of claims 11 to 16, wherein the sample used in detecting an arteriosclerosis-associated disease or in evaluating the degree of progression of arteriosclerosis is selected from the group consisting of: serum, plasma, blood, urine and saliva.

18. Use of a marker according to any one of claims 11 to 13 for screening a prophylactic or therapeutic drug for an arteriosclerosis-associated disease or arteriosclerosis.

19. Use of a marker according to any one of claims 11 to 13 for in vitro detection or screening for recurrence of an arteriosclerosis-associated disease.

20. Use of a marker according to any one of claims 11 to 13 for in vitro detection or screening of a subject at risk of developing an arteriosclerosis-associated disease in the future.

Technical Field

The present invention relates to the technical field of clinical diagnostic agents for aiding in the diagnosis of arteriosclerosis-associated diseases and arteriosclerosis. More specifically, the present invention relates to biomarkers for arteriosclerosis-related diseases, biomarkers that reflect the state of arteriosclerosis (hereinafter also referred to as "degree of progression"), measurement reagents and kits using the biomarkers, methods for detecting arteriosclerosis-related diseases, and methods for evaluating the degree of progression of arteriosclerosis.

Background

Arteriosclerosis is a general term for a state in which various substances are deposited inside an artery to thicken and harden the inner wall of the artery, thereby losing elasticity and narrowing the lumen. Although there are 3 types of atherosclerosis (hereinafter referred to as atherosclerosis), mesocalcified plaque, and arteriosclerotic disease, atherosclerosis generally refers to atherosclerosis in most cases. Atherosclerosis is arteriosclerosis caused in a relatively thick artery such as an aorta, a cerebral artery, and a coronary artery, and an atheromatous plaque (atherosclerotic lesion) is formed by accumulating a porridge-like substance composed of fat such as cholesterol in an intima of the artery, and the lumen of the artery is narrowed by the gradual thickening. Further, unstable atheroma formed peels off to form thrombus, which becomes a cause of various infarctions, and thus, it is known that arteriosclerosis causes diseases such as myocardial infarction, peripheral arterial disease, aortic aneurysm, aortic dissection, stroke (atherosclerotic cerebral infarction, transient ischemic attack, and the like), renal artery stenosis, internal carotid artery stenosis, angina pectoris, and the like, and has recently increased with the aging of the population and the change in lifestyle. Arteriosclerosis develops asymptomatically, and if it reaches the onset of myocardial infarction, cerebral infarction, aortic aneurysm, etc., it is a serious disease that is life-threatening. However, since the onset of the disease can be avoided by performing appropriate treatment, it is very important to detect arteriosclerosis, arteriosclerotic diseases at an early stage and to manage the degree of progression of arteriosclerosis.

For example, thoracic aortic aneurysms caused by arteriosclerosis and the like are often found by chance in chest X-ray examinations such as physical examinations, abdominal aortic aneurysms are often found by palpating the abdomen with a tapping to diagnose gastrointestinal diseases such as gastric ulcer and cholelithiasis, or by chance in abdominal ultrasound (ultrasonic) examinations such as gastrointestinal tract, kidney, and prostate, or in thoracic and abdominal MRI examinations.

As markers for arteriosclerosis and diseases associated with arteriosclerosis, lipid markers such as LDL cholesterol and oxidized LDL, which are used for risk evaluation; inflammatory markers such as CRP and pentaxin 3 (non-patent document 1). However, they are not sufficient in terms of sensitivity, specificity and disease specificity.

Further, as a method for non-invasively grasping the state of arteriosclerosis, measurement of Intima-Media Thickness (hereinafter, referred to as IMT) by carotid ultrasound examination, measurement of the Velocity difference of arterial waves between hands and feet (upper-ankle Pulse Wave Velocity) (hereinafter, referred to as baPWV), and the like are known (non-patent document 2). These examinations can be performed in many hospitals because they require time within ten and several minutes and do not perform an invasive, simple investigation of the degree of progress of arteriosclerosis, but are clearly unsuitable compared to blood examinations and the like in terms of unacceptable examinations if not performed in hospitals with equipment in order, and in terms of throughput for screening examinations for groups with high risk of arteriosclerosis from large-scale populations.

Documents of the prior art

Patent document

Non-patent document 1: pingshan Zhemen et al, Vascular Medicine, 10(1), 10-15, 2014

Non-patent document 2: diagnosis と therapy に Seki するガイドライン (2011-2012 contract study shift ) value method に Seki するガイドライン, 2013 of blood vessel invasion (diagnosis and treatment guideline for cardiovascular disease (2011-2012 combined study group report) non-invasive evaluation guideline for blood vessel function, 2013)

Disclosure of Invention

Problems to be solved by the invention

Blood tests such as blood pressure, LDL cholesterol, and HDL cholesterol, which have been used as risk factors for screening arteriosclerosis in physical examination and the like, are numerical values at the time of examination, and do not reflect the state of tissues and cells of the blood vessel wall under various risk factors or exposure of different pressure and different time periods. Based on the above-described circumstances, if a biomarker which is closely related to the evaluation method of arteriosclerosis in carotid ultrasound and baPWV examinations and can be used for screening is developed, it is expected that a new situation will be brought about in the prevention of arteriosclerosis, the diagnosis of the onset of arteriosclerosis-related diseases, the evaluation of the degree of progression, the development of therapeutic drugs, and the like.

The problem to be solved by the present invention is to provide a novel biomarker which can reflect the state of arteriosclerosis and can detect arteriosclerosis-related diseases and evaluate the degree of progression of arteriosclerosis in a simple and highly sensitive manner.

