JP2013064616A - Method for detecting onset risk of advanced arterial sclerosis and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means for more easily screening arterial sclerosis patient.SOLUTION: A method for detecting the onset risk of advanced arterial sclerosis for an analyte includes a measurement step of measuring the concentration of secreted GRP78 protein in a biological specimen obtained from the analyte. Furthermore, a kit for detecting the onset risk of advanced arterial sclerosis for an analyte is provided with at least measurement means for measuring the concentration of secreted GRP78 protein in a biological specimen obtained from the analyte. The measurement means is an antibody that specifically recognizes secreted GRP78 protein.

Description

  The present invention relates to a method for detecting the possibility of developing advanced arteriosclerosis and use thereof. More specifically, the present invention relates to a method, kit, and marker for detecting the possibility of developing arteriosclerosis with an arterial stenosis degree of 75% or more.

  The number one cause of death in the world (developed countries) is arteriosclerotic disease. It has been reported that various proteins can be used as markers for arteriosclerosis. Examples of such proteins include sICAM (soluble intercellular adhesion molecule) -1, MMP (matrix metalloproteinase) -1,2,3,9, TIMP (tissue inhibitor of metalloproteinase) -1,2, and high sensitivity CRP (C -reactive protein) (Non-patent Document 1 and Non-patent Document 2).

  By the way, the inventors have so far reported that an endoplasmic reticulum stress response occurs in an atherosclerotic lesion, and as a result, the expression of GRP78 protein, which is an endoplasmic reticulum-related molecule, increases in the atherosclerotic lesion. Specifically, it has been reported that the expression of GRP78 protein is increased in arteriosclerotic tissues, particularly in places where stenosis of blood vessels is strong or easily ruptured (Non-patent Document 3).

  Another group reports that the GRP78 protein moves to the cell surface by endoplasmic reticulum stress and is secreted extracellularly (Non-Patent Documents 4 to 6).

  Furthermore, since the anticancer drug becomes less effective when the expression of GRP78 protein increases in tumor cells, GRP78 protein is used as a marker for resistance to anticancer drug treatment in tumor cells (Non-patent Documents 7 and 8).

Masami Ogura et al., “Inflammatory Response Biomarker for the Prevention of Cardiovascular Events”, Heart View Vol. 13, No. 6 (2009) p.8-14 Linda Hermus et al., "Carotid plaque formation and serum biomarkers", Atherosclerosis 213 (2010) p.21-29 Masafumi Myoishi et al., "Increased Endoplasmic Reticulum Stress in Atherosclerotic Plaques Associated With Acute Coronary Syndrome", Circulation 116 (2007) p.1226-1233 Yi Zhang et al., "Cell Surface Relocalization of the Endoplasmic Reticulum Chaperone and Unfolded Protein Response Regulator GRP78 / BiP", Journal of Biological Chemistry Vol. 285, No. 20 (2010) p.15065-15075 Nicolas Chignard et al., "Cleavage of Endoplasmic Reticulum Proteins in Hepatocellular Carcinoma: Detection of Generated Fragments in Patient Sera", Gastroenterology 130 (2006) p.2010-2022 David L. Wiest et al., "Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of" ER-resident "molecular chaperones", Proc. Natl. Acad. Sci. USA 94 (1997) p.1884 -1889 Eunjung Lee et al., "GRP78 as a Novel Predictor of Responsiveness to Chemotherapy in Breast Cancer", Cancer research 66 (16) (2006) p.7849-7853 Paul J. Mintz et al., "Fingerprinting the circulating repertoire of antibodies from cancer patients", Nature biotechnology 21 (2003) p.57-63

  If various arteriosclerosis markers as described above are used, it is possible to detect the possibility that the subject has developed arteriosclerosis by a relatively simple method, but there is no marker that can be confirmed with reproducibility. . Therefore, in order to determine whether or not the subject from which the arteriosclerosis marker is detected is an advanced arteriosclerosis patient (a high-risk patient whose arterial stenosis is 75% or more) that really needs treatment. Further, it is necessary to directly confirm the degree of stenosis of the artery and the presence or absence of vulnerability in the sclerosis site of the artery by performing an image diagnosis (for example, ultrasonic examination, computed tomography (CT)). Image diagnosis has the problem that the examination requires labor and cost, but the only way to screen patients with advanced arteriosclerosis is to perform image diagnosis.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a means that can more easily screen patients with advanced arteriosclerosis.

  As a result of intensive studies to achieve the above object, the present inventors have significantly increased the concentration of secreted GRP78 protein in blood in patients with carotid arterial stenosis (stenosis of 75% or more). In patients with severe carotid artery stenosis, the concentration of secreted GRP78 protein in blood increases with increasing arterial stenosis, and the present invention is completed based on such new findings. It came to.

  Arteriosclerosis markers such as sICAM-1, MMP-1, 2, 3, and 9 reported so far pay attention to the mechanism of arteriosclerosis (for example, the mechanism of unstable plaque). It was discovered by searching for molecules related to. On the other hand, the present inventors have found a secreted GRP78 protein as an arteriosclerosis marker having a completely new feature that has never been seen by focusing on endoplasmic reticulum stress, which is a completely new viewpoint. It was. It has not been reported so far that a patient with advanced arteriosclerosis can be screened using a biological sample such as blood, and the present inventors disclose for the first time here.

That is, the method according to the present invention is a method for detecting the possibility that the subject has developed advanced arteriosclerosis,
It includes a measurement step of measuring the concentration of secreted GRP78 protein in a biological sample obtained from the subject.

  According to the method for detecting the possibility that the subject according to the present invention has developed atherosclerosis (hereinafter referred to as “the method for detecting the possibility of developing atherosclerosis of the present invention”). Based on the concentration of the secreted GRP78 protein measured in the measurement step, the boundary is set in advance with reference to the concentration of the secreted GRP78 protein in a biological sample obtained from a patient who has developed advanced arteriosclerosis. When the value is higher than the value, it can be determined that the subject may develop advanced arteriosclerosis. As described above, conventionally, the detection of arteriosclerosis patients and the screening of patients with advanced arteriosclerosis are respectively performed by using two separate means: (i) detection of arteriosclerosis markers in biological samples and (ii) imaging diagnosis. According to the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention, the concentration of secreted GRP78 protein in a biological sample obtained from a subject is relatively Easily both arteriosclerosis patient discovery and advanced arteriosclerosis patient screening can be performed.

