JPWO2012067150A1 - Method for examining cerebral infarction with Interleukin-6 receptorsubunitbeta protein - Google Patents

Abstract

A test method for diagnosing cerebral infarction in the animal, comprising a step of measuring the amount of Interleukin-6 receptor subunit beta protein in a blood sample collected from the test animal, and a test for diagnosing the cerebral infarction disease type And testing methods for predicting the prognosis of cerebral infarction.

Description

  The present invention relates to a method for examining cerebral infarction, a method for evaluating cerebral infarction treatment or prevention effect, a method for screening for cerebral infarction preventive or therapeutic agent, and cerebral infarction examination, cerebral infarction treatment effect evaluation or cerebral infarction prevention / treatment. The present invention relates to a drug candidate compound screening kit.

  Stroke is a general term for cerebrovascular disorders such as cerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage. Of these stroke types, cerebral infarction has been relatively increasing recently. Thrombolytic therapy, anticoagulant therapy, antiplatelet therapy, administration of cerebral protective drugs, etc. are used as drug therapy in the acute phase of cerebral infarction. Surgical surgery is performed for patients with severe or hemorrhagic cerebral infarction. Is done. Cerebral infarction symptoms are strongest in the acute phase and then gradually improve. This is because the necrotic brain tissue is swollen and the surrounding brain tissue is also compressed and damaged. As the swelling subsides, the surrounding tissues recover their function and the symptoms are fixed. However, since free radicals released from cerebral ischemic sites have the function of necrosing surrounding tissues, continuous treatment is required to improve the functional prognosis even after the acute phase.

The clinical types of cerebral infarction include lacunar infarction that develops when the intracerebral small artery is occluded, atherothrombotic cerebral infarction that develops when the intracerebral aorta is occluded by atheroma, and a thrombus in the heart becomes an obturator. Examples include cardiogenic cerebral embolism that develops by blocking an artery. Since appropriate treatment methods for the acute and chronic phases differ depending on these clinical types, a method for rapidly diagnosing the clinical type of patients with cerebral infarction has been demanded.
As a method of classifying clinical types of cerebral infarction, clinical symptom observation and echocardiography, MRI, MRA, cervical vascular echo, etc. are performed, and a flowchart of clinical diagnosis of cerebral infarction based on TOAST classification (Non-patent Document 1), etc. As a result of simultaneous measurement of molecular markers such as CRP, D-Dimer, RAGE, MMP-9, S100B, BNP within 24 hours of cerebral infarction, Although it has been disclosed that cardiogenic cerebral embolism can be classified as another disease type by setting a specific cutoff value with BNP and D-Dimer (Non-patent Document 2), etc. There was a need for molecular markers that could be diagnosed.
The prognosis of cerebral infarction is to evaluate the degree of movement disorder in daily life by the modified Rankin Scale (Non-patent Document 3) or Barthel Index (Non-patent Document 4) between 3 months or 1 year after onset. It is judged by. Predicting the prognosis in advance is important for early determination of a treatment method for cerebral infarction. Although it has been disclosed that death can be predicted by setting a specific cutoff value with BNP in plasma measured at the time of admission (Non-patent Document 5), a molecular marker for predicting the degree of injury when alive is required It was.

Lee, L.J. et al., Stroke, 1081-1089 (2000) Montaner et al., Stroke, 2280-2287 (2008) van Swieten, J.C et al., Stroke, 604-607 (1988) Mahoney, F.L et al., Maryland State Med J, 61-65 (1965) Shibazaki, K et al., Inter Med, 1601-1604 (2009)

  In view of the above problems, the present invention has an object to provide a method for quickly and surely inspecting cerebral infarction using serum or plasma of patients with cerebral infarction and the like, and a method for evaluating cerebral infarction treatment effects. To do.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that Interleukin-6 receptor subunit beta protein in patient plasma differs between its concentrations in plasma of patients with different disease types and prognosis. Thus, the present inventors have found that the amount of Interleukin-6 receptor subunit beta protein is a useful index for the examination of cerebral infarction, and have completed the present invention.

