Kit and method for assessing dementia or brain function

Abstract

One or more embodiments of the present invention provide: a method capable of determining dementia in an early stage with a simple operation; and a method capable of determining brain function with a simple operation.　One or more embodiments of the present invention relate to a method for obtaining indices for determining dementia or determining brain function in a subject, said method including measuring gene expression or enzymatic activities of one or more proteins involved in energy metabolism reactions in a sample derived from a subject.

Description

[Technical field]

[0001] The present invention relates to a method for obtaining an index for diagnosing dementia or brain function in a subject. The present invention also relates to a kit for determining dementia or brain function. The present invention also relates to a method for obtaining an index for determining the degree of aging phenomenon in a subject. The present invention also relates to a kit for determining the degree of aging phenomena. [Background technology]

[0002] As the aging population advances, the incidence of dementia is increasing, and research and development of therapeutic drugs is actively being conducted. Dementia can be classified into Alzheimer's type, vascular type, Lewy body type, and frontotemporal type. Of these, Alzheimer's type dementia has the largest number of patients, accounting for more than 60% of the total. In 1999, donepezil hydrochloride (brand name Aricept, etc.) (improves memory impairment and cognitive impairment and inhibits the progression of the disease) was released for Alzheimer's type dementia, and in 2011, galantamine (Reminyl), rivastigmine (Exelon patch, Rivastigmine patch), and memantine (Memantine) were released. These medicines slow the progression of dementia. However, there are currently no therapeutic drugs for dementia, and development is still difficult. Therefore, currently, efforts are being made to treat dementia through early detection and lifestyle improvements.

[0003] PET (positron emission tomography) and SPECT (single photon emission computed tomography) are known as imaging tests for diagnosing dementia. However, these imaging tests are expensive and can only detect symptoms when they have progressed, so they are not suitable for early diagnosis of dementia.

[0004] In addition, cognitive function tests such as the Hasegawa Dementia Assessment Scale are known to be used to identify dementia, but these tests are used after the patient recognizes the progression of symptoms, and cannot be used to determine dementia or brain function in the early stages of onset. Additionally, cognitive function tests have the problem that the results are ambiguous.

[0005] There is also a known method of diagnosing dementia by testing for proteins in cerebrospinal fluid, but this method has the drawback of being highly invasive and cannot be used for early diagnosis.

[0006] A method for comprehensive analysis of blood microRNAs of subjects has also been developed to predict the onset of dementia. However, there are issues with this method, such as the complicated procedure for extracting blood microRNAs as pretreatment of the test sample, and the need for an expensive measurement system.

[0007] In addition, with the progress of aging, maintaining the health of the elderly has become an urgent issue, and expectations for anti-aging medicine are rising. For example, visual assessment of the appearance has been used to determine the degree of various aging phenomena such as skin spots and wrinkles. However, since it is difficult to obtain objective and quantitative results from visual assessment, a search for new biomarkers that can appropriately evaluate the degree of aging phenomena is underway.

[0008] Examples of patent documents disclosing methods for determining brain dysfunction based on gene expression levels (biomarkers) of specific proteins include Patent Documents 1 to 3.

[0009] Patent Document 1 describes a method for testing for Alzheimer's disease, which comprises detecting in vitro a decrease in the level or function of at least one factor in the insulin / IGF signaling pathway, such as insulin, in central nervous system tissue derived from a subject.

[0010] Patent Document 2 describes a method for detecting phosphorylation of at least one substrate protein, such as MARCKS, in a subject, and determining that the subject is suffering from or at risk of developing Alzheimer's disease if the level of phosphorylation is higher than that in a normal sample.

[0011] Patent Document 3 describes a biomarker for detecting cognitive dysfunction. The biomarker described in Patent Document 3 is described as a protein that depends on Complement C4, Prothrombin, Complement C3, Gelsolin, and the like.

[0012] Furthermore, examples of patent documents that disclose a method for determining the degree of aging phenomena based on the gene expression level of a specific protein (biomarker) include Patent Documents 4 to 6.