  In the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention, since the concentration of secreted GRP78 protein is measured, the cutoff value (boundary value) is clear. That is, in a subject who does not develop advanced arteriosclerosis (a normal subject or a subject whose arterial stenosis is lower than 75%), an increase in blood concentration of secreted GRP78 protein is not observed, Even if recognized, it is not a significant rise. On the other hand, the increase in the blood concentration of the secreted GRP78 protein is remarkable in the subject who develops advanced arteriosclerosis (the subject whose arterial stenosis degree is 75% or more). For this reason, there is no overlap between the range of the concentration of secreted GRP78 protein in the subject who develops advanced arteriosclerosis and the range of the concentration of secreted GRP78 protein in the subject who does not develop advanced arteriosclerosis. It is easy to determine whether or not the patient is an arteriosclerosis patient. Therefore, there is no risk of detecting false positives or false negatives, and the obtained results are highly reliable.

  Furthermore, in the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention, it is only necessary to measure the concentration of secreted GRP78 protein using a biological sample such as blood. It can be handled and is efficient. For this reason, compared with the conventional image diagnosis, it is possible to reduce the labor and cost for screening patients with advanced arteriosclerosis. As a result, it can contribute to reduction of medical expenses.

  Further, in the image diagnosis, when a contrast medium is used, complications such as allergic symptoms or shock symptoms to the contrast medium may occur. In contrast, in the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention, the concentration of secreted GRP78 protein in a biological sample (eg, blood) obtained from a subject may be measured. There is no risk of complications with contrast agents that are a concern in diagnostic imaging. Therefore, it can be said that this is a safer method than image diagnosis.

  The method for detecting the possibility that the subject according to the present invention has developed advanced arteriosclerosis can be used to detect the possibility that the sclerosis site in the artery of the subject has vulnerability.

  As described above, the method of detecting the possibility that the subject according to the present invention has developed advanced arteriosclerosis is performed by measuring the concentration of secreted GRP78 protein in a biological sample obtained from the subject. It is possible to detect the possibility that the specimen has developed advanced arteriosclerosis. In other words, the method for detecting the possibility that the subject according to the present invention has developed advanced arteriosclerosis is obtained by measuring the concentration of the secreted GRP78 protein in a biological sample obtained from the subject. It can be used to detect not only the degree of stenosis of the artery but also the possibility that the sclerosing site in the artery of the subject has vulnerability.

  Furthermore, since the concentration of secreted GRP78 protein in the blood increases as the hardening site becomes more fragile, by measuring the concentration of secreted GRP78 protein in the blood, not only the degree of stenosis of the subject's artery It is also possible to predict the degree (degree) of arterial vulnerability.

  In advanced arteriosclerosis, blood flow is reduced due to stenosis of blood vessels, but it is also a state where thrombosis is likely to occur due to the fragility of the arteriosclerosis site. Or cerebral infarction is likely to develop. Therefore, detecting the possibility that the sclerosis site in the artery of the subject is fragile leads to detecting the possibility of developing myocardial infarction and cerebral infarction, which is very effective.

  Conventionally, there is only a method for directly confirming the sclerosis site by image diagnosis for the sclerosis site vulnerability in the artery of the subject, and it has not been reported to detect the presence or absence of the sclerosis site using a specific protein as a marker. . According to the method of detecting the possibility that the subject according to the present invention has developed advanced arteriosclerosis, it is possible to detect the presence or absence of the fragility of the sclerosis site by a relatively simple method. It is possible to reduce the labor and cost for detecting the presence or absence of sex and contribute to the reduction of medical costs.

  The method of detecting the possibility that the subject according to the present invention has developed advanced arteriosclerosis can be used to detect the possibility that the subject has developed severe stenosis in the artery.

  As described above, the method of detecting the possibility that the subject according to the present invention has developed advanced arteriosclerosis is performed by measuring the concentration of secreted GRP78 protein in a biological sample obtained from the subject. It is possible to detect the possibility that the specimen has developed advanced arteriosclerosis. In other words, the method of detecting the possibility that the subject according to the present invention has developed advanced arteriosclerosis is obtained by measuring the concentration of secreted GRP78 protein in a biological sample obtained from the subject. It can be used to detect the possibility of developing severe stenosis in an artery.

  Furthermore, since the concentration of secreted GRP78 protein in blood increases as the degree of arterial stenosis increases, the degree of arterial stenosis can also be predicted by measuring the concentration of secreted GRP78 protein in blood. .

  In the method for detecting the possibility that the subject according to the present invention has developed atherosclerosis, the secretory GRP78 protein may be a fragment of the GRP78 protein.

  In the method for detecting a possibility that the subject according to the present invention has developed advanced arteriosclerosis, the biological sample may be blood.

The kit according to the present invention is a kit for detecting the possibility that a subject has developed advanced arteriosclerosis,
It is characterized by comprising at least measuring means for measuring the concentration of secreted GRP78 protein in a biological sample obtained from the subject.

  If it is the said structure, since the density | concentration of the secretory GRP78 protein in the biological sample acquired from the test object can be measured using the measurement means with which the kit was provided, the said test object developed advanced arteriosclerosis. The possibility of being present can be easily detected.

  In the kit according to the present invention, the measurement means is preferably an antibody that specifically recognizes secreted GRP78 protein.

  The kit according to the present invention includes an antibody that specifically recognizes secreted GRP78 protein as a measurement means, so that immunology such as ELISA (enzyme-linked immunosorbent assay) method, RIA (radio immunoassay) method, Western blot method and the like can be used. It is possible to measure the concentration of secreted GRP78 protein in a biological sample obtained from a subject by an automatic measurement method. Therefore, it is possible to easily detect the possibility that the subject has developed advanced arteriosclerosis.

  Further, by using the kit according to the present invention, it is possible to easily detect not only the degree of stenosis of the subject's artery but also the possibility that the sclerosing site in the artery of the subject has vulnerability. .

  The present invention is the use of secreted GRP78 protein as a marker for advanced arteriosclerosis.

  In addition, the present invention is an advanced arteriosclerosis marker comprising a secreted GRP78 protein.

  By using the secreted GRP78 protein as a marker for advanced arteriosclerosis, both the discovery of arteriosclerosis patients and the screening of patients with advanced arteriosclerosis can be performed relatively easily.

  In addition, secretory GRP78 protein develops advanced arteriosclerosis, whereas the expression level of the secreted GRP78 protein is markedly increased in subjects who develop advanced arteriosclerosis (subjects with arterial stenosis of 75% or more). In non-examined subjects (normal subjects or subjects whose arterial stenosis is lower than 75%), even if an increase in the expression of secreted GRP78 protein is observed or not, it is not a marked increase. For this reason, if the secretory GRP78 protein is used as an advanced arteriosclerosis marker, the cutoff value (boundary value) is clear, so there is no possibility of detecting false positives or false negatives. Therefore, the reliability of the obtained result is high.