Technical scheme for solving problems

In order to achieve the object of providing an excellent marker for arteriosclerotic diseases reflecting the state of arteriosclerosis, the present inventors carried out proteome analysis using arteriosclerotic thoracic aortic aneurysm tissue as a disease that develops as a disease caused by arteriosclerosis. Arteriosclerotic thoracic aortic aneurysms are very suitable for the analysis of arteriosclerotic diseases using tissues because they occur in cases of arteriosclerosis and their disease sites are clearly distinguished. The obtained protein spectrum can be classified into tissues according to the degree of progression of the disease, and these classifications can well explain the changes in known structural proteins, proteases, and the like. Proteomic analysis of healthy aortic tissue was also performed by the same method. From the data obtained, proteome data of healthy aortic tissue was compared with tissues of patients with arteriosclerotic thoracic aortic aneurysm to determine factors associated with onset of disease. In addition, in order to determine factors related to disease progression, tissue proteome data between each classification of arteriosclerotic thoracic aortic aneurysm patients was compared. Factors that co-vary in both comparisons are presumed to be related to the onset and progression of the disease. Among the co-varying factors, factors that fluctuate also in blood were sought, and a novel marker that accurately reflects the onset and progression of arteriosclerosis, that is, the onset and progression of arteriosclerosis-related diseases was found, thereby completing the present invention.

That is, the present invention includes the following configurations:

(1) a method for detecting an arteriosclerosis-associated disorder, the method comprising: determining, in a sample obtained from a subject, the steps of 1) and/or 2) below:

1) NPC2(niemann-pick disease type C2) protein;

2) IGFBP7(insulin-like growth factor binding protein 7) protein.

(2) A method for detecting or screening for recurrence of an arteriosclerosis-associated disease, the method comprising: determining the following steps 1) and/or 2) in a sample obtained from a subject at risk of recurrence of an arteriosclerosis-associated disease:

1) NPC2(niemann-pick disease type C2) protein;

2) IGFBP7(insulin-like growth factor binding protein 7) protein.

(3) A method for detecting or screening a subject at risk of developing an arteriosclerosis-associated disease in the future, the method comprising: determining, in a sample obtained from a subject, the steps of 1) and/or 2) below:

1) NPC2(niemann-pick disease type C2) protein;

2) IGFBP7(insulin-like growth factor binding protein 7) protein.

(4) A method for evaluating the effect of a prophylactic or therapeutic agent for arteriosclerosis-associated diseases or arteriosclerosis, which comprises: a step of measuring the following 1) and/or 2) in a sample obtained from a subject in need of prevention or treatment of arteriosclerosis-associated diseases or arteriosclerosis, before administration of a preventive or therapeutic agent for arteriosclerosis-associated diseases or arteriosclerosis; a step of determining the following 1) and/or 2) in a sample obtained from the subject after administration of an arteriosclerosis-associated disease or a preventive or therapeutic agent for arteriosclerosis; alternatively, the method comprises: the step of determining the following 1) and/or 2) from samples obtained at two or more time points in chronological order from a subject in need of prevention or treatment of arteriosclerosis-associated diseases or arteriosclerosis after administration of a preventive or therapeutic agent for arteriosclerosis-associated diseases or arteriosclerosis:

1) NPC2(niemann-pick disease type C2) protein;

2) IGFBP7(insulin-like growth factor binding protein 7) protein.

(5) The method according to any one of (1) to (4), wherein the arteriosclerosis-associated diseases are selected from the group consisting of: myocardial infarction, peripheral arterial disease, aortic aneurysm, aortic dissection, atherosclerotic cerebral infarction, transient ischemic attack, renal artery stenosis, internal carotid artery stenosis, angina pectoris.

(6) A method for assessing the degree of progression of arteriosclerosis, the method comprising: determining, in a sample obtained from a subject, the steps of 1) and/or 2) below:

1) NPC2(niemann-pick disease type C2) protein;

2) IGFBP7(insulin-like growth factor binding protein 7) protein.

(7) The method according to any one of (1) to (6), wherein the sample is selected from the group consisting of: serum, plasma, blood, urine and saliva.

(8) The method according to any one of (1) to (7), wherein the subject is an animal in need of prevention or treatment of arteriosclerosis-associated diseases or arteriosclerosis, and the method evaluates or judges the effect of a prophylactic or therapeutic agent for arteriosclerosis-associated diseases or arteriosclerosis using the NPC2 protein and/or IGFBP7 protein as an index.

(9) The method according to any one of (1) to (8), wherein the subject is human.

(10) The method according to any one of (1) to (9), wherein the measurement method is an immunoassay or mass spectrometry.

(11) A marker for detecting an arteriosclerosis-associated disease, comprising the following 1) and/or 2):

1) NPC2 protein;

2) IGFBP7 protein.

(12) The detection marker according to (11), wherein the arteriosclerosis-associated diseases are selected from the group consisting of: myocardial infarction, peripheral arterial disease, aortic aneurysm, aortic dissection, atherosclerotic cerebral infarction, transient ischemic attack, renal artery stenosis, internal carotid artery stenosis, angina pectoris.

(13) A marker for evaluating the degree of progression of arteriosclerosis, comprising the following 1) and/or 2):

1) NPC2 protein;

2) IGFBP7 protein.

(14) An assay reagent for detecting an arteriosclerosis-associated disease or for evaluating the degree of progression of arteriosclerosis, which comprises an antibody or an aptamer for use in assaying the marker of any one of (11) to (13).

(15) The assay reagent according to (14), wherein the antibody is a monoclonal antibody and/or a polyclonal antibody.

(16) A kit for detecting an arteriosclerosis-associated disease or for evaluating the degree of progression of arteriosclerosis, which comprises the measuring reagent of (14) or (15).

(17) The marker, the measurement reagent, or the kit according to any one of (11) to (16), wherein the sample used for detecting the arteriosclerosis-associated disease or for evaluating the degree of progression of arteriosclerosis is selected from the group consisting of: serum, plasma, blood, urine and saliva.

(18) Use of the marker according to any one of (11) to (13) for screening a prophylactic or therapeutic drug for arteriosclerosis-associated diseases or arteriosclerosis.

(19) Use of the marker according to any one of (11) to (13) for in vitro detection or screening for recurrence of arteriosclerosis-associated disease.