[0013] Patent Document 4 describes a method for determining the degree of skin aging, which includes measuring the expression and / or gene expression of a secretory protein and / or an intracellular protein in skin cells and / or skin tissue, and the expression of the secretory protein and / or the intracellular protein changes depending on the degree of skin aging. The biomarkers described in Patent Document 4 are Kallikrein 7, Keratin 7, etc.

[0014] Patent document 5 describes a method for evaluating the degree of aging of a cell and / or an individual using the expression level of GREM1 protein and / or GREM2 protein, or the expression level of the gene encoding GREM1 protein and / or the gene encoding GREM2 protein in a biological sample as an indicator.

[0015] Patent Document 6 describes a method for evaluating the state of cellular senescence of fibroblasts, which is characterized by including a step of determining the expression level e of the gene products of OLFML2A and / or CRLF1 in a biological sample. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] Special Publication No. 2008-522199 [Patent Document 2] International Publication WO2015 / 099094 [Patent Document 3] International Publication WO2019 / 012671 [Patent Document 4] JP 2012-189602 A [Patent Document 5] JP 2019-007759 A [Patent Document 6] JP 2013-116088 A Summary of the Invention [Problem to be solved by the invention]

[0017] One or more embodiments of the present invention aim to provide a method and a kit therefor that can diagnose dementia at an early stage by a simple operation, and a method and a kit therefor that can assess brain function by a simple operation. Another object of one or more embodiments of the present invention is to provide a method and a kit therefor that can determine the degree of aging phenomenon at an early stage by a simple operation. [Means for solving the problem]