  Furthermore, by using the secreted GRP78 protein as a marker for advanced arteriosclerosis, the concentration of the secreted GRP78 protein contained in the subject-derived sample is used as an index, not only the degree of stenosis of the subject's artery but also the subject. It is possible to detect the possibility that the sclerosis site in the artery of the specimen has vulnerability.

The method of monitoring the fragility of the sclerosis site in the artery of the subject according to the present invention,
It includes a measurement step of measuring the concentration of secreted GRP78 protein in a biological sample obtained from the subject.

  According to the method for monitoring the fragility of a sclerosis site in an artery of a subject according to the present invention, by measuring the concentration of secreted GRP78 protein in a biological sample obtained from the subject over time, It is possible to monitor the degree of vulnerability of the cured part. Specifically, the concentration of secreted GRP78 protein in a biological sample obtained from a subject is measured over time, and when the concentration of secreted GRP78 protein is lower than the previous measurement, the fragility of the hardening site in the artery Can be expected to be reduced. On the other hand, when the concentration of the secreted GRP78 protein is higher than that at the previous measurement, it can be predicted that the fragility of the sclerosing site in the artery is enhanced. As described above, having a fragile arterial sclerosis site means that the subject is in a pathological condition in which myocardial infarction or cerebral infarction is likely to occur. Therefore, monitoring the fragility of the sclerosis site in the artery of the subject is very effective because it also leads to monitoring the possibility of developing myocardial infarction and cerebral infarction.

  Furthermore, according to the method for monitoring the fragility of the sclerosis site in the artery of the subject according to the present invention, for example, the efficacy of the subject to which a prophylactic or therapeutic agent for arteriosclerosis is administered can be confirmed. .

  Note that Non-Patent Document 3 merely discloses that an increase in the expression of GRP78 protein in atherosclerotic lesions (ie, tissues) was observed. In general, even if protein expression is observed in a particular tissue, the protein is not necessarily secreted into the blood at a detectable concentration. So far, it has been reported that the GRP78 protein moves to the cell surface by endoplasmic reticulum stress and is secreted extracellularly (Non-Patent Documents 4 to 6), but it is reported that it is secreted into the blood. There is no. For this reason, just because the expression of GRP78 protein was observed in arteriosclerotic lesions, it cannot be predicted that secreted GRP78 protein is detected in the blood of patients with advanced arteriosclerosis. Furthermore, it is not predictable that the concentration of secreted GRP78 protein in the blood increases with increasing arterial stenosis and fragility of arterial sclerosis in patients with advanced arteriosclerosis.

  According to the present invention, both the discovery of arteriosclerosis patients and the screening of patients with advanced arteriosclerosis can be performed relatively easily by measuring the concentration of secreted GRP78 protein in a biological sample obtained from a subject. There is an effect.

  In patients with advanced arteriosclerosis, when the arterial stenosis or the fragility of the arteriosclerotic site increases, the concentration of secreted GRP78 protein in the blood increases accordingly. Therefore, by comparing the reference value set in advance based on the degree of arterial stenosis and the degree of vulnerability of the arterial sclerosis site with the concentration of secreted GRP78 protein in the biological sample, the degree of arterial stenosis in the subject Predicting the degree of fragility of the sclerosis site, or monitoring the fragility level of the sclerosis site in the artery of the subject by measuring the concentration of secreted GRP78 protein in the biological sample obtained from the subject over time There is an effect that can be.

  Furthermore, since the cutoff value is clear, the present invention does not have the possibility of detecting false positives or false negatives, and the results obtained are highly reliable. Moreover, since it is only necessary to measure the concentration of secreted GRP78 protein using a biological sample such as blood, a larger number of samples can be handled at a time, which is efficient.

  As described above, according to the present invention, it is possible to easily screen patients with advanced arteriosclerosis that really need treatment, which can contribute to reduction of medical costs.

It is a figure showing the result of having calculated the density | concentration of GRP78 protein contained in a serum sample, (a) is a test curve, (b) is calculating the density | concentration of GRP78 protein contained in each serum sample. It is a graph showing the result of having performed.

  Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to this, and can be implemented in a mode in which various modifications are made within the described range. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, “A to B” indicating a numerical range means “A or more and B or less”.

(1. Method for detecting the possibility of development of advanced arteriosclerosis)
The method according to the present invention for detecting the possibility that a subject has developed advanced arteriosclerosis (hereinafter referred to as “the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention”). This is a configuration including at least a measurement step of measuring the concentration of secreted GRP78 protein in a biological sample obtained from a specimen.

  Here, in the present specification, the “GRP78 protein” refers to heat shock 70 kDa protein 5 (Heat shock 70 kDa protein 5). The “GRP78 protein” may also be referred to as a 78 kDa glucose-regulated protein (GRP-78) or an immunoglobulin heavy chain-binding protein (BiP). is there.

  The “GRP78 protein” has been reported to have a function of stabilizing protein folding (see Non-Patent Document 3). New proteins such as membrane proteins and secreted proteins are folded and sugar chain modified in the endoplasmic reticulum, which is one of the organelles, and acquire a function as a protein. The GRP78 protein is present in the endoplasmic reticulum and promotes protein folding. Moreover, it has been reported that the “GRP78 protein” is secreted extracellularly (Non-Patent Documents 4 to 6). In the present specification, the above-mentioned “GRP78 protein” secreted extracellularly is specifically referred to as “secreted GRP78 protein” and is distinguished from “GRP78 protein” existing in the cell or on the cell membrane.

  Therefore, in the present specification, the “secreted GRP78 protein” is intended to be one in which the GRP78 protein present on the cell membrane is secreted outside the cell. That is, all fragments of the GRP78 protein that are secreted outside the cell and existed in a released state from the cell are included in the category of the “secreted GRP78 protein”.

  The amino acid sequence of GRP78 protein is registered in the database for various biological species. For example, human (Homo sapiens) GRP78 protein (Genbank, HSPA5 protein, 655 amino acids, Accession: AAI12964); rat (Rattus norvegicus) GRP78 protein (Genbank, Heat shock protein 5, 654 amino acids, Accession: AAH62017); mouse (Mus musculus) ) GRP78 protein (Genbank, heat shock 70kD protein 5 (glucose-regulated protein), 655 amino acids, Accession: CAM24607); Xenopus laevis GRP78 protein (Genbank, Hspa5 protein, 655 amino acids, Accession: AAH41200); zebrafish ( Danio rerio) GRP78 protein (Genbank, Heat shock protein 5, 650 amino acids, Accession: AAH63946); Bovine (Bos taurus) GRP78 protein (Genbank, heat shock 70 kDa protein 5, 655 amino acids, Accession: ABS45042) .