That is, the gist of the present invention is as follows.
[1] A test method for diagnosing cerebral infarction in an animal, comprising a step of measuring the amount of Interleukin-6 receptor subunit beta protein in a blood sample collected from the test animal.
[2] The blood sample collected from the test animal is collected from the test animal at any time between immediately after the onset of cerebral infarction-like symptoms and the 20th day after the onset. [1] The examination method for diagnosis of cerebral infarction according to [1].
[3] A test method for diagnosing the type of cerebral infarction in an animal, comprising a step of measuring the amount of Interleukin-6 receptor subunit beta protein in a blood sample collected from the test animal.
[4] The examination method according to [3], wherein the disease type diagnosis is one of atherothrombotic, cardiogenic cerebral embolism, and Branch atheromatous disease (BAD).
[5] A test method for predicting the prognosis of cerebral infarction in the animal, comprising a step of measuring the amount of Interleukin-6 receptor subunit beta protein in a blood sample collected from the test animal.
[6] The prediction of the prognosis of cerebral infarction according to [5], wherein the blood sample collected from the test animal is collected from the test animal on the seventh day of the onset of cerebral infarction Inspection method.
[7] including a step of measuring the amount of Interleukin-6 receptor subunit beta protein in a blood sample collected from an animal in need of prevention or treatment of cerebral infarction administered with a prophylactic or therapeutic agent for cerebral infarction, A method for evaluating the effect of preventing or treating cerebral infarction in the animal.
[8] A prophylactic agent for cerebral infarction comprising a step of contacting a subject with a candidate compound for a prophylactic or therapeutic agent for cerebral infarction and then measuring the amount of Interleukin-6 receptor subunit beta protein in the subject, A screening method for therapeutic drugs.
[9] A kit for screening for cerebral infarction examination, for evaluating cerebral infarction treatment, or for candidate compounds for preventing or treating cerebral infarction, comprising a reagent capable of measuring the amount of Interleukin-6 receptor subunit beta protein.

  According to the present invention, an interleukin-6 receptor subunit beta protein whose plasma concentration in a cerebral infarction patient is different from that in a patient with a different disease type or prognosis is a molecular marker for cerebral infarction examination (in the present specification, This is sometimes referred to as “IL-6ST protein marker”). By using IL-6ST protein marker alone or in combination with other cerebral infarction molecular markers, it becomes possible to quickly and surely diagnose cerebral infarction, or to classify cerebral infarction and predict prognosis. .

Hereinafter, embodiments of the present invention will be described in detail.
(1) Cerebral infarction examination method The first of the present invention is a method for examining cerebral infarction in an animal, comprising the step of measuring the amount of Interleukin-6 receptor subunit beta protein in a blood sample collected from the subject animal. is there.

  In the present specification, “cerebral infarction” is a disease in which the cerebral artery is blocked by a thrombus or embolus and becomes ischemic, and the brain tissue is necrotized and damaged, and a lacunar infarction that develops due to blocking of a small intracerebral artery. Atherothrombotic cerebral infarction caused by occlusion of intracerebral aorta due to atheroma, cardiogenic cerebral embolism caused by occlusion of intracerebral artery due to thrombosis in heart, from ascending aorta to aortic arch Aortic cerebral embolism, which is an embolism that develops when the obturator dissociates from the atherosclerotic lesion and occludes the intracerebral artery, and a plaque formed in the main artery obstructs the entrance of the penetrating branch “Branch atheromatous disease” (hereinafter sometimes referred to as “BAD”), an infarction of the penetrating branch region that occurs, arterial dissection where the intima of the cerebral blood vessel is damaged and blood enters the arterial wall to occlude the cerebral blood vessel Any of these are included. In addition, cerebral infarction includes any of acute phase, subacute phase, recovery phase (chronic phase) and the like.

  In this specification, the Interleukin-6 receptor subunit beta protein (hereinafter sometimes referred to as “IL-6ST protein”) refers to Interleukin-6, which is a target for measuring the content in a blood sample in the test method of the present invention. It means a receptor subunit beta protein and may be any protein derived from a test animal. Specific examples of human-derived proteins include proteins represented by the specific amino acid sequences described below. Furthermore, fragments, derivatives, and mutants of proteins having the same functions as these are also included.

  In the above examination method, the “test animal” may be any animal that may cause cerebral infarction, and specifically includes rodents such as humans, monkeys, and rats. Of these, the method for examining cerebral infarction of the present invention is particularly preferably performed in humans suspected of having cerebral infarction, humans after onset of cerebral infarction, or the like.