[0018] As a result of extensive investigations, the present inventors have completed the following invention. (1) A method for obtaining an index for assessing dementia or brain function in a subject, comprising: A method comprising measuring gene expression or enzymatic activity of one or more proteins involved in energy metabolic reactions in a sample derived from a subject. (2) The method according to (1), wherein the assessment of dementia is a assessment of the presence or absence of dementia, a assessment of the risk of developing dementia, or a assessment of the severity of dementia. (3) The sample is a body fluid sample or a bone marrow sample, comparing the measured values ​​of gene expression or enzymatic activity of the one or more proteins to a reference value; The method according to (2), wherein a measured value of gene expression or enzymatic activity of the one or more proteins that is higher than the reference value indicates that the subject suffers from dementia, that the subject is at risk of developing dementia, or the severity of dementia in the subject. (4) The method according to (3), wherein the ratio of the measured value of gene expression or enzyme activity of the one or more proteins to the reference value is greater than or equal to 1.1 and less than or equal to 50.0. (5) The sample is a central nervous tissue sample, comparing the measured values ​​of gene expression or enzymatic activity of the one or more proteins to a reference value; The method according to (2), wherein a measured value of gene expression or enzymatic activity of the one or more proteins that is lower than the reference value indicates that the subject suffers from dementia, that the subject is at risk of developing dementia, or the severity of dementia in the subject. (6) The assessment of brain function includes assessment of whether or not there is a decline in brain function, assessment of the risk of a decline in brain function, assessment of the degree of decline in brain function, assessment of whether or not there is an autonomic nervous system disorder, assessment of the risk of an autonomic nervous system disorder, assessment of the degree of an autonomic nervous system disorder, assessment of whether or not there is a memory disorder, assessment of the risk of a memory disorder, assessment of the degree of a memory disorder, assessment of whether or not there is a motor disorder, assessment of the risk of a motor disorder, assessment of the degree of a motor disorder, assessment of whether or not there is a coordination disorder, assessment of the risk of a coordination disorder, assessment of the degree of a coordination disorder, assessment of whether or not there is an involuntary movement disorder, assessment of the risk of an involuntary movement disorder, assessment of the degree of an involuntary movement disorder, assessment of whether or not there is a sensory disorder, assessment of the risk of a sensory disorder, assessment of the degree of a sensory disorder, assessment of whether or not there is a visual disorder, assessment of the risk of a visual disorder, assessment of the degree of a visual disorder, assessment of whether or not there is an olfactory disorder, assessment of the risk of an olfactory disorder, assessment of the degree of an olfactory disorder, assessment of whether or not there is a hearing disorder, assessment of the risk of a hearing disorder, assessment of the degree of a hearing disorder, The method according to (1), which is for determining the presence or absence of a disturbance in balance, determining the risk of a disturbance in balance, determining the degree of a disturbance in balance, determining the presence or absence of a sleep disorder, determining the risk of a sleep disorder, determining the degree of a sleep disorder, determining the presence or absence of a speech disorder, determining the risk of a speech disorder, determining the degree of a speech disorder, determining the presence or absence of an emotional dysregulation disorder, determining the risk of an emotional dysregulation disorder, determining the degree of an emotional dysregulation disorder, determining the presence or absence of a headache, determining the risk of a headache, determining the degree of a headache, determining the presence or absence of convulsions, determining the risk of convulsions, determining the degree of convulsions, determining the presence or absence of tremors, determining the risk of tremors, determining the degree of tremors, determining the presence or absence of hallucinations, determining the risk of hallucinations, determining the degree of hallucinations, determining the presence or absence of delusions, determining the risk of delusions, determining the degree of delusions, determining the presence or absence of illusions, determining the risk of illusions, determining the degree of illusions, determining the presence or absence of abnormal behavior, determining the risk of abnormal behavior, determining the degree of abnormal behavior, determining the presence or absence of depression, determining the risk of developing depression, or determining the severity of depression. (7) The method according to any one of (1) to (6), wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3, and PDK1. (8) A kit for assessing dementia or brain function, comprising: A kit comprising a reagent for measuring gene expression or enzyme activity of one or more proteins involved in energy metabolic reactions in a sample derived from a subject. (9) The kit described in (8), wherein the assessment of dementia is a assessment of the presence or absence of dementia, a assessment of the risk of developing dementia, or a assessment of the severity of dementia. (10) The assessment of brain function includes assessment of whether or not there is a decline in brain function, assessment of the risk of decline in brain function, assessment of the degree of decline in brain function, assessment of whether or not there is an autonomic nervous system disorder, assessment of the risk of an autonomic nervous system disorder, assessment of the degree of autonomic nervous system disorder, assessment of whether or not there is a memory disorder, assessment of the risk of a memory disorder, assessment of the degree of memory disorder, assessment of whether or not there is a motor disorder, assessment of the risk of a motor disorder, assessment of the degree of a motor disorder, assessment of whether or not there is a coordination disorder, assessment of the risk of a coordination disorder, assessment of the degree of a coordination disorder, assessment of whether or not there is an involuntary movement disorder, assessment of the risk of an involuntary movement disorder, assessment of the degree of an involuntary movement disorder, assessment of whether or not there is a sensory disorder, assessment of the risk of a sensory disorder, assessment of the degree of a sensory disorder, assessment of whether or not there is a visual disorder, assessment of the risk of a visual disorder, assessment of the degree of a visual disorder, assessment of whether or not there is an olfactory disorder, assessment of the risk of an olfactory disorder, assessment of the degree of an olfactory disorder, assessment of whether or not there is a hearing disorder, assessment of the risk of a hearing disorder, assessment of the degree of a hearing disorder, The kit according to (8), which is for determining the presence or absence of a disturbance in balance, determining the risk of a disturbance in balance, determining the degree of a disturbance in balance, determining the presence or absence of a sleep disorder, determining the risk of a sleep disorder, determining the degree of a sleep disorder, determining the presence or absence of a speech disorder, determining the risk of a speech disorder, determining the degree of a speech disorder, determining the presence or absence of an emotional dysregulation disorder, determining the risk of an emotional dysregulation disorder, determining the degree of an emotional dysregulation disorder, determining the presence or absence of a headache, determining the risk of a headache, determining the degree of a headache, determining the presence or absence of convulsions, determining the risk of convulsions, determining the degree of convulsions, determining the presence or absence of tremors, determining the risk of tremors, determining the degree of tremors, determining the presence or absence of hallucinations, determining the risk of hallucinations, determining the degree of hallucinations, determining the presence or absence of delusions, determining the risk of delusions, determining the degree of delusions, determining the presence or absence of illusions, determining the risk of illusions, determining the degree of illusions, determining the presence or absence of abnormal behavior, determining the risk of abnormal behavior, determining the degree of abnormal behavior, determining the presence or absence of depression, determining the risk of developing depression, or determining the severity of depression. (11) The kit according to any one of (8) to (10), wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3, and PDK1. (12) The kit according to any one of (8) to (11), wherein the reagent is a primer pair for amplifying a nucleic acid containing a gene for the one or more proteins, a probe that hybridizes with a nucleic acid containing a gene for the one or more proteins, or an antibody that specifically binds to the one or more proteins. (13) A method for obtaining an index for determining the degree of aging in a subject, comprising the steps of: A method comprising measuring gene expression or enzymatic activity of one or more proteins involved in energy metabolic reactions in a sample derived from a subject. (14) The method according to (13), wherein the determination of the degree of aging is determination of age, determination of the degree of aging in brain tissue, determination of the degree of aging in peripheral blood, or determination of the degree of aging in bone marrow cells. (15) The method according to (13) or (14), wherein the sample is a body fluid sample, a bone marrow sample, or a central nervous tissue sample. (16) The method according to any one of (13) to (15), wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3, and PDK1. (17) A kit for determining the degree of aging in a subject, comprising: A kit comprising a reagent for measuring gene expression or enzyme activity of one or more proteins involved in energy metabolic reactions in a sample derived from a subject. (18) The kit according to (17), wherein the determination of the degree of aging is determination of age, determination of the degree of aging in brain tissue, determination of the degree of aging in peripheral blood, or determination of the degree of aging in bone marrow cells. (19) The kit according to (17) or (18), wherein the sample is a body fluid sample, a bone marrow sample, or a central nervous tissue sample. (20) The kit according to any one of (17) to (19), wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3, and PDK1. (21) The kit according to any one of (17) to (20), wherein the reagent is a primer pair for amplifying a nucleic acid containing a gene for the one or more proteins, a probe that hybridizes with a nucleic acid containing a gene for the one or more proteins, or an antibody that specifically binds to the one or more proteins.