  In addition, the amino acid sequence of GRP78 protein is not limited to the amino acid sequence illustrated above, The variant of these amino acid sequences is also contained. That is, mutant proteins that can be generated by mutation are also included in the GRP78 protein in the description of the present invention. Specifically, the amino acid sequence exemplified above has an amino acid sequence in which one or several amino acids are deleted, substituted and / or added, and has an activity of stabilizing protein folding. Mutant proteins are also included in the category of GRP78 protein in the description of the present invention. Whether or not the mutant protein has an activity of stabilizing protein folding is measured by, for example, a known in vitro folding experiment. In this method, the protein of interest denatured with urea or the like is diluted in a large excess of buffer solution, and at this time, by adding GRP78 protein or mutant GRP78 protein to the buffer solution, the effect on folding (folding) is exerted. (See “Endo Toshiya et al.,“ Maturation, Transport, and Quality Control of Protein Lifetime Concentrated Master Cells ”, Yodosha Publishing, February 2007, page 29”). For example, when the target protein is an enzyme, it can be determined whether or not the mutant protein has an activity of stabilizing protein folding by confirming the recovery of the enzyme activity of the target protein.

  Here, the above “one or several amino acids have been deleted, substituted and / or added” means deletion, substitution and / or addition by a known mutant peptide production method such as site-directed mutagenesis. As many amino acids as possible (for example, 20 or less, preferably 10 or less, more preferably 7 or less, further preferably 5 or less, particularly preferably 3 or less) are deleted, substituted and / or added. Means that. The site at which one or several amino acids are deleted, substituted and / or added may be the site if the protein after the deletion, substitution and / or addition of amino acids has activity as a GRP78 protein. Any site in the amino acid sequence may be used.

  Preferred variants have conservative or non-conservative amino acid substitutions, deletions or additions. Silent substitution, addition and deletion are preferred, and conservative substitution is particularly preferred. These mutations do not change the activity of the protein.

  Typically seen as conservative substitutions are substitutions of one amino acid for another in the aliphatic amino acids Ala, Val, Leu, and Ile, exchange of hydroxyl residues Ser and Thr, acidic residues Asp and Glu exchange, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe, Tyr.

  The “secreted GRP78 protein” is not particularly limited as long as it is a fragment of the GRP78 protein that is secreted outside the cell and is released from the cell. For example, it is detected in human serum. The “secreted GRP78 protein” can be a fragment of the above-mentioned human GRP78 protein (Genbank, Accession: AAI12964) or a mutant protein thereof. More specifically, it is a protein that specifically binds to an antibody provided in a commercially available GRP measurement kit ELISA (Uscn E92343Hu, Life Science Inc, Wuhan, China) used in Examples described later.

  For example, the amino acid sequence of the “secreted GRP78 protein” detected in human serum is, for example, an antibody that specifically recognizes GRP78 protein using human serum (for example, the commercially available GRP used in the Examples). It is possible to perform immunoprecipitation using the antibody provided in the measurement kit ELISA) and then identify using mass spectrometry.

  In the present specification, the term “protein” is used interchangeably with “polypeptide” or “peptide”.

  Further, in the present specification, the above-mentioned “advanced arteriosclerosis” intends arteriosclerosis in which the degree of arterial stenosis is 75% or more.

  The degree of arterial stenosis is usually measured by methods such as ultrasonic examination, CT contrast examination, and arteriography. In the image data obtained by these methods, it can be determined that the degree of stenosis of the artery is 75% or more when the blood vessel lumen is in a state of being constricted by 75% or more. In the present specification, a stenosis having a stenosis degree of 75% or more is particularly referred to as “high stenosis”.

  Furthermore, in the method according to the present invention for detecting the possibility that the subject has developed advanced arteriosclerosis, the concentration of the secreted GRP78 protein contained in the subject-derived sample is used as an index, and the artery of the subject is examined. It is possible to detect not only the degree of stenosis but also the possibility that the sclerosis site in the artery of the subject has vulnerability (vulnerability).

  Vulnerability in arterial sclerosis is confirmed by ultrasound, magnetic resonance imaging (MRI), positron-emission tomography (PET), and intravascular ultrasound as an invasive method. Attempts to depict unstable plaques using optical coherence tomography (OCT), vascular endoscopes, etc. have been actively carried out (“Takaya Norihide et al.,“ Diagnosis of arteriosclerosis ”, (See Angiology Vol. 48, 2008, P456-461). Vulnerability at arteriosclerotic sites is usually measured by carotid echo and can be assessed using echo intensity inside the carotid plaque, plaque surface morphology, plaque stability, plaque mobility, etc. as indicators (“Ozaki (See Toshiya, “Practical Carotid Echo”, Thrombostasis, 19 (1): 35-38, 2008)).

  A fragile atherosclerotic lesion with a relatively large lipid core covered with a pathologically thin fibrous cap is referred to as an “unstable plaque”. In addition, the above-mentioned fibrous capsule is very thin, and a plaque with partial inflow of blood flow is called “moile plaque”, and the fibrous capsule is very thin and has a stick-like or pedunculated shape. Plaques exhibiting a special form are called “floating plaques”. The “plaque” refers to “a patchy hypertrophic lesion of the intima present at the arteriosclerotic site”.

  The site that develops advanced arteriosclerosis is not particularly limited. For example, constriction of blood vessels in the carotid artery, coronary artery, aorta, etc. is contemplated.

  The subject to be detected by the method for detecting the possibility of developing advanced arteriosclerosis of the present invention is not particularly limited as long as it is a subject capable of developing arteriosclerosis. For example, dogs, cats, Examples include non-human mammals such as sheep, goats, mice, rats, rabbits, cows, horses, pigs, and humans in general. In addition, the subject to be detected by the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention may be a person who has already been diagnosed as having developed arteriosclerosis. It may be a person who has not yet been diagnosed as having developed the disease. By applying the method for detecting the possibility of developing advanced arteriosclerosis of the present invention to a person who has already been diagnosed as having developed arteriosclerosis, the arteriosclerosis in which the subject has developed is highly advanced. Whether or not it is arteriosclerosis can be detected. Further, by applying the method for detecting the possibility of developing arteriosclerosis of the present invention to a person who has not yet been diagnosed as having developed arteriosclerosis, the subject develops arteriosclerosis. It can be detected whether or not.

  The method for detecting the possibility of developing advanced arteriosclerosis according to the present invention is based on the possibility that the sclerosing site in the artery of the subject is vulnerable and / or the subject develops severe stenosis in the artery. It can be suitably used to detect the possibility of Further, it may be a method for determining the possibility of development of advanced arteriosclerosis or a method for acquiring data for determining the possibility of development of advanced arteriosclerosis. When the present invention is a method for acquiring data for determining the possibility of development of advanced arteriosclerosis, a determination step by a doctor is not included.