(20) Use of the marker according to any one of (11) to (13) for in vitro detection or screening of a subject at risk of developing an arteriosclerosis-associated disease in the future.

Effects of the invention

By using NPC2 or IGFBP7 found in the present invention as a marker for arteriosclerosis-associated diseases (i.e., a marker for detecting arteriosclerosis-associated diseases) or an arteriosclerosis marker (i.e., a marker for evaluating the degree of progression of arteriosclerosis), the onset of arteriosclerosis-associated diseases and arteriosclerosis can be detected with high sensitivity. Further, NPC2 and IGFBP7 are considered as markers for capturing different side surfaces of arteriosclerosis, respectively, as shown in the examples, and it is possible to obtain more detailed information on the degree of progression of arteriosclerosis by using these combinations.

In addition, the measurement reagent and the measurement kit using the marker for detecting arteriosclerosis-related diseases or the marker for evaluating the degree of progression of arteriosclerosis of the present invention can be used for early diagnosis of the onset of arteriosclerosis-related diseases and arteriosclerosis, and for grasping (monitoring) the progression state of arteriosclerosis, and can contribute to prevention of the progression of diseases by early detection of these diseases.

The marker for detecting a disease associated with arteriosclerosis or the marker for evaluating the degree of progression of arteriosclerosis of the present invention can also be used for the development and evaluation of preventive and therapeutic drugs for these diseases. Furthermore, recurrence of arteriosclerosis-associated diseases can also be detected or screened, as well as subjects at risk of developing arteriosclerosis-associated diseases in the future.

Drawings

FIG. 1-1 is a graph showing the results of correlation analysis between the NPC2 value and maxIMT value in a serum sample of a patient with arteriosclerosis-related diseases (lower limb arteriosclerotic obliteration, myocardial infarction) in example 1.

FIGS. 1-2 are graphs showing the results of trend analysis of NPC2 values and maxIMT values in serum samples of patients with aortic aneurysms (including thoracic aortic aneurysm, abdominal aortic aneurysm, and thoracoabdominal complex-onset aortic aneurysm) in example 1. "NS" means "not significant difference".

FIGS. 1 to 3 are graphs showing the results of comparative analyses of the NPC2 value and the presence or absence of carotid plaque in serum samples of patients with arteriosclerosis-related diseases (aortic aneurysm, aortic dissection, lower limb arteriosclerotic obliteration, myocardial infarction) in example 1.

FIG. 2-1 is a graph showing the results of comparative analysis of NPC2 value and baPWV value (cut-off value 1400(cm/s)) in serum samples of patients with arteriosclerosis-associated diseases (aortic aneurysm, aortic dissection, lower limb arteriosclerotic obliteration, myocardial infarction) in example 2.

Fig. 2-2 is a graph showing the results of comparative analysis of NPC2 value and baPWV value (critical value 1700(cm/s)) in serum samples of patients with arteriosclerosis-related diseases (aortic aneurysm, aortic dissection, lower limb arteriosclerotic occlusion, and myocardial infarction) in example 2.

FIG. 3-1 is a graph showing the results of correlation analysis between the value of IGFBP7 and the value of maxIMT in a serum sample of a patient with arteriosclerosis-related diseases (lower limb arteriosclerotic obliteration, myocardial infarction) in example 3.

FIG. 3-2 is a graph showing the results of trend analysis of IGFBP7 values versus maxIMT values in serum samples from patients with aortic aneurysms (including thoracic aortic aneurysms, abdominal aortic aneurysms, and thoracoabdominal complex-onset aortic aneurysms) in example 3.

FIG. 3-3 is a graph showing the results of comparative analysis of the IGFBP7 value and the presence or absence of carotid plaque in a serum sample of a patient with arteriosclerosis-related diseases (aortic aneurysm, aortic dissection, lower limb arteriosclerotic obliteration, myocardial infarction) in example 3. "NS" means "not significant difference".

FIG. 4-1 is a graph showing the results of correlation analysis between the CRP value and the maxIMT value in a serum sample of a patient with arteriosclerosis-related diseases (lower limb arteriosclerotic occlusion, myocardial infarction) in comparative example 1.

FIG. 4-2 is a graph showing the results of trend analysis of CRP value and maxIMT value in a serum sample of a patient with aortic aneurysm (including thoracic aortic aneurysm, abdominal aortic aneurysm and thoraco-abdominal complex-onset aortic aneurysm) in comparative example 1. "NS" means "not significant difference".

FIG. 4-3 is a graph showing the results of comparative analysis of the CRP value and the presence or absence of carotid plaque in serum samples of patients with arteriosclerosis-associated diseases (aortic aneurysm, aortic dissection, lower limb arteriosclerotic occlusion, and myocardial infarction) in comparative example 1. "NS" means "not significant difference".

FIG. 5-1 is a graph showing the results of comparative analysis of the CRP value and the baPWV value (cut-off value 1400(cm/s)) in serum samples of patients with arteriosclerosis-associated diseases (aortic aneurysm, aortic dissection, lower limb arteriosclerotic occlusion, and myocardial infarction) in comparative example 2.

Fig. 5-2 is a graph showing the results of comparative analysis of the CRP value and the baPWV value (critical value 1700(cm/s)) in the serum sample of the patient with arteriosclerosis-associated diseases (aortic aneurysm, aortic dissection, lower limb arteriosclerotic occlusion, myocardial infarction) in comparative example 2. "NS" means "not significant difference".

FIG. 6-1 is a graph showing the measurement results of NPC2 values in serum samples of healthy subjects, non-arteriosclerotic thoracic aortic aneurysm patients, and arteriosclerotic thoracic aortic aneurysm patients in example 4. "HC" means health control; "TAA-NAS" means "Thoracic aotomysm-non atherosclerotic (non-arteriosclerotic Thoracic aortic aneurysm)"; "TAA-AS" means "Thoracic oral and systemic-atheroclerotic (arteriosclerotic Thoracic aortic aneurysm)"; "NS" means "not significant difference".