[0019] (22) The method according to any one of (1) to (7) and (13) to (16), wherein measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction in a sample derived from the subject comprises the following (a) and (b): (a) obtaining a mononuclear cell-containing sample by isolating mononuclear cells from said sample; (b) measuring the gene expression or enzymatic activity of said one or more proteins in said mononuclear cell-containing sample. (23) The kit according to any one of (8) to (12) and (17) to (21), further comprising a reagent and / or a column for separating mononuclear cells from the sample.

[0020] (24) A method for assessing dementia or brain function in a subject, comprising: Measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolism reaction in a sample from a subject; and Diagnosing dementia or brain function in the subject based on the measured values ​​of gene expression or enzyme activity of the one or more proteins. The method includes: The subject herein is preferably a human or a non-human animal. The sample herein is preferably a sample isolated from the subject. (25) The method according to (24), wherein the assessment of dementia is assessment of the presence or absence of dementia, assessment of the risk of developing dementia, or assessment of the severity of dementia. (26) The sample is a body fluid sample or a bone marrow sample, comparing the measured values ​​of gene expression or enzymatic activity of the one or more proteins to a reference value; 26. The method of claim 25, wherein a measured value of gene expression or enzymatic activity of the one or more proteins that is higher than the reference value indicates that the subject suffers from dementia, that the subject is at risk of developing dementia, or that the subject has a severity of dementia. (27) The method according to (26), wherein the ratio of the measured value of gene expression or enzyme activity of the one or more proteins to the reference value is greater than or equal to 1.1 and less than or equal to 50.0. (28) The sample is a central nervous tissue sample, comparing the measured values ​​of gene expression or enzymatic activity of the one or more proteins to a reference value; 26. The method of claim 25, wherein a measured value of gene expression or enzymatic activity of the one or more proteins lower than the reference value indicates that the subject suffers from dementia, that the subject is at risk of developing dementia, or the severity of dementia in the subject. (29) The method described in (24), wherein the assessment of brain function is the assessment described in (6). (30) A method for determining a degree of aging in a subject, comprising the steps of: Measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolism reaction in a sample from a subject; and determining the degree of aging in said subject based on the measured values ​​of gene expression or enzymatic activity of said one or more proteins; The method includes: The subject herein is preferably a human or a non-human animal. The sample herein is preferably a sample isolated from the subject. (31) The method according to (30), wherein the assessment of the degree of the aging phenomenon is the assessment according to (14). (32) The method according to (30) or (31), wherein the sample is a body fluid sample, a bone marrow sample, or a central nervous tissue sample. (33) The method according to any one of (24) to (32), wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3, and PDK1. (34) The method according to any one of (24) to (33), wherein measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction in a sample derived from the subject comprises the following (a) and (b): (a) obtaining a mononuclear cell-containing sample by isolating mononuclear cells from said sample; (b) measuring the gene expression or enzymatic activity of said one or more proteins in said mononuclear cell-containing sample.