(I) Measurement Step In the measurement step, a method for measuring the concentration of secreted GRP78 protein in a biological sample obtained from a subject (hereinafter referred to as “subject-derived sample”) is a secreted type in the subject-derived sample. The method is not particularly limited as long as it can measure the concentration of GRP78 protein, and any conventionally known method can be used.

  For example, a substance that specifically interacts with the secreted GRP78 protein in the sample derived from the subject, for example, an antibody that specifically recognizes (binds) the secreted GRP78 protein (hereinafter referred to as “anti-secreted GRP78 antibody”). And using a known immunological measurement method such as ELISA, RIA, Western blot, immunoprecipitation, antibody array, etc. as appropriate.

  Of the above measurement methods, the ELISA method can be suitably used because it is more sensitive and simple. The specific conditions for these measurement methods are matters that can be appropriately set by those skilled in the art.

  In the ELISA method, the protein contained in the specimen-derived sample is immobilized on a multiwell plate (also referred to as “microtiter plate”), and then the secreted GRP78 protein is detected by an antibody that specifically recognizes the secreted GRP78 protein. This is a method for detecting the concentration. An antibody that specifically recognizes the secreted GRP78 protein may be detected using, for example, an anti-IgG antibody labeled with alkaline phosphatase or peroxidase as a secondary antibody. The ELISA method may be a sandwich method.

The “anti-secretory GRP78 antibody” can be produced by using various known methods using the secreted GRP78 protein as an antigen, and the production method is not particularly limited. The above-mentioned “antibody” means an immunoglobulin (IgA, IgD, IgE, IgY, IgG, IgM and their Fab fragment, F (ab ′) ₂ fragment, Fc fragment). Although it may be an antibody, it is preferably a monoclonal antibody.

  In the present specification, the “biological sample obtained from the subject (subject-derived sample)” is not particularly limited as long as it can detect the secreted GRP78 protein. For example, blood (serum, plasma) In addition, lymph fluid, urine, pathological specimen samples (for example, samples obtained by carotid artery detachment), and the like can be used. As a method for preparing the specimen-derived sample, a known method may be appropriately performed. For example, when preparing serum, blood is obtained from a subject, blood is centrifuged (for example, 1500 × g, 10 minutes), and the supernatant may be obtained.

  The inventors of the present invention have a marked increase in the expression of secreted GRP78 protein in patients who develop severe arteriosclerosis in the carotid artery (patients whose arterial stenosis is 75% or more). In a subject who has not developed arteriosclerosis (a normal subject or a subject whose arterial stenosis is lower than 75%), even if an increase in the expression of secreted GRP78 protein is observed or not, it is remarkable. I found that it was not a rise. Based on this knowledge, it is possible to determine whether or not the subject may develop advanced arteriosclerosis using the concentration of secreted GRP78 protein contained in the subject-derived sample as an index.

  Furthermore, in the method according to the present invention for detecting the possibility that the subject has developed advanced arteriosclerosis, the concentration of the secreted GRP78 protein contained in the subject-derived sample is used as an index, and the artery of the subject is examined. It is possible to detect not only the degree of stenosis but also the possibility that the sclerosing site in the artery of the subject has vulnerability.

  A method for determining whether or not the subject may develop advanced arteriosclerosis based on the concentration of secreted GRP78 protein measured in the measurement step is not particularly limited. A boundary value (cut-off value) that can determine whether or not there is a possibility of developing sclerosis is set in advance, and the secretory GRP78 protein contained in the sample derived from the subject is more than the boundary value. Whether or not the subject is likely to develop advanced arteriosclerosis can be determined based on whether or not the concentration is high.

  Note that the “boundary value” may vary depending on the species, sex, lifestyle, type of sample used, and the like, and therefore needs to be set in advance accordingly. In the present invention, the “boundary value” can be set by measuring the concentration of secreted GRP78 protein in a biological sample obtained from a patient who has developed advanced arteriosclerosis and referring to the measured value. As a specific method for setting the “boundary value”, for example, “mean value ± standard deviation of the concentration of secreted GRP78 protein in a biological sample obtained from a patient who has developed advanced arteriosclerosis” or ROC (Receiver Operating Characteristic) analysis.

  According to the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention, the boundary value thus set is compared with the concentration of secreted GRP78 protein in the sample derived from the subject. When the type GRP78 protein concentration is higher than the boundary value, it can be determined that there is a possibility that the subject has developed advanced arteriosclerosis. Conversely, if the concentration of secreted GRP78 protein in the subject-derived sample is lower than the boundary value, it can be determined that there is no possibility that the subject has developed advanced arteriosclerosis.

  In addition, according to the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention, the sclerosing site in the artery of the subject is vulnerable based on the concentration of the secreted GRP78 protein measured by the measuring step. It can be determined whether or not there is a possibility. The determination method is not particularly limited. For example, a boundary value that can be used to determine whether or not there is a possibility that the sclerosis site in the artery of the subject has vulnerability is set in advance. Determining whether or not there is a possibility that the sclerosis site in the artery of the subject may be vulnerable depending on whether the concentration of the secreted GRP78 protein contained in the subject-derived sample is high Can do. For example, the boundary value is compared with the concentration of the secreted GRP78 protein in the subject-derived sample, and when the concentration of the secreted GRP78 protein is higher than the boundary value, the hardening site in the artery of the subject Can be determined to have a vulnerability. Conversely, if the concentration of the secreted GRP78 protein in the subject-derived sample is lower than the boundary value, it is determined that there is no possibility that the sclerosis site in the artery of the subject has vulnerability. be able to.

  In addition, according to the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention, the subject has developed severe stenosis in the artery based on the concentration of secreted GRP78 protein measured by the measurement step. It can be determined whether there is a possibility. Although the determination method is not particularly limited, for example, a boundary value that can be used to determine whether or not the subject may develop severe stenosis in an artery is set in advance, and the subject is more than the boundary value. Whether or not there is a possibility that the subject has developed severe stenosis in the artery can be determined based on whether or not the concentration of the secreted GRP78 protein contained in the derived sample is high.

  Further, according to the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention, the concentration of the secreted GRP78 protein measured in the measurement step is set to the degree of vulnerability and / or the degree of stenosis of the artery. It is also possible to predict the degree of fragility of the sclerosis site in the arteries of advanced arteriosclerosis patients and / or the degree of stenosis of the arteries in patients with advanced arteriosclerosis by comparing with reference values set in advance.