FIG. 6-2 is a graph showing the results of measurement of IGFBP7 values in serum samples of healthy subjects, non-arteriosclerotic thoracic aortic aneurysm patients, and arteriosclerotic thoracic aortic aneurysm patients in example 4. "HC" means "health control"; "TAA-NAS" means "Thoracic aotomysm-non atherosclerotic (non-arteriosclerotic Thoracic aortic aneurysm)"; "TAA-AS" means "Thoracic oral and systemic-atheroclerotic (arteriosclerotic Thoracic aortic aneurysm)".

Detailed Description

In order to identify a marker reflecting the arteriosclerotic state, the present inventors performed proteome analysis as follows using aortic mesodermal smooth muscle layer tissues collected from 14 aortic tissues and 29 tissues of patients with arteriosclerotic thoracic aortic aneurysm of healthy subjects.

■ tissue disruption and Trypsin digestion

After collection, the frozen tissue was disrupted by a bead breaker, and Lys-C (Lysyl Endopeptidase) and Trypsin (Trypsin) were added thereto, followed by digestion by overnight incubation at 37 ℃. The resulting digested peptide was desalted using a C18 (octadecylsilyl) column and used as an analytical sample.

■ analysis

Analysis samples were separated by Nano LC (Hitachi Nano Frontier, Hitachi high tech Co.) and then subjected to MS/MS analysis by means of an attached tripleTOF 5600(SCIEX Co.). Proteins were identified from the measurement data by Mascot database search (Matrix science), quantified using integrated LC-MS data and comparative quantitative proteome Analysis software 2 digital (two-Dimensional Image conversion Analysis by Liquid chromatography-mass spectrometry, mitsui), classified and intra-classified comparison of the arteriosclerotic thoracic aortic aneurysm group, and compared between the normal aortic group and the arteriosclerotic thoracic aortic aneurysm group, and differential proteins were searched. As a result, NPC2 and IGFBP7 were identified as proteins showing a significant increase in the classification of the arteriosclerotic thoracic aortic aneurysm group and in the normal aortic and arteriosclerotic thoracic aortic aneurysm groups, and the present invention was completed.

In the present invention, the marker of the present invention was found by selecting a candidate factor not only for comparison between a healthy subject and an arteriosclerotic thoracic aortic aneurysm, but also by referring to comparison data between a non-arteriosclerotic thoracic aortic aneurysm and an arteriosclerotic thoracic aortic aneurysm.

As described above, both NPC2 and IGFBP7 were observed to show a significant increase in expression in the mesenteric aorta tissue from a patient with atherosclerotic thoracic aortic aneurysm, which develops as a disease due to arteriosclerosis, as compared with the mesenteric aorta tissue of a healthy patient. By using NPC2 and IGFBP7 as markers alone, it is possible to detect diseases related to arteriosclerosis and arteriosclerosis, but as shown in examples described later, both markers are more closely related to different arteriosclerosis indexes (as shown in examples 1 to 2 described later, NPC2 is a marker more closely related to the presence or absence of carotid plaque and baPWV value, and as shown in example 3, IGFBP7 is a marker more closely related to the maximum IMT value of carotid bulbus (hereinafter, referred to as maxmt value)), so that by combining NPC2 and IGFBP7, an excellent detection effect of reflecting the state (degree of progression) of arteriosclerosis can be exhibited more accurately.

The present invention is characterized by using a marker for detecting an arteriosclerosis-associated disease or a marker for evaluating the degree of progression of arteriosclerosis using NPC2 and/or IGFBP 7.

In the present invention, a "marker" or a "biomarker" refers to a molecule used as a target for measurement of a sample obtained from a subject.

Markers of the invention comprise NPC2 and/or IGFBP 7. Human NPC2 (i.e., Niemann-Pick disease type C2 protein, Niemann-Pick disease type C2 protein) is a secreted protein represented by UniProt accession number P61916 (http:// www.uniprot.org/UniProt/P61916). The amino acid sequence of human NPC2 is shown in SEQ ID NO. 1. 1, the amino acid sequence from 1 st to 19 th is a signal peptide, and the amino acid sequence from 20 th to 151 th is a mature protein. NPC2 is a glycoprotein consisting of 132 amino acids in humans, is present in high amounts in the epididymis, but is also present in many other tissues. NPC2 has cholesterol binding capacity and is believed to be involved in cholesterol efflux from liposomes together with NPC 1. A functional deficiency of NPC2 results in Niemann's disease type C, which is manifested by abnormal deposition of cholesterol in liposomes (Marie T. Vanier, Gilles Millat. Structure and function of the NPC2 protein. Biochimica et Biophysica Acta 2004; 1685: 14-21).

In the present invention, the NPC2 protein includes proteins which do not have a sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, more preferably 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, most preferably 100% to the amino acid sequence from position 20 to position 151 of the amino acid sequence shown in SEQ ID NO. 1, and which have cholesterol binding activity and/or cholesterol excretion activity from liposomes, or functional defect mutants, and also includes multimers (dimers and/or more) of such proteins.

Human IGFBP7 (i.e., Insulin-like growth factor-binding protein7, Insulin-like growth factor binding protein 7), on the other hand, is a secreted protein represented by UniProt accession number Q16270 (http:// www.uniprot.org/UniProt/Q16270). The amino acid sequence of human IGFBP7 is shown in SEQ ID NO. 2. In the amino acid sequence shown in SEQ ID NO. 2, the amino acid sequences from 1 st to 26 th are signal peptides, and the amino acid sequences from 27 th to 282 th are mature proteins. IGFBP7 is expressed in many tissues such as heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, and colon. Unlike other IGFBP proteins, IGFBP7 is known to bind insulin weakly and strongly to insulin-like growth factor (IGF). IGFBP7 is reported to be associated with cell proliferation control, apoptosis, cell aging, and angiogenesis (Shuzhen Zhu, growing Xu, hanging Zhang, Wenjing Ruan, Maode Lai. insulin-like growth factor binding protein-related protein1and cancer. clinical Chimica Acta 2014; 431: 23-32).