[0021] (35) A biomarker for assessing dementia or brain function, comprising one or more proteins involved in an energy metabolic reaction, or a nucleic acid comprising a base sequence encoding the amino acid sequence of the one or more proteins. (36) A biomarker for determining the degree of aging, comprising one or more proteins involved in an energy metabolic reaction, or a nucleic acid comprising a base sequence encoding the amino acid sequence of the one or more proteins. (37) The biomarker according to (35) or (36), wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3, and PDK1. (38) The biomarker according to any one of (35) to (37), wherein the nucleic acid is mRNA or cDNA prepared from the mRNA.

[0022] (39) In the manufacture of kits for diagnosing dementia or assessing brain function, Use of the reagent to measure gene expression or enzymatic activity of one or more proteins involved in energy metabolism reactions. (40) The use according to (39), wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3, and PDK1. (41) The use according to (39) or (40), wherein the reagent is a primer pair for amplifying a nucleic acid containing a gene for the one or more proteins, a probe that hybridizes with a nucleic acid containing a gene for the one or more proteins, or an antibody that specifically binds to the one or more proteins. (42) The use according to any one of (39) to (41), wherein the kit has the features of the kit according to any one of (8) to (12) and (23).

[0023] (43) In the manufacture of a kit for determining the degree of aging phenomena, Use of the reagent to measure gene expression or enzymatic activity of one or more proteins involved in energy metabolism reactions. (44) The use according to (43), wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3, and PDK1. (45) The use according to (43) or (44), wherein the reagent is a primer pair for amplifying a nucleic acid containing a gene for the one or more proteins, a probe that hybridizes with a nucleic acid containing a gene for the one or more proteins, or an antibody that specifically binds to the one or more proteins. (46) The use according to any one of (43) to (45), wherein the kit has the features of the kit according to any one of (17) to (21) and (23).

[0024] (47) A method for measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction in a subject, comprising the steps of: A method comprising measuring gene expression or enzymatic activity of one or more proteins involved in energy metabolic reactions in a sample derived from a subject. The subject herein is preferably a human or a non-human animal, more preferably a human. Here, the subject is preferably a subject suspected of dementia, a subject suffering from dementia, a subject receiving treatment for dementia, a subject desiring or needing a determination of the risk of developing dementia, a subject suspected of declining brain function, a subject with declining brain function, a subject receiving treatment to treat declining brain function, a subject desiring or needing a determination of brain function, a subject suspected of aging, a subject having an aging phenomenon, a subject receiving treatment to reverse an aging phenomenon, or a subject desiring or needing a determination of the extent of an aging phenomenon. The sample is preferably a sample isolated from the subject. (48) The method according to (47), wherein the sample is a body fluid sample, a bone marrow sample, or a central nervous tissue sample. (49) The method according to (47) or (48), further comprising comparing the measured values ​​of gene expression or enzyme activity of the one or more proteins with a reference value. (50) The method according to any one of (47) to (49), wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3, and PDK1. (51) The method according to any one of (47) to (50), wherein measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction in a sample derived from the subject comprises the following (a) and (b): (a) obtaining a mononuclear cell-containing sample by isolating mononuclear cells from said sample; (b) measuring the gene expression or enzymatic activity of said one or more proteins in said mononuclear cell-containing sample.