  As described above, the fragility of the arteriosclerotic site may be caused by ultrasound, MRI, PET, and attempts to depict unstable plaque using intravascular ultrasonography, OCT, vascular endoscope, etc. as invasive methods. (Refer to “Norihide Takatani et al.,“ Image diagnosis of arteriosclerosis ”, Angiology Vol. 48, 2008, P456-461”). Vulnerability at arteriosclerotic sites is usually measured by carotid echo and can be assessed using echo intensity inside the carotid plaque, plaque surface morphology, plaque stability, plaque mobility, etc. as indicators (“Ozaki (See Toshiya, “Practical Carotid Echo”, Thrombostasis, 19 (1): 35-38, 2008)).

  For example, in contrast to the pathological tissue, the echo intensity is considered to be atheroma or hematoma in the case of low brightness, fibrous tissue in the case of equal brightness, calcified lesion in the case of high brightness, and hardening in the case of low brightness. It is judged that the vulnerability of the part is high. That is, for example, when measured by carotid artery echo, when the echo intensity of the plaque is high in contrast to the pathological tissue, the degree (degree) of vulnerability of the arterial sclerosis site (also referred to as “vulnerability”). ) Is judged to be “mild”, and the degree of vulnerability of the arteriosclerotic site is judged to be “medium” when the echo intensity of the plaque is equal in contrast to the pathological tissue. When the plaque echo intensity is low in contrast to the tissue, the degree of vulnerability of the arteriosclerotic site is determined to be “severe”.

  The degree of arterial sclerosis vulnerability is related to the likelihood of thrombosis and, in turn, the likelihood of developing myocardial and cerebral infarction. It is effective to predict the vulnerability level.

  As described above, in patients with advanced arteriosclerosis, when the degree of arterial stenosis and the degree of arteriosclerotic site fragility increase, the concentration of secreted GRP78 protein in the blood increases accordingly. By comparing the concentration of the secreted GRP78 protein to a preset reference value based on sclerosis site fragility and / or arterial stenosis degree, and / or sclerosis site fragility in arteries of advanced arteriosclerosis patients and / or Alternatively, the degree of arterial stenosis in patients with advanced arteriosclerosis can be predicted.

  Specifically, when predicting the degree of sclerosis in the arteries of patients with advanced arteriosclerosis, measure the concentration of secreted GRP78 protein in biological samples obtained from patients with various arteriosclerosis having various fragility levels. The reference value may be set for each degree of vulnerability of the cured portion with reference to the measured value. When predicting the degree of arterial stenosis in patients with advanced arteriosclerosis, the concentration of secreted GRP78 protein in biological samples obtained from patients with advanced arteriosclerosis having various degrees of stenosis is measured, and the measured values are referred to. A reference value may be set for each degree of arterial stenosis. The above “reference value” can vary depending on the type of specimen, sex, lifestyle, type of sample used, etc., and can vary depending on the degree of vulnerability or stenosis of the assumed hardening site. It is necessary to set in advance. A specific method for setting the “reference value” can be described by replacing “boundary value” with “reference value” in the description of the method for setting the “boundary value”.

  In the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention, the reference value thus set for each fragility of the arterial sclerosis site and the secreted GRP78 protein in the sample derived from the subject are used. For example, if the concentration of the secreted GRP78 protein is higher than the reference value where the degree of sclerosis of the arterial sclerosis site is set as “mild”, the sclerosis site of the arteries of the subject It can be determined that there is a possibility that the vulnerability is “mild” or higher (that is, “mild”, “medium”, or “severe”). In addition, when the concentration of the secreted GRP78 protein is higher than the reference value at which the sclerosis degree of the arterial sclerosis site is set as “medium”, the fragility of the arterial sclerosis site of the subject is “medium” ”Degree” or more (ie, “medium” or “severe”), and the sclerosis level of the arterial sclerosis site is higher than the reference value set as “severe” It can be determined that the fragility of the arterial sclerosis site of the subject may be “severe”. Conversely, the concentration of the secreted GRP78 protein in the sample derived from the subject is lower than the reference value where the fragility of the arterial sclerosis site is set as “severe”, and the fragility of the arterial sclerosis site is “moderate” ”Is higher than the reference value set as”, it can be determined that the vulnerability of the arterial sclerosis site of the subject may be “moderate”, Is lower than the reference value set as “medium” and the vulnerability of the arterial stiffening site is higher than the reference value set as “mild”, the vulnerability of the arterial stiffening site of the subject Can be determined to be “mild”.

  Similarly, in the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention, as described above, the reference value set for each degree of stenosis of the artery and the secretory GRP78 in the sample derived from the subject. When the concentration of the secreted GRP78 protein is higher than a reference value set as an arterial stenosis degree of 80% or higher, the subject has a stenosis degree of 80% or higher. It can be determined that there is a possibility of developing advanced arteriosclerosis. Further, when the concentration of the secreted GRP78 protein is higher than the reference value set as the arterial stenosis degree of 90% or more, the subject develops advanced arteriosclerosis having a stenosis degree of 90% or more. It can be determined that there is a possibility. Conversely, the concentration of the secreted GRP78 protein in the sample derived from the subject is lower than the reference value set as the arterial stenosis degree of 90% or higher and higher than the reference value set as the arterial stenosis degree of 80% or higher. In this case, it can be determined that there is a possibility that the subject has developed atherosclerosis having a stenosis degree of 80% or more and less than 90%.

(Ii) Others The method for detecting the possibility of developing advanced arteriosclerosis according to the present invention includes, in addition to the above steps, (i) secretory GRP78 in a biological sample obtained from a patient developing advanced arteriosclerosis. A step of measuring a protein concentration and setting a boundary value with reference to the measured value; (ii) a secreted GRP78 protein in a biological sample obtained from a patient with advanced arteriosclerosis having various arteriosclerotic site fragility levels; A step of measuring the concentration and referring to the measured value to set a reference value capable of determining the fragility of the arterial sclerosis site; (iii) in a biological sample obtained from patients with advanced arteriosclerosis having various degrees of stenosis A step of measuring the concentration of secreted GRP78 protein and referring to the measured value to set a reference value from which the degree of arterial stenosis can be determined; (iv) the secreted GRP78 protein measured in the measuring step The method may further include a step of comparing the quality concentration with the boundary value, and (iv) a step of comparing the concentration of the secreted GRP78 protein measured in the measurement step with the reference value. The method for setting the “boundary value” and the “reference value” is as described in the section “(i) Measurement step”.

  Since the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention has the above-described configuration, it is possible to detect the possibility that the subject has developed advanced arteriosclerosis. Therefore, according to the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention, it is possible to screen highly arteriosclerotic patients (high risk patients) with high accuracy and simply. As a result, an appropriate treatment can be performed on a patient with advanced arteriosclerosis that really needs treatment, so that medical costs can be reduced.