  The “blood sample” collected from the test animal is not particularly limited as long as it contains IL-6ST protein and can measure its concentration. Specifically, EDTA plasma, citrate Any of plasma such as plasma, serum, and whole blood may be used, but among these, EDTA plasma can be easily collected, is easily stored, and is preferably used because of its large collection amount. The timing for collecting the blood sample from the test animal may be any timing as long as the diagnosis of cerebral infarction is performed. Since cerebral infarction changes in symptoms and pathology from onset to onset, it will be described in detail below.

  In the test method of the present invention, the Interleukin-6 receptor subunit beta protein, which is a target for measuring the content in a blood sample collected from a test animal, is the amino acid represented by UniProtKB Entry Name IL6RB_HUMAN in the case of humans. A protein consisting of a sequence. For example, sequence information can be obtained by inputting the EntryName in a National Center for Biotechnology Information (NCBI) database or the like. The amino acid sequence is human, but when the test animal is different, a homologous protein derived from the animal becomes the protein to be measured.

  The method for measuring the content of the protein in the blood sample is not particularly limited as long as the content of the protein can be measured. For example, an existing immunoassay method using an antibody of the protein or a chromatographic method. All components captured on a chromatographic carrier (eg, cation exchanger, anion exchanger, hydrophobic chromatographic carrier, metal ion, etc.) under certain conditions using a combination of holographic techniques and time-of-flight mass spectrometry (TOF-MS) For example, a method of measuring the mass of the solution at once, a two-dimensional gel electrophoresis method, or the like is used. As the immunoassay, a method of quantitative detection according to an enzyme immunoassay, a method of measurement by a fluorescence immunoassay, a chemiluminescence immunoassay, or the like is preferable. The enzyme immunoassay method is a detection method using an enzyme as a labeling substance among the labeled immunoassay methods. It is particularly preferable to select an enzyme-linked immunosorbent assay (ELISA) method using an immunosolvent. Among the ELISA methods, the sandwich method is mentioned as one of the particularly preferable measurement modes in consideration of the convenience of operation, economic convenience, and general versatility in clinical examination. These measurement methods include, for example, the New Chemistry Laboratory (Edited by the Japanese Biochemical Society; Tokyo Kagaku Dojin), Molecular Cloning, A Laboratory Manual (T. Maniatis et al., Cold Spring Harbor Laboratory (2001)), Antibodies-A Laboratory. The method can be performed according to a method described in a general experiment such as Manual (E. Harlow, et al., ColdSpring Harbor Laboratory (1988)) or the like.

  Antibodies and the like (including antibody fragments) used for the above measurement can be obtained by a known method using the target IL-6ST protein as an antigen. However, it does not have to be produced using the target IL-6ST protein as an antigen, and any protein may be used as long as it exhibits at least cross-reactivity with the protein and can measure its content. As such an immunoassay and an antibody used therefor, for example, an antibody attached to Human soluble gp130 Immunoassay (catalog No. DGP00) manufactured by R & D is preferably used.

  As a blood sample used in the method of the present invention, a blood sample immediately after being collected from a test animal is preferably used for the measurement, but a stored sample may be used. The method for storing the blood sample is not particularly limited as long as the amount of molecular marker for cerebral infarction examination does not change. For example, low temperature conditions such as 0 to 10 ° C. that do not freeze, dark conditions, and no vibration conditions. preferable. In the case of unavoidably freezing, a method capable of avoiding marker molecule decomposition, oxidation reaction, etc., such as deep freezing, is preferable.

  In the test method of the present invention, the content of the IL-6ST protein marker in a blood sample collected from a test animal (in this specification, this may be referred to as “specimen”) is measured. Using this as an index, cerebral infarction can be examined. In addition, it is a composite index that combines the contents of specimens such as CRP and D-Dimer, which are existing cerebral infarction markers, and links the observation of clinical symptoms with the results of echocardiography, MRI, MRA, cervical vascular echo, etc. It is possible to inspect more accurately by using.

  When performing the cerebral infarction test using the content of the molecular marker for cerebral infarction examination as an index, the absolute value of the content of the molecular marker for cerebral infarction examination in the specimen is defined as an appropriate cutoff value. It may be a method of inspecting.