In the present invention, IGFBP7 protein includes a protein which is composed of an amino acid sequence having a sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, more preferably 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, and most preferably 100% to the amino acid sequence from position 27 to position 282 in the amino acid sequence shown in SEQ ID NO. 2, and which has insulin binding activity or does not have such activity, and also includes multimers (dimers and/or more) of such proteins.

The marker of the present invention may be used in combination with known markers, such as LDL cholesterol, HDL cholesterol, denatured LDL such as oxidized LDL, sdLDL (small dense LDL), C-reactive protein (CRP), pentraxin3, IL-6, which have been reported to be involved in arteriosclerotic diseases.

In the present invention, the "arteriosclerosis-related diseases" mean diseases which develop as a disease caused by arteriosclerosis. The disease detectable by the arteriosclerosis-associated disease marker of the present invention is not particularly limited as long as it is a disease caused by arteriosclerosis. Specific examples thereof include: myocardial infarction, peripheral arterial disease, aortic aneurysm, aortic dissection, stroke (atherosclerotic cerebral infarction, transient ischemic attack, etc.), renal artery stenosis, internal carotid artery stenosis, angina pectoris, etc. In the present specification, arteriosclerosis and related diseases are sometimes collectively referred to as "arteriosclerotic diseases". In addition, the various arteriosclerosis-associated diseases described above may be referred to as arteriosclerosis-associated diseases, and they can be diagnosed, examined, evaluated or judged by the marker of the present invention.

In the present invention, the method for detecting a marker for arteriosclerosis-associated diseases and the method for determining an arteriosclerosis marker (i.e., the method for determining a marker for evaluating the degree of progression of arteriosclerosis) comprise the step of determining the NPC2 protein and/or the IGFBP7 protein. When NPC2 protein and/or IGFBP7 protein in a sample is measured, NPC2 and/or IGFBP7 can be measured as glycoproteins, and the amino acid sequence portion of NPC2 and/or IGFBP7 can be measured after post-translational modifications such as sugar chains and phosphates are removed by pretreatment.

The method for detecting arteriosclerosis-associated diseases can detect arteriosclerosis-associated diseases in a subject by measuring the marker of the present invention in a sample obtained from the subject. Therefore, a method of detecting an arteriosclerosis-related disease may be referred to as a method of diagnosing, examining, evaluating, or judging the arteriosclerosis-related disease, a method of collecting in vitro data for detecting the arteriosclerosis-related disease, or the like.

As shown in examples 1 to 3 described later, it was confirmed that the marker of the present invention has close correlation with carotid artery ultrasound, baPWV examination, and the like, which are indicators of arteriosclerosis, and a method of evaluating the degree of progression of arteriosclerosis, that is, a method of evaluating the degree of progression of arteriosclerosis, enables grasping information of the same degree as the degree of progression of arteriosclerosis of a blood vessel obtained by carotid artery ultrasound, baPWV examination, and the like, only by testing the marker of the present invention in a sample obtained from a subject. Therefore, the method of evaluating the degree of progression of arteriosclerosis may be referred to as a method of diagnosing, examining, evaluating, or judging arteriosclerosis, a method of collecting data in vitro for detecting arteriosclerosis, or the like.

Examples of animals that can be subjects to be used in the method for detecting a disease associated with arteriosclerosis and the method for evaluating the degree of progression of arteriosclerosis include: mammals such as human, monkey, cow, pig, horse, dog, cat, sheep, goat, rabbit, hamster, guinea pig, mouse, and rat, and preferably human.

Examples of the sample used in the method for detecting a disease associated with arteriosclerosis and the method for evaluating the degree of progression of arteriosclerosis include: serum, plasma, blood, urine, saliva, etc.

In the present invention, for example, in a sample collected from a patient suspected of arteriosclerosis, the possibility of the current arteriosclerosis-related disease or the progression of arteriosclerosis, the risk of the onset (possibility) of the future arteriosclerosis-related disease or the recurrence of the arteriosclerosis-related disease can be judged, detected or screened by measuring the NPC2 protein level and/or the IGFBP7 protein level in vitro. When the NPC2 protein level and/or the IGFBP7 protein level in a patient sample is higher than that in a healthy subject, it can be judged that the possibility that arteriosclerosis-related diseases are currently progressing, the risk of (possibility of) the onset of arteriosclerosis-related diseases in the future, or the possibility that arteriosclerosis-related diseases are relapsing is high, when the NPC2 protein level and/or the IGFBP7 protein level in the patient sample is compared with that in the healthy subject.

Specifically, according to the method for detecting an arteriosclerosis-associated disease and the method for evaluating the degree of progression of arteriosclerosis, the present invention also includes the following methods:

a method for detecting or screening for the recurrence of an arteriosclerosis-associated disease by testing a marker of the present invention in a sample obtained from a subject at risk of the recurrence of the arteriosclerosis-associated disease;

a method for detecting or screening a subject at risk of developing an arteriosclerosis-associated disease in the future by testing a marker of the present invention in a sample obtained from the subject.

The threshold value of each marker for determining that the possibility of the onset of the disease related to arteriosclerosis or the possibility of the progression of arteriosclerosis is high at present is not limited, and the following examples are given. The following critical values were calculated from ROC curves plotted from the data shown in the examples.

(i) Cutoff value for NPC2 based on example 1 (comparative analysis for the presence or absence of carotid plaque): 2.8ng/mL (sensitivity 68.0%, specificity 22.2%);

critical value of NPC2 based on example 2 (Critical value of baPWV 1400 cm/s): 2.3ng/mL (sensitivity 75.0%, specificity 29.0%);

critical value of NPC2 based on example 2 (Critical value of baPWV 1700 cm/s): 3.4ng/mL (sensitivity 65.0%, specificity 35.0%).

(ii) Cut-off based on IGFBP7 of example 3 (comparative analysis for the presence or absence of carotid plaque): 178.0ng/mL (sensitivity 53.3%, specificity 22.2%).