  Note that the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention may be performed in combination with conventional image diagnosis (for example, ultrasonic examination, CT). The accuracy of diagnosis can be improved by performing further image diagnosis on patients with advanced arteriosclerosis screened by the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention. In addition, since the method for detecting the possibility of developing advanced arteriosclerosis according to the present invention can be used to screen patients with advanced arteriosclerosis by a simple method, the number of specimens used for image diagnosis can be reduced as compared with the prior art. it can. For this reason, the effort and cost concerning image diagnosis can be reduced, and as a result, the medical cost can be reduced.

(2. Kit for detecting the possibility of development of advanced arteriosclerosis)
A kit (hereinafter referred to as “kit of the present invention”) for detecting the possibility that a subject according to the present invention has developed atherosclerosis is a secreted GRP78 protein in a biological sample obtained from the subject. It is the structure provided with the measurement means for measuring the density | concentration of at least.

  The “measuring means” is not particularly limited as long as it is a substance that specifically interacts with the secreted GRP78 protein. As such a substance, for example, an antibody (anti-secretory GRP78 antibody) that specifically recognizes (binds) a secretory GRP78 protein can be mentioned. The concentration of secreted GRP78 protein can be measured by carrying out known methods such as ELISA, RIA, Western blot, immunoprecipitation, and antibody array using such a measuring means. For a specific method for measuring the concentration of secreted GRP78 protein in a “secreted GRP78 protein”, “antisecretory GRP78 antibody”, and a sample derived from a subject, the above-mentioned “1. As described in the section "How to do".

  One type of the “measuring means” may be provided alone, or two or more types may be provided in combination. For example, when the concentration of secreted GRP78 protein in a sample derived from a subject is measured by sandwich ELISA, the kit according to the present invention uses two types of “recognition means” that recognize different epitopes of secreted GRP78 protein. An antisecretory GRP78 antibody may be provided.

  When the secreted GRP78 protein is measured by an immunological technique (for example, ELISA, Western blot, etc.), the kit of the present invention includes a configuration for performing the immunological technique, for example, a labeled secondary antibody. In addition, one or more coloring reagents, washing buffer solutions, and the like may be combined and included.

  Since the kit according to the present invention has the above-described configuration, by using the kit according to the present invention, it is possible to easily detect the possibility that the subject has developed advanced arteriosclerosis.

  The kit according to the present invention can be suitably used to detect the possibility that a sclerosing site in an artery of a subject has vulnerability. Moreover, the kit according to the present invention can be suitably used for detecting the possibility that the subject has developed severe stenosis in the artery.

(3. Use of advanced arteriosclerosis marker and secretory GRP78 protein as advanced arteriosclerosis marker)
The advanced arteriosclerosis marker according to the present invention is composed of a secretory GRP78 protein.

  The present invention also provides the use of secreted GRP78 protein as a marker for advanced arteriosclerosis.

  In the present specification, the “advanced arteriosclerosis marker” is a substance that serves as an index for detecting the possibility of developing advanced arteriosclerosis, and the secretory GRP78 protein corresponds to this substance. Since the secretory GRP78 protein can be used as a marker for advanced arteriosclerosis, whether or not the subject may develop advanced atherosclerosis by measuring the concentration of the secreted GRP78 protein in the subject-derived sample. Can be detected. For example, a boundary value that can be used to determine whether or not the subject has developed atherosclerosis is set in advance, and the concentration of the secreted GRP78 protein contained in the subject-derived sample is higher than the boundary value. Whether or not the subject is likely to develop advanced arteriosclerosis can be detected.

  The specific method for measuring the concentration of the secreted GRP78 protein in the “secreted GRP78 protein” and the sample derived from the subject is as described in the above section “1. Method for detecting the onset of advanced arteriosclerosis”. It is.

  As described above, since the secretory GRP78 protein has a marked increase in expression in a subject developing advanced arteriosclerosis, the subject can be used as a marker for advanced arteriosclerosis by using the secreted GRP78 protein as a highly arteriosclerosis marker. The possibility of developing sclerosis can be easily detected.

  Furthermore, since the secreted GRP78 protein can be used as a “marker of the fragility of the sclerosis site in arteries”, by measuring the concentration of the secreted GRP78 protein in the subject-derived sample, It can be detected whether there is a possibility that the cured portion has vulnerability. In addition, since the secreted GRP78 protein can be used as an “arterial stenosis marker”, the subject develops severe stenosis in the artery by measuring the concentration of the secreted GRP78 protein in the subject-derived sample. It is possible to detect whether or not there is a possibility.

(4. Method of monitoring the degree of vulnerability of arterial sclerosis)
According to the present invention, the method for monitoring the fragility of a sclerosis site in an artery of a subject (hereinafter referred to as “the monitoring method of the present invention”) measures the concentration of secreted GRP78 protein in a biological sample obtained from the subject. It is the structure which includes the measurement process to perform at least. The “degree of fragility of the hardening site in the artery of the subject” means “the degree (degree) of the fragility of the hardening site in the artery of the subject”. The method for determining the degree of fragility of the sclerosis site in the artery of the subject is as described in the section “1. Method for detecting the onset of advanced arteriosclerosis”.

  The method for measuring the concentration of secreted GRP78 protein in the sample derived from the subject is as described in the section “1. Method for detecting the onset of advanced arteriosclerosis”.

  In patients with advanced arteriosclerosis, when the fragility of the sclerosis site in the artery increases, the amount of GRP78 protein secreted in the blood increases, so the subject to be monitored for the fragility of the sclerosis site in the artery is derived from the subject. By measuring the concentration of the secreted GRP78 protein in the sample a plurality of times over time, it becomes possible to monitor the fragility of the sclerosis site in the artery of the subject.

  Specifically, the concentration of secreted GRP78 protein in a biological sample obtained from a subject to be monitored for the degree of vulnerability of the sclerosis site in the artery is measured, and the concentration of secreted GRP78 protein in the sample derived from the subject is measured at the previous measurement. If it is reduced from the previous measurement, it can be determined that the degree of fragility of the sclerosis site in the artery has decreased since the previous measurement. Conversely, if the concentration of secreted GRP78 protein in the sample derived from the subject is higher than that at the previous measurement, it can be determined that the degree of fragility of the sclerosis site in the artery has increased since the previous measurement.

  By performing such monitoring, it is possible to easily select a therapeutic agent for advanced arteriosclerosis, determine its efficacy, and the like in a patient diagnosed with advanced arteriosclerosis. Further, in patients who have developed arteriosclerosis but have not yet been diagnosed with advanced arteriosclerosis, it is possible to easily select a prophylactic agent for advanced arteriosclerosis, determine its efficacy, and the like.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by an Example.