  The cut-off value is a value determined when determining a target disease group and a non-disease group (a group other than the target disease) by paying attention to a certain substance. When determining the target disease and non-disease, it is negative if it is below the cut-off value, positive if it is above the cut-off value, or positive if it is below the cut-off value, and if it is above the cut-off value The disease can be determined as negative (Kanai Masamitsu, Clinical Laboratory Proposal Kanehara Publishing Co., Ltd.).

  The index used for the purpose of evaluating the clinical usefulness of the cutoff value includes sensitivity and specificity. A certain population is determined using a cut-off value, and among the diseased patients, a (true positive) is positive in the determination, and b (false negative) is negative in the determination while being a disease patient. A / (a + b) when c (false positive) indicates that the patient is not a disease patient but is positive in the determination and d (true negative) indicates that the patient is not a disease patient and is negative in the determination. Can be represented as sensitivity (true positive rate) and d / (c + d) as specificity (true negative rate).

  The distribution of measured values between the target disease group and the non-disease group usually partially overlaps. Therefore, sensitivity and specificity change by raising or lowering the cutoff value. Decreasing the cut-off value increases the sensitivity, but the specificity decreases, and increasing the cut-off value decreases the sensitivity, but increases the specificity. As a determination method, it is preferable that both sensitivity and specificity are higher. A determination method in which the values of sensitivity and specificity do not exceed 0.5 is not recognized as useful.

  The method for setting the cut-off value is to set 95% of the distribution of the non-disease group as one of the values at both ends from the center as the cut-off value. When the distribution of the non-disease group shows a normal distribution And a method of setting an average value + 2 times standard deviation (SD) or an average value−2SD as a cut-off value.

  In general, in order to determine whether or not a diagnostic method is useful, the sensitivity and specificity vary depending on the cutoff value set as described above, so the sensitivity and specificity at a certain cutoff value are simply used. Rather than evaluating, it is desirable to evaluate with an index that maintains high sensitivity and specificity when the cut-off value is raised or lowered, for example, the AUC value of the ROC curve. The AUC value of the ROC curve becomes 1 when a cutoff value exists such that both sensitivity and specificity are 100%, and approaches 0.5 when the diagnostic performance is not good. Therefore, when the performance of a certain diagnostic method is judged by the AUC value of the ROC curve, it can be evaluated that the method is suitable as a diagnostic method if it is 0.7 or more. Since the IL-6ST protein marker described in the present invention also has an ROC curve AUC value of 0.7 or more, it is used in the cerebral infarction examination method described later.

(1-1) Cerebral infarction type examination method using IL-6ST protein marker As a specific method of the cerebral infarction examination method using IL-6ST protein marker of the present invention, a method for distinguishing cerebral infarction type Examples include inspection methods. The disease type means a clinically distinct disease type known per se, specifically “atherothrombotic cerebral infarction” (herein referred to as “atherothrombotic”). Or “cardiogenic cerebral embolism” (sometimes referred to herein as “cardiogenic cerebral embolism”), or by a plaque formed in the main artery blocking the entrance of the penetrating branch Examples thereof include “Branch atheromatous disease” (hereinafter sometimes referred to as “BAD”), which is an infarction of the penetrating branch region.

  In order to distinguish the type of cerebral infarction by measuring the amount of IL-6ST protein contained in the sample, first, the cutoff value that can distinguish each disease type in the amount of IL-6ST protein in the blood sample Is determined by the above-described method, and the amount of IL-6ST protein in the sample is compared with the cut-off value to determine which disease type group is entered.

  As a disease type that can be distinguished by the amount of IL-6ST protein contained in the blood sample of the present invention, a cut-off value that can distinguish between the two disease type groups by the IL-6ST protein content can be set. If there is no particular limitation, specifically, for example, as shown in Table 1, "Atherothrombotic cerebral infarction" and "BAD", "Cardiogenic cerebral embolism" and "BAD", "BAD" And other types of disease.

  The timing of obtaining the specimen when performing these measurements is not particularly limited immediately after the onset of cerebral infarction-like symptoms, but Table 1 shows preferred timings.