Based on these cut-off values, when the NPC2 protein level and/or IGFBP7 protein level in the patient sample is higher than the cut-off values, it can be judged that the possibility of the onset of the arteriosclerosis-associated disease or the possibility that arteriosclerosis is progressing is high.

The marker of the present invention can also be used for screening a prophylactic or therapeutic agent for arteriosclerosis-associated diseases or arteriosclerosis, and for evaluating and judging the effect of a prophylactic or therapeutic agent for arteriosclerosis-associated diseases or arteriosclerosis. For example, when evaluating and judging the effect of a prophylactic or therapeutic agent for arteriosclerosis-related diseases or arteriosclerosis, the following method can be used: samples are taken from the subject animal before and after administration of the corresponding prophylactic or therapeutic drug to the subject animal in need of prevention or treatment of an arteriosclerosis-associated disease or arteriosclerosis, or samples are taken from the subject animal at two or more time points in chronological order after administration of the prophylactic or therapeutic drug, and changes in NPC2 protein levels and/or IGFBP7 protein levels in the samples are studied over time. By administering the corresponding prophylactic or therapeutic agent, the NPC2 protein level and/or IGFBP7 protein level in a sample taken from the subject animal shows a tendency to be inhibited or stabilized against the formation and progression of arteriosclerotic lesions being inhibited over time. On the other hand, the NPC2 protein level and/or IGFBP7 protein level in a sample taken from the subject animal shows a tendency to decrease with time when the development, progression or progression of arteriosclerotic lesions is improved by administration of the corresponding prophylactic or therapeutic drugs.

As a method for measuring the marker protein of the present invention, any known method such as immunoassay and mass spectrometry can be used as long as it is a method for specifically measuring NPC2 protein or IGFBP7 protein. In the reagent used for measuring the marker protein of the present invention, an antibody, an aptamer, or the like can be used as a detection agent.

The immunoassay method is not particularly limited, and examples thereof include various enzyme immunoassay methods, Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), double monoclonal antibody sandwich immunoassay, monoclonal polyclonal antibody sandwich immunoassay, immunostaining method, immunofluorescence method, western blotting method, biotin-avidin method, immunoprecipitation method, colloidal gold agglutination method, immunochromatography method, latex agglutination method (LA), immunoturbidimetry (TIA), and the like.

As reagents used in the immunoassay, an anti-NPC 2 antibody, an anti-IGFBP 7 antibody which have been already sold as a detection agent, may be used, and antibodies may also be prepared by a conventional method based on the well-known amino acid sequence of NPC2 (SEQ ID NO:1) and the amino acid sequence of IGFBP7 (SEQ ID NO: 2). The antibody may specifically recognize the amino acid sequence structures of NPC2 and IGFBP7, or may specifically recognize the entire structure including sugar chains and post-translational modifications such as disulfide bonds and phosphorylation.

The animal species and clone from which the protein NPC2 or IGFBP7 is derived are not particularly limited as long as they are antibodies that can detect the protein. Examples thereof include antibodies derived from rabbit, goat, mouse, rat, guinea pig, horse, sheep, camel, chicken, etc., and both monoclonal antibodies and polyclonal antibodies may be used. In addition, antibodies suitable for specifically binding to all subclasses of NPC2 protein, IGFBP7 protein may be used. Of course, fragments such as recombinant antibodies, Fab 'or F (ab')2 fragments may also be used.

As the agent for detecting an arteriosclerosis-related disease or the agent for evaluating arteriosclerosis of the present invention, an aptamer having binding to NPC2 protein or IGFBP7 protein can be used. In the production of aptamers, DNA or RNA that can bind to DNA or RNA or their derivatives, i.e., aptamers and aptamers composed of amino acids, can be synthesized and used by a known method from the known amino acid sequence of NPC2 (SEQ ID NO:1) and the amino acid sequence of IGFBP7 (SEQ ID NO: 2). In the measurement, the binding of the aptamer can be detected by a luminescence method, a fluorescence method, or a surface plasmon resonance method.

Mass spectrometry is not particularly limited, and mass spectrometry can be used in which an ion source using electrospray ionization (ESI), Matrix Assisted Laser Desorption Ionization (MALDI), Surface Enhanced Laser Desorption Ionization (SELDI), or the like is combined with a time-of-flight analyzer (TOF), an ion trap analyzer (IT), a fourier transform analyzer (FT), or the like. LC-MS and CE-MS, etc., in which a mass spectrometer is connected to a separation device such as high-performance liquid chromatography (HPLC) or Capillary Electrophoresis (CE), etc., can be used. Further, examples of the method for obtaining mass spectrum data include: data Independent Analysis (DIA), Data Dependent Analysis (DDA), multiple reaction monitoring method (MRM), etc. The mass spectrum also includes a case where a sample is labeled with a stable isotope such as iTRAQ reagent (SCIEX).

Various reagents required for the detection of the arteriosclerosis-related disease or the evaluation of the degree of progression of arteriosclerosis of the present invention may be packaged in a kit. For example, the following are provided as kits: (i) a monoclonal or polyclonal antibody specific for the marker protein of the invention as a capture antibody; (ii) an enzyme-labeled monoclonal antibody or polyclonal antibody specific to the marker protein of the present invention as a detection antibody; (iii) substrate solution, and the like.

Examples

Hereinafter, examples of the present invention are shown, and the present invention is not limited to these examples.

[ example 1 ]

Relationship between serum NPC2 value, maxIMT value, and plaque

The subjects were treated with aortic aneurysm, aortic dissection, lower limb arteriosclerotic obliteration, and myocardial infarction, and compared with the serum concentration of NPC2 measured by ELISA, the maxmt value of carotid artery ball examined by ultrasound, and plaque discovery (presence or absence of plaque) examined by carotid artery ultrasound. NPC2 was measured using NPC2 ELISA kit (Aviva Systems Biology Co.) according to the protocol attached to the kit. The maxmt value is the maximum wall thickness including the carotid artery plaque measured by Aplio series manufactured by canon medical systems co. In the ultrasonic examination, the group in which the presence of Plaque was confirmed was defined as a Plaque (Plaque) group, and the group in which no significant Plaque was confirmed was defined as a Plaque-free group, thereby determining the presence or absence of Plaque.