(1) Preparation of serum sample Venous blood of carotid artery echo case (6 cases) was collected and centrifuged (3000 rpm (1500 × g), 10 minutes), and then serum was obtained. The obtained serum samples were stored at −80 ° C. until used for experiments.

(2) Experimental method All kits and serum samples were thawed on ice before the experiment. A GRP78 control solution, solution A, solution B, and wash solution were prepared based on a procedure manual attached to a commercially available GRP measurement kit ELISA (Uscn E92343Hu, Life Science Inc, Wuhan, China).

(Preparation of GRP78 control solution)
Standard (GRP78 protein) was dissolved in a diluent (100 ng / ml) and allowed to stand at room temperature for 10 minutes. Thereafter, GRP78 control solutions with various protein concentrations were prepared in order (10 ng / ml, 5 ng / ml, 2.5 ng / ml, 1.25 ng / ml, 0.625 ng / ml, 0.312 ng / ml). , 0.156 ng / ml).

(Production of liquid A)
The 2 × A solution was diluted 2 times with distilled water, and the 100 × A reagent was diluted 100 times with the A solution.

(Production of liquid B)
The 2 × B solution was diluted 2 times with distilled water, and the 100 × B reagent was diluted 100 times with the B solution.

(Preparation of wash solution)
The 30 × wash solution was diluted 30 times with distilled water.

  Serum samples and GRP78 control solution were added in 100 μL each to a 96-well plate and incubated at 37 ° C. for 2 hours. Thereafter, the serum sample and the control solution were aspirated, 100 μL of solution A was added, and the mixture was further incubated at 37 ° C. for 1 hour. After the A liquid was aspirated, it was washed 3 times with 350 μL of Wash liquid. Thereafter, 100 μL of solution B was added and kept at 37 ° C. for 30 minutes. The liquid B was aspirated and washed 5 times with 350 μL of Wash liquid. 90 μL of a luminescent solution (TMB substrate) was added and incubated at 37 ° C. for 15-25 minutes. 50 μL of the reaction stop solution attached to the ELISA kit was added, and immediately the absorbance was measured with a Multi Spectrometer (Viento, Dainippon Pharmaceutical CO. Ltd, Osaka, Japan) (450 nm). Next, a calibration curve was prepared based on the measurement result of the absorbance of the GRP78 control solution. Based on the obtained calibration curve, the concentration of GRP78 protein contained in the serum sample was calculated. The results are shown in Table 1 and FIG.

  FIG. 1 is a diagram showing the results of calculating the concentration of GRP78 protein contained in a serum sample, (a) is a test curve, and (b) is the GRP78 protein contained in each serum sample. It is a graph showing the result of having calculated the density | concentration of.

  In cases 4 to 6 diagnosed as having high arteriosclerosis (stenosis of 75% or more) by carotid echo, it was confirmed that the amount of GRP78 protein secreted into the blood was increased. On the other hand, secretion of GRP78 protein into the blood was not observed in cases 1 and 2 diagnosed as having no arteriosclerosis (degree of stenosis 0%) by carotid artery echo. In case 3 with a stenosis of 65%, GRP78 protein was slightly secreted into the blood, but the amount of GRP78 protein secreted was significantly increased compared to cases 1 and 2 that did not develop arteriosclerosis. I didn't think it was. Specifically, in Non-Patent Document 3 (Masafumi Myoishi et al., “Increased Endoplasmic Reticulum Stress in Atherosclerotic Plaques Associated With Acute Coronary Syndrome”, Circulation 116 (2007) p.1226-1233), the present inventors When the coronary artery stenosis is moderate, the expression of GRP78 protein at the arteriosclerosis site hardly increases, whereas the histology of the basic experiment shows that it has high stenosis + vulnerability (vulnerability) Have reported a rapid increase in the expression of the GRP78 protein. Based on such basic experimental data, the concentration of secreted GRP78 protein in serum increases rapidly in cases of severe stenosis, but in the case of 65% stenosis with moderate stenosis, secreted GRP78 protein in serum. It was judged that the concentration of was not increased.

  Furthermore, it was confirmed that in patients with advanced arteriosclerosis, when the degree of arterial stenosis increases, the amount of GRP78 protein secreted into the blood tends to increase.

  From this result, it was confirmed that by measuring the amount of GRP78 protein secreted into the blood, it is possible to select patients with arteriosclerosis, particularly those with advanced arteriosclerosis that require treatment. Furthermore, in patients with advanced arteriosclerosis, when the degree of stenosis of the arteries increases, the amount of GRP78 protein secreted into the blood tends to increase. Therefore, by measuring the amount of GRP78 protein secreted in the blood, This suggests the possibility of predicting the degree of stenosis.

  As described above, in the method of detecting the possibility that the subject according to the present invention has developed advanced arteriosclerosis, the concentration of the secreted GRP78 protein in the biological sample obtained from the subject is measured. Can accurately acquire data for determining the possibility of developing advanced arteriosclerosis. For this reason, patients with advanced arteriosclerosis that really need treatment can be selected from patients with arteriosclerosis. Moreover, the marker and kit according to the present invention are useful for detecting the possibility that the subject according to the present invention has developed advanced arteriosclerosis. Therefore, the present invention can be widely used not only in the field of diagnostic medicine such as medical devices and diagnostic kits but also in the industry in the field of health medicine.

  In addition, the method for monitoring the degree of arterial stenosis in a subject according to the present invention can be used for development of therapeutic agents and treatment methods for advanced arteriosclerosis. Therefore, the present invention can be widely used in industries in the life science field including the pharmaceutical field.

Claims (10)

A method for detecting the possibility that a subject has developed atherosclerosis,
A method comprising a measurement step of measuring the concentration of secreted GRP78 protein in a biological sample obtained from the subject.   The method according to claim 1, wherein the method is used to detect a possibility that a sclerosis site in an artery of the subject has vulnerability.   The method according to claim 1, wherein the subject is used to detect a possibility that the artery has developed severe stenosis.   The method according to any one of claims 1 to 3, wherein the secreted GRP78 protein is a fragment of GRP78 protein.   The method according to claim 1, wherein the biological sample is blood. A kit for detecting the possibility that a subject has developed atherosclerosis,
A kit comprising at least measuring means for measuring the concentration of secreted GRP78 protein in a biological sample obtained from the subject.   The kit according to claim 6, wherein the measuring means is an antibody that specifically recognizes a secreted GRP78 protein.   Use of secreted GRP78 protein as a marker for advanced arteriosclerosis.   An advanced arteriosclerosis marker comprising a secreted GRP78 protein. A method for monitoring the fragility of a sclerosis site in an artery of a subject,
A method comprising a measurement step of measuring the concentration of secreted GRP78 protein in a biological sample obtained from the subject.

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