  In the measurement method of the present invention, the amount of IL-6ST protein in a blood sample of a patient clinically determined as the above-mentioned distinct disease type is measured in advance, and the two disease type groups are distinguished by the above method. Set the cutoff value. The content of the IL-6ST protein marker in the collected specimen is measured, and the absolute value is compared with the cut-off value set by the method described above, so that the specimen from which the specimen is collected for any disease type. Whether an examiner (animal) is included can be determined. The method for clinically confirming various cerebral infarctions is not particularly limited, but examples include methods for confirming from device diagnostic methods such as head X-ray CT, head MRI, MRA, cerebral angiography, cervical vascular echo, etc. It is done.

  In addition, when testing for the above-mentioned disease types, it is possible to combine the contents in specimens such as CRP and D-Dimer, which are existing cerebral infarction markers, and to observe clinical symptoms and echocardiography, MRI, MRA, cervical By using a composite index that correlates results such as blood vessel echoes, it is possible to inspect more accurately.

(1-2) Test method for predicting prognosis of cerebral infarction using IL-6ST protein marker As a specific method of the cerebral infarction test method using IL-6ST protein marker of the present invention, the brain of a subject (animal) is used. Examples include an inspection method for predicting the prognosis of infarction. The prognosis of cerebral infarction is indicated by the degree of injury due to cerebral infarction of the patient after a certain period of time after the onset of cerebral infarction. The degree of failure can be assessed using an already established indicator known per se. Indicators include, for example, Japan's modified Rankin Scale (mRS) criteria (Yukito Shinohara et al., MRS Reliability Research Group, Study on Reliability of Modified Rankin Scale-Japanese Version Criteria and Questionnaire, Stroke 2007; 29: 6-13) etc. are used.

  After a certain period of time, it may be any time after onset, but from the viewpoint of assessing the state of rehabilitation of a subject (animal), for example, it is generally evaluated from 3 months to 1 year after the onset Is. In the prediction of prognosis, considering the usefulness of the prediction method, the amount of IL-6ST protein in the blood sample obtained from the subject for about 2 weeks immediately after the onset, and the subject 3 months after the onset A method for predicting the state is preferable.

  In the measurement method of the present invention, the amount of IL-6ST protein in a blood sample at the time of measurement for each patient who has been clinically determined in advance at the time of the above-mentioned mRS is measured in advance, and mRS to be distinguished at the time of the prediction purpose. In the meantime, the cutoff value which distinguishes both mRS groups by the above-mentioned method is set. More specifically, for example, in a case where a patient with mRS of 0 to 1 after 3 months of onset is distinguished from a patient with 2 to 5 of mRS, a patient with mRS of 0 to 5 3 months after onset (during prediction purposes) 7 days after the onset of illness (at the time of measurement), the amount of IL-6ST protein in the blood sample is measured, and after 3 months, between the measured value of patients with mRS 0-1 and the measured value of patients 2-5 Set the cutoff value. Thereafter, a sample at the time of measurement is collected, the content of the IL-6ST protein marker in the sample is measured, and the absolute value is compared with the cut-off value set by the above method to predict. The state of the subject (animal) at the time of purpose can be predicted.

  In the prediction method of the present invention, particularly preferably, about 2 weeks after onset, more preferably around 3 months after onset by measuring the amount of IL-6ST protein in a sample collected from a subject on the 7th day of onset And a method for predicting whether mRS of the subject is 2 to 5 or 1 or less.

  In addition, another preferable prediction method of the present invention is to measure the amount of IL-6ST protein in a specimen collected from a subject collected from a subject on the 7th day after onset for about 2 weeks, more preferably onset 3 For example, a method for predicting whether the mRS of the subject around 3 months will be 3 to 5 or less than 2 is mentioned.

  Thus, a subject (animal) diagnosed with cerebral infarction by the method of the present invention has a very good prognostic course and therapeutic effect by receiving treatment suitable for each disease.