[0025] (52) A method for assessing dementia or brain function in a subject, and improving or treating dementia or brain function in the subject, comprising: Measuring gene expression or enzyme activity of one or more proteins involved in energy metabolism reactions in a sample from a subject; Diagnosing dementia or brain function in the subject based on the measured values ​​of gene expression or enzyme activity of the one or more proteins; and Administering a treatment for improving or treating dementia or brain function to the subject for whom the result of the determination indicates a need for improving or treating dementia or brain function. The method includes: The subject herein is preferably a human or a non-human animal. The sample herein is preferably a sample isolated from the subject. (53) The method according to (52), wherein the assessment of dementia is assessment of the presence or absence of dementia, assessment of the risk of developing dementia, or assessment of the severity of dementia. (54) The sample is a body fluid sample or a bone marrow sample, comparing the measured values ​​of gene expression or enzymatic activity of the one or more proteins to a reference value; 53. The method of claim 53, wherein a measured value of gene expression or enzymatic activity of said one or more proteins higher than said reference value is indicative of said subject suffering from dementia, said subject being at risk of developing dementia, or the severity of dementia in said subject. (55) The method according to (54), wherein the ratio of the measured value of gene expression or enzyme activity of the one or more proteins to the reference value is greater than or equal to 1.1 and less than or equal to 50.0. (56) The sample is a central nervous tissue sample, comparing the measured values ​​of gene expression or enzymatic activity of the one or more proteins to a reference value; 53. The method of claim 52, wherein a measured value of gene expression or enzymatic activity of said one or more proteins lower than said reference value is indicative of said subject suffering from dementia, said subject being at risk of developing dementia, or the severity of dementia in said subject. (57) The method described in (52), wherein the assessment of brain function is the assessment described in (6). (58) A method for determining a degree of an aging phenomenon in a subject and improving or treating the aging phenomenon in the subject, comprising: Measuring gene expression or enzyme activity of one or more proteins involved in energy metabolism reactions in a sample from a subject; determining the degree of aging in the subject based on the measured gene expression or enzymatic activity of the one or more proteins; and administering a treatment for improving or treating the aging phenomenon to the subject for whom the result of the determination indicates a need for improving or treating the aging phenomenon; The method includes: The subject herein is preferably a human or a non-human animal. The sample herein is preferably a sample isolated from the subject. (59) The method according to (58), wherein the assessment of the degree of the aging phenomenon is the assessment according to (14). (60) The method according to (58) or (59), wherein the sample is a body fluid sample, a bone marrow sample, or a central nervous tissue sample. (61) The method according to any one of (52) to (60), wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3, and PDK1. (62) The method according to any one of (52) to (61), wherein measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction in a sample derived from the subject comprises the following (a) and (b): (a) obtaining a mononuclear cell-containing sample by isolating mononuclear cells from said sample; (b) measuring the gene expression or enzymatic activity of said one or more proteins in said mononuclear cell-containing sample.

[0026] This specification includes the disclosures of Japanese Patent Application No. 2020-038991, which is the priority basis of this application. Effect of the Invention

[0027] According to one or more embodiments of the present invention, dementia can be diagnosed by a simple operation. Early diagnosis of dementia is possible. According to one or more embodiments of the present invention, brain function can be determined with a simple operation. According to one or more embodiments of the present invention, the degree of aging phenomenon can be determined by a simple operation. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows the average relative expression level of each target gene to the expression level of the 18S ribosomal RNA gene in the body fluid samples of each mouse in Example 1 and Comparative Example 1. [Diagram 2] FIG. 2 shows the average relative expression level of each target gene to the expression level of the 18S ribosomal RNA gene in the body fluid samples of each mouse in Example 2. [Diagram 3] FIG. 3 shows the relationship between the average relative expression level of each target gene in the body fluid samples of the mice of Example 2 and the age of the mice in weeks. FIG. 3A shows the relationship between the average relative expression level of the Glut1 gene in the body fluid samples of the mice of Example 2 and the age of the mice in weeks. FIG. 3B shows the relationship between the average relative expression level of the Glut3 gene in the body fluid samples of the mice of Example 2 and the age of the mice in weeks. FIG. 3C shows the relationship between the average relative expression level of the MCT4 gene in the body fluid samples of the mice of Example 2 and the age of the mice in weeks. FIG. 3D shows the relationship between the average relative expression level of the PHD3 gene in the body fluid samples of the mice of Example 2 and the age of the mice in weeks. FIG. 3E shows the relationship between the average relative expression level of the PDK1 gene in the body fluid samples of the mice of Example 2 and the age of the mice in weeks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] <1.Terminology> <1.1. Samples derived from subjects> As used herein, the term "subject" refers to a human or non-human animal that is the subject of assessment of the degree of dementia, brain function, or aging. Examples of non-human animals include non-human mammals such as primates, rats, mice, gerbils, guinea pigs, hamsters, ferrets, rabbits, cows, horses, pigs, goats, dogs, and cats.