In detail, the assay of NPC2 concentration by ELISA was performed by adding diluted serum samples to a solid phase plate of anti-NPC 2 antibody and incubating. Then, the sample solution was removed, and a biotin-labeled anti-NPC 2 antibody was added and incubated. After the incubation, a washing operation was performed, and HRP (horse radish peroxidase) labeled avidin was added and incubated. Subsequently, washing was performed, a TMB (3,3',5,5' -tetramethylbenzidine, 3,3',5,5' -tetramethylbenzidine) solution was added to the reaction mixture to develop a color, and then a stop solution was added thereto, and the measurement was performed using a microplate reader (SpectraMax M2e, Molecular Devices) at a main wavelength of 450nm and a sub-wavelength of 540 nm. The concentration of NPC2 was measured by calculating the concentration of each standard solution from a calibration curve prepared from the measurement data of the respective standard solutions measured simultaneously.

The analyses of correlation between the serum NPC2 value and maxmt value were performed on 23 cases of arteriosclerosis-related diseases (14 cases of lower limb arteriosclerotic occlusion and 9 cases of myocardial infarction) as subjects. As a result: the correlation coefficient between the serum NPC2 value and maxmt value was 0.151(p ═ 0.491) (fig. 1-1: B _ maxmt on the horizontal axis indicates Bifurcation (carotid bulb) maxmt).

Trend analysis of serum NPC2 values versus maxmt values was performed as follows: 48 cases of aortic aneurysms (including thoracic aortic aneurysm, abdominal aortic aneurysm, and thoracoabdominal complex-onset aortic aneurysm. the same applies to all the following examples) were divided into 4 groups of 12 cases with a low value group (low group), 12 cases with a medium value 1 group (medium 1 group), 12 cases with a medium value 2 group (medium 2 group), and 12 cases with a high value group (high group) in order from the maxmt low value side, and serum NPC2 values of the groups were examined. The significance of the differences in trends was examined using the Jonckheere-Terpsra test. As shown in FIG. 1-2, it was confirmed that the higher the maxIMT value, the higher the serum NPC2 value.

Comparative analysis of serum NPC2 values with the presence or absence of carotid plaques was performed as follows: for a total of 84 cases (52 cases of aortic aneurysm, 10 cases of aortic dissection, 14 cases of lower limb arteriosclerotic occlusion, and 8 cases of myocardial infarction), 2 groups were divided into no plaque and plaque, and the serum NPC2 values of each group were examined. The U test of Mann-Whitney was used for statistical processing. As shown in fig. 1-3, in carotid plaque groups, serum NPC2 values were significantly high (p ═ 0.013).

[ example 2 ]

Relationship between serum NPC2 value and baPWV value

The serum concentration of NPC2 was measured by ELISA using subjects of aortic aneurysm, aortic dissection, lower limb arteriosclerotic obliteration, and myocardial infarction, and compared with the baPWV value. The serum concentration of NPC2 was measured in the same manner as described in example 1. The baPWV value was measured using BP-203RPEII (approved) manufactured by COLIN corporation of Japan.

Comparative analysis of serum NPC2 values to baPWV values was performed as follows: for a total of 85 cases (49 cases of aortic aneurysm, 11 cases of aortic dissection, 8 cases of lower limb arteriosclerotic occlusion, and 17 cases of myocardial infarction), the critical value of baPWV was classified into 2 groups as 1400(cm/s) or 1700(cm/s), and the serum NPC2 values of each group were examined. The U test of Mann-Whitney was used for statistical processing. As shown in fig. 2-1 and 2-2, in the group of high baPWV values, the serum NPC2 value was significantly high (p ═ 0.014 at cutoff value 1400(cm/s) and 0.031 at cutoff value 1700 (cm/s)).

The presence of carotid plaque, a significant increase in the baPWV high value group (examples 1and 2), suggests that serum NPC2 values may be a marker for the development of atherosclerosis and diseases associated with atherosclerosis.

[ example 3 ]

Correlation between serum IGFBP7 value, maxIMT value, and plaque Presence/absence

The serum concentration of IGFBP7 was measured by ELISA on the same patient as in example 1, and compared with maxmit value of carotid artery ball examined by ultrasound and plaque finding (plaque presence or absence) examined by carotid artery ultrasound. IGFBP7 was assayed using an ELISA kit for insulin-like growth factor-binding protein7 (cloud-clone) according to the protocol attached to the kit. The maxmtt value measurement and the judgment of the presence or absence of the plaque were performed by the method described in example 1.

Specifically, in the measurement of the concentration of IGFBP7 by ELISA, a diluted serum sample was added to a solid phase plate of anti-IGFBP 7 antibody and incubated. Then, the sample solution was removed, and the detection reagent solution A was added and incubated. After the incubation, a washing operation was performed, and a detection reagent B solution was added and incubated. Next, washing was performed, a TMB solution was added to the reaction mixture to perform a color reaction, and then a stop solution was added thereto, followed by measurement using a microplate reader (SpectraMax M2e, Molecular Devices Co., Ltd.) at a main wavelength of 450nm and a sub-wavelength of 540 nm. The concentration of IGFBP7 was measured by calculating the concentration against the data from a calibration curve prepared from the measurement data of each standard solution measured simultaneously.

Results of correlation analysis of serum IGFBP7 values with maxmt values, and correlation of serum IGFBP7 values with maxmt values with significance of correlation coefficient R0.503 (p 0.015) (fig. 3-1: B _ maxmt on horizontal axis indicates bifurcation (carotid bulbus) maxmt). Furthermore, the trend analysis results of serum IGFBP7 values versus maxmt values are shown in fig. 3-2, which indicates that the higher the maxmt value, the more significant the serum IGFBP7 value (p < 0.05). Furthermore, the results of comparative analysis of serum IGFBP7 values with the presence or absence of carotid plaque are shown in FIGS. 3-3, which confirmed that serum IGFBP7 tended to be high in the carotid plaque group.