(2) Kit for measuring cerebral infarction The present invention further provides a kit for use in the above-described cerebral infarction examination, or evaluation of the prevention or treatment effect of cerebral infarction described below, or a screening method for a cerebral infarction prevention or treatment drug. The kit is also included. The contents of the kit are configured by a combination of equipment or reagents. However, the configuration or form of the kit is not limited as long as it includes a substance that is essentially the same as each component described below, or a substance that is essentially the same as a part thereof. Even if they are different, they are included in the kit of the present invention. As a reagent, for example, when measuring an IL-6ST protein marker by an immunoassay, an anti-IL-6ST protein marker antibody is included. In addition, a biological sample diluent, an antibody-immobilized solid phase, a reaction buffer, a washing solution, a labeled secondary antibody or an antibody fragment thereof, a labeled detection reagent, a standard substance, and the like are also included as necessary. Examples of the diluted solution of the biological sample include an aqueous solution containing a protein such as BSA or casein in a surfactant or a buffer.

  As the antibody-immobilized solid phase, a material in which various polymer materials are shaped to suit the application and anti-molecular marker antibodies or antibody fragments thereof are solid-phased is used. Shapes are tubes, beads, plates, fine particles such as latex, sticks, etc., and materials are polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, polyethylene terephthalate, etc. Polymer materials, glass, ceramics and metals. Examples of the method for immobilizing an antibody include known methods such as a physical method and a chemical method, or a combination method thereof. For example, a 96-well polystyrene immunoassay microterplate prepared by subjecting an antibody or antibody fragment to a hydrophobic solid phase can be used.

  Any reaction buffer may be used as long as it provides a solvent environment for the binding reaction between the antibody on the antibody-immobilized solid phase and the antigen in the biological sample. However, surfactants, buffers, BSA, Reaction buffers containing proteins such as casein, preservatives, stabilizers, reaction accelerators and the like can be mentioned.

  As the labeled secondary antibody or antibody fragment thereof, the antibody or antibody fragment used in the present invention is labeled with a labeling enzyme such as horseradish peroxidase (HRP), bovine small intestine alkaline phosphatase, β-galactosidase, or a buffering agent. , BSA, casein and other proteins, preservatives and the like are used.

  As a reagent for detecting a labeled substance, for example, horseradish peroxidase according to the labeling enzyme, a substrate for absorption measurement such as tetramethylbenzidine or orthophenylenediamine, or a fluorescence such as hydroxyphenylpropionic acid or hydroxyphenylacetic acid. If the luminescent substrate such as a substrate or luminol is alkaline phosphatase, an absorbance measurement substrate such as 4-nitrophenyl phosphate, a fluorescent substrate such as 4-methylumbelliferyl phosphate, and the like can be given.

(3) Method for evaluating effect of prevention or treatment of cerebral infarction The second aspect of the present invention is blood collected from an animal in need of prevention or treatment of cerebral infarction administered with a cerebral infarction preventive or therapeutic agent. A method for evaluating the effect of preventing or treating cerebral infarction in an animal, comprising a step of measuring the amount of an IL-6ST protein marker in a sample.

  Specifically, an animal that requires prevention or treatment of cerebral infarction is a human who exhibits symptoms of cerebral infarction or an animal described in (1) (these may be simply referred to as “subjects”), Examples include patients diagnosed with cerebral infarction by the method (1) above.

  The cerebral infarction preventive or therapeutic agent may be any as long as it is used as a cerebral infarction preventive or therapeutic agent. For example, thrombolytic agents such as urokinase and tissue plasminogen activator, antiparin such as heparin, etc. Examples include anticoagulants such as coagulants, cyclooxygenase inhibitors, phosphodiesterase inhibitors, thromboxen A2 (TXA2) synthesis inhibitors, and brain protective agents such as free radical scavengers.

  Whether the amount of IL-6ST protein marker in blood samples collected from animals in need of prevention or treatment of cerebral infarction administered cerebral infarction preventive or therapeutic agent is increased or decreased compared to the amount before administration Alternatively, when approaching the amount in a control animal that does not develop cerebral infarction, it can be determined that there was a prophylactic or therapeutic effect.

  Since the cerebral infarction preventive or therapeutic agent to be treated varies depending on the disease type, it is possible to appropriately treat the patient if the disease type is diagnosed with the IL-6ST protein marker. For example, if a lacunar infarction is diagnosed, it can be treated with a thromboxen A2 (TXA2) synthesis inhibitor and a cyclooxygenase inhibitor can prevent recurrence of cerebral infarction. In addition, when the possibility of poor prognosis can be predicted with the marker, the prognosis is improved by using several kinds of therapeutic agents such as free radical scavengers from the early stage of treatment, or by enhancing the content of rehabilitation. It is possible to perform such treatment.

(4) Screening method for cerebral infarction preventive or therapeutic agent The third aspect of the present invention is to contact a subject with a candidate compound for a prophylactic or therapeutic agent for cerebral infarction, and then contact IL in the blood sample of the subject. It is a screening method for a prophylactic or therapeutic agent for cerebral infarction, comprising a step of measuring the amount of -6ST protein marker.

As the subject, a non-human animal individual, tissue, cell, or the like in which the content of the IL-6ST protein marker exhibits the same abnormality as in the cerebral infarction state is used. Examples of animals having cerebral infarction include non-human cerebral infarction animals in which a cerebral infarction state is formed by a surgical technique or the like (Yuji Kuge, Kazuo Minematsu et al. Nylon Monofilament for Intraluminal Middle Cerebral Artery Occlusionin Rats. Stroke , 26, 1655 (1995)). In addition to the subject, it is also preferable to use an individual, tissue, cell, etc. of the same kind as the subject that has an IL-6ST protein marker content similar to that in the normal state (normal) as a control.
Based on the IL-6ST protein marker, if a pathological condition similar to a certain disease type in humans can be constructed in a non-human animal with cerebral infarction, an experimental system for developing a therapeutic agent in a patient with cerebral infarction of a certain disease type Can be established. In addition, by monitoring the prognosis of a non-human animal individual with the marker, it is possible to develop a therapeutic agent effective for the prognosis of human cerebral infarction.

Examples of candidate compounds for preventing or treating cerebral infarction (hereinafter sometimes referred to as “candidate compounds”) to be brought into contact with these subjects include peptides, proteins, non-peptidic compounds, low-molecular synthetic compounds, and the like. These compounds may be novel compounds or known compounds. Moreover, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like containing these compounds may be used.
The dose, administration method, treatment time and the like may be appropriately selected according to the subject to be used.

The method for measuring the content of the IL-6ST protein marker in the subject after contacting with the candidate compound can be performed by a method suitable for each subject. For example, when the subject is a cell, it may be prepared as a cell extract according to a known method and used as a sample, or the cell may be immobilized on a culture plate or a slide glass and used as a sample. Also good. For example, when a cell extract is prepared and used as a sample, detection can be performed by ELISA or the like. It is preferable that these samples have the same number of cells used for extraction, or the amount of purified RNA or the amount of extracted protein so that the numerical values obtained as a result of detection can be compared and analyzed accurately.
If the amount of IL-6ST protein marker in a subject contacted with a candidate compound increases or decreases compared to the amount before contact, or approaches the amount in a control subject, the candidate compound is It can be determined that it has an effect of preventing or treating infarction.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Evaluation of Test Method for Discriminating Cerebral Infarction Disease Type Using IL-6ST Protein Marker Patients with confirmed diagnosis for each type of cerebral infarction, specifically, “Lucuna Infarction” (Table "Atrac thrombotic cerebral infarction" (referred to as "Atherothrombotic" in the table), "Cardiogenic cerebral embolism" (referred to as "Cardiogenic cerebral embolism" in the table) "Aortogenic cerebral embolism" (referred to in the table as "Aortogenic cerebral embolism"), "Branch atheromatous disease" (referred to as "BAD" in the table), "Arterial dissection" (Table In patients with a disease type (referred to as “non-classifiable type” in the table) that do not fall under any of these conditions (referred to as “arterial dissection”), they are described immediately after the onset of cerebral infarction (“DAY0” in the table). Blood samples were collected (number of people listed in Table 2), and each obtained EDTA plasma. . The concentration of IL-6ST protein in EDTA plasma was measured by an immunoassay using R &D's Human soluble gp130 Immunoassay (Catalog No. DGP00). Specifically, a calibration curve was prepared using IL-6ST protein, which is a standard product attached to the kit, and the concentration of IL-6ST protein was calculated from this calibration curve.

  Next, in order to evaluate the discrimination performance at each blood sampling time point, the AUC value of the ROC curve was calculated according to the method described in Fawcett, T. (2004) ROC Graphs: Notes and Practical Considerations for Researchers. The results are shown in Table 2. It is shown that a value exceeding 0.7 at each time of blood collection is useful as a test method for determining the disease type of a cerebral infarction patient.