As described above, the serum IGFBP7 value significantly correlated with the carotid bulbar maxmt value, and the higher the carotid bulbar maxmt value, the more significantly the serum IGFBP7 value was, thus indicating the possibility of being a marker for the development of arteriosclerosis and arteriosclerosis-related diseases. In addition, the data of examples 1 to 3 show that NPC2 and IGFBP7 have stronger correlation with different indicators of arteriosclerosis progression. That is, by performing examination by combining NPC2 and IGFBP7, more detailed information on the degree of progression of arteriosclerosis is expected.

[ comparative example 1 ]

Relationship between serum CRP value and maxIMT value, and whether plaque exists or not

The serum concentration of CRP was measured by ELISA using the same patient as in example 1, and compared with maxmt value of carotid bulbus examined by ultrasound and plaque finding (presence or absence of plaque) examined by carotid ultrasound. As a reagent for measuring CRP, a C-reactive protein kit CRP-latex X2 "student" NX (Denka Ministry of Japan) was used, and as a measuring device, a LABOSPECT 008 Hitachi automatic analyzer and a 7180 Hitachi automatic analyzer (Hitachi high tech Co., Ltd.) were used. The maxmtt value measurement and the judgment of the presence or absence of plaque were performed by the method described in example 1.

The correlation coefficient between the serum CRP value and the maxmit value was-0.274 (p ═ 0.207) as a result of the correlation analysis between the serum CRP value and the maxmt value (fig. 4-1: B _ maxmt on the horizontal axis represents the bifurcation (carotid bulbar) maxmt). In addition, the results of trend analysis of serum CRP values and maxmit values are shown in fig. 4-2, and no trend was confirmed between serum CRP values and maxmit values. Furthermore, the results of comparative analysis of serum CRP values with the presence or absence of carotid plaque are shown in FIGS. 4 to 3, and no correlation was observed between serum CRP values and the presence or absence of carotid plaque.

[ comparative example 2 ]

Relationship between serum CRP value and baPWV value

The serum concentration of CRP was measured by ELISA method using the same patient as in example 2 as a subject, and compared with the baPWV value. Serum concentration of CRP was measured according to the method described in comparative example 1, and baPWV value was measured according to the method described in example 2.

As shown in fig. 5-1 and 5-2, at the cutoff value 1400(cm/s), the serum CRP value was a significantly high value (p ═ 0.024) among the baPWV high value group, but at the cutoff value 1700(cm/s), no correlation was confirmed.

The above examples 1 to 3 and comparative examples 1and 2 show that NPC2 and IGFBP7 are useful markers as markers for diseases associated with the progression of arteriosclerosis and arteriosclerosis, compared to CRP.

[ example 4 ]

Comparison of NPC2 and IGFBP7 in serum samples from patients with non-arteriosclerotic thoracic aortic aneurysm and patients with arteriosclerotic thoracic aortic aneurysm (ELISA method)

To confirm the usefulness as markers of diseases related to arteriosclerosis such as NPC2 and IGFBP7, NPC2 and IGFBP7 values in the serum of patients with non-arteriosclerotic thoracic aortic aneurysm (genetic connective tissue disease) and arteriosclerotic thoracic aortic aneurysm patients were measured. NPC2 was measured according to the method described in example 1, and IGFBP7 was measured according to the method described in example 3. StatFlex ver6.0(Artec Inc.) was used for statistical treatment, and Mann-Whitney U test was used for the significance test.

As shown in FIG. 6-1, NPC2 was found to be 2.91. + -. 2.01ng/mL, 3.02. + -. 3.21ng/mL, and 9.03. + -. 4.37ng/mL in healthy subjects (44 cases), non-arteriosclerotic thoracic aortic aneurysm patients (20 cases), and arteriosclerotic thoracic aortic aneurysm patients (29 cases), respectively, and was found to be higher in arteriosclerotic thoracic aortic aneurysm patients than in non-arteriosclerotic thoracic aortic aneurysm patients. Thus, NPC2 was shown to be a useful marker for arteriosclerosis-related diseases.

As shown in FIG. 6-2, IGFBP7 was 139.20. + -. 28.47ng/mL, 187.25. + -. 58.97ng/mL, and 291.96. + -. 316.83ng/mL in healthy subjects (44 cases), non-arteriosclerotic thoracic aortic aneurysm patients (20 cases), and arteriosclerotic thoracic aortic aneurysm patients (29 cases), respectively, and was also high in the arteriosclerotic thoracic aortic aneurysm patients as compared with the non-arteriosclerotic thoracic aortic aneurysm patients. Thus, IGFBP7 was shown to also be a useful marker for arteriosclerosis-related diseases.

Industrial applicability of the invention

By using NPC2 and/or IGFBP7 as a novel marker for arteriosclerosis-related diseases and arteriosclerosis progression of the present invention, the progression of arteriosclerosis and the onset of arteriosclerosis-related diseases can be detected with high sensitivity. Therefore, the measurement reagent and kit using the arteriosclerosis-associated disease marker or arteriosclerosis progression marker of the present invention can be used for screening and early diagnosis of the onset of arteriosclerosis-associated disease or arteriosclerosis, monitoring of the degree of arteriosclerosis progression, evaluation of risk of recurrence of arteriosclerosis-associated disease, evaluation of risk of future onset, development and evaluation of preventive and therapeutic drugs, and the like, and further, the present invention can be utilized in the industry for producing the measurement reagent and kit.

All publications, patents and patent applications cited in this specification are herein incorporated in their entirety by reference.

Sequence listing

<110> Rongyan chemical Co., Ltd

National Center for research and development of circulatory diseases

<120> markers for arteriosclerosis and arteriosclerosis-related diseases

<130> PH-7840-PCT

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<170> PatentIn version 3.5

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