  In other words, by comparing the concentrations of IL-6ST protein in the plasma of each disease type patient immediately after the onset, it is possible to discriminate between cerebral infarction patients with the disease types of "Atherothrombotic cerebral infarction" and "Branch atheromatous disease" In the same comparison, it is also possible to distinguish between patients with cerebral infarction of “cardiogenic cerebral embolism” and “Branch atheromatous disease”, and “Branch atheromatousdisease” and other types of cerebral infarction. It was found that a test can be performed to identify the patient.

Example 2 Evaluation of a test method for predicting the prognosis of cerebral infarction using an IL-6ST protein marker The prognosis of cerebral infarction is determined after the onset of cerebral infarction, after a certain period of time, to the extent of the cerebral infarction of the patient. Shown. Japanese version of modified Rankin Scale (mRS) criteria (Yukito Shinohara et al., MRS Reliability Study Group, Study on Reliability of Modified Rankin Scale-Japanese Version Criteria and Questionnaire) Blood was collected 7 days after the onset of cerebral infarction (indicated as “DAY7” in the table) for patients classified as 0-5 using the introduction of Stroke 2007; 29: 6-13) (each in Table 2) EDTA plasma was obtained.
The concentration of IL-6ST protein in the plasma was measured in the same manner as in Example 1, and the AUC value of the ROC curve was calculated from the measured values in the same manner as in Example 1. The results are shown in Table 3.
It is shown that a value exceeding 0.7 at each time of blood collection is useful as a test method for determining the prognosis of a cerebral infarction patient.

  Specifically, by comparing the concentration of IL-6ST protein in the plasma on the 7th day after the onset of the patients who become each rank after 3 months, the degree of injury (prognosis) 3 months after the onset of cerebral infarction 3 months after the onset, it was found that a test for discriminating between patients with rank 2 to 5, patients with rank 0 to 1, patients with rank 3 to 5, and patients with rank 0 to 2 could be performed.

  According to the method of the present invention, diagnosis of cerebral infarction, prognosis prediction test and evaluation of treatment / prevention effect can be performed, which is useful in the fields of medicine and diagnosis. Moreover, according to the method of the present invention, a therapeutic / prophylactic agent for cerebral infarction can be screened, which is also useful in the pharmaceutical field.

Claims (9)

  A test method for diagnosing cerebral infarction in an animal, comprising a step of measuring the amount of Interleukin-6 receptor subunit beta protein in a blood sample collected from the test animal.   The blood sample collected from the test animal is collected from the test animal at any time between immediately after the onset of cerebral infarction-like symptoms and 20 days after the onset. Test method for diagnosis of cerebral infarction as described in 1.   A test method for diagnosing a cerebral infarction disease in the animal, comprising a step of measuring the amount of Interleukin-6 receptor subunit beta protein in a blood sample collected from the test animal.   The test method according to claim 3, wherein the disease type diagnosis is any one of atherothrombotic, cardiogenic cerebral embolism, and Branch atheromatous disease.   A test method for predicting the prognosis of cerebral infarction in an animal, comprising a step of measuring the amount of Interleukin-6 receptor subunit beta protein in a blood sample collected from the test animal.   The test method for predicting the prognosis of cerebral infarction according to claim 5, wherein the blood sample collected from the test animal is collected from the test animal on the 7th day of the onset of cerebral infarction.   Measuring the amount of Interleukin-6 receptor subunit beta protein in a blood sample collected from an animal in need of prevention or treatment of cerebral infarction administered with a prophylactic or therapeutic agent for cerebral infarction, A method for evaluating the effect of prevention or treatment of cerebral infarction.   An agent for preventing or treating cerebral infarction, comprising the step of contacting a subject with a candidate compound for a prophylactic or therapeutic agent for cerebral infarction and then measuring the amount of Interleukin-6 receptor subunit beta protein in the subject. Screening method.   A kit for screening for cerebral infarction examination, for evaluating cerebral infarction treatment effects, or for candidate compounds for preventing or treating cerebral infarction, comprising a reagent capable of measuring the amount of Interleukin-6 receptor subunit beta protein.