[0030] As samples derived from a subject, body fluid samples such as peripheral blood, saliva, urine, sputum, sweat, pharyngeal swabs, and nasal swabs, bone marrow samples, and central nervous tissue samples such as brain tissue can be used. In particular, from the viewpoint of non-invasiveness during sample collection, body fluid samples or bone marrow samples are preferred, and body fluid samples, particularly peripheral blood samples, are most preferred. As peripheral blood samples, various forms of peripheral blood samples such as serum, plasma, and whole blood can be used. As bone marrow samples, bone marrow fluid samples can be used. Samples derived from a subject are preferably samples isolated from the subject. As shown in the examples, it has been confirmed that the gene expression levels or enzyme activities of proteins described below are higher in body fluids such as peripheral blood or bone marrow in subjects suffering from dementia, subjects who may develop dementia in the future, subjects with reduced brain function, or subjects whose brain function may be reduced in the future, compared to normal samples, and that they are lower in central nervous tissues such as brain tissue, although not shown in the examples. In addition, as shown in the Examples, it has been confirmed that the gene expression level or enzyme activity of the protein described later is higher in body fluids such as peripheral blood or bone marrow in elderly subjects compared to young subjects, and is lower in central nervous tissues such as brain tissue, although not shown in the Examples. In addition, as shown in the Examples, it has been confirmed that the gene expression level or enzyme activity of the protein described later is higher in body fluids such as peripheral blood or bone marrow in subjects with brain tissue aging, subjects with possible brain tissue aging, subjects with peripheral blood aging, subjects with bone marrow cell aging, or subjects with possible bone marrow cell aging, compared to normal subjects, and is lower in central nervous tissues such as brain tissue, although not shown in the Examples.

[0031] In addition, a mononuclear cell-containing sample containing mononuclear cells (MNCs) separated from the subject's body fluid or bone marrow can be used as a body fluid sample or bone marrow sample derived from a subject. In this specification, "mononuclear cells" refers to lymphocytes and / or monocytes. In addition, in this specification, "leukocytes" refers collectively to neutrophils, basophils, eosinophils, lymphocytes and monocytes.

[0032] <1.2. Dementia> As used herein, "dementia" refers to any of Alzheimer's dementia, vascular dementia, Lewy body dementia, and frontotemporal dementia, with Alzheimer's dementia being particularly preferred.

[0033] Diagnosing dementia includes determining whether or not a subject is afflicted with dementia, determining the risk of developing dementia in a subject, determining the severity of dementia in a subject, and the like.

[0034] <1.3. Brain function> As used herein, "brain function" refers to functions of brain tissue involved in memory, movement, sensation, sleep, language, and the like.

[0035] Determining brain function includes determining whether or not brain function has declined in the subject, determining the risk of decline in brain function in the subject, determining the degree of decline in brain function in the subject, determining whether or not there is an autonomic nervous system disorder in the subject, determining the risk of autonomic nervous system disorder in the subject, determining the degree of autonomic nervous system disorder in the subject, determining whether or not there is a memory disorder in the subject, determining the risk of memory disorder in the subject, determining the degree of memory disorder in the subject, determining whether or not there is a motor disorder in the subject, determining the risk of motor disorder in the subject, determining the degree of motor disorder in the subject, determining whether or not there is a coordination disorder in the subject, determining the risk of coordination disorder in the subject, determining the degree of coordination disorder in the subject, determining whether or not there is an involuntary movement disorder in the subject, determining the risk of involuntary movement disorder in the subject, determining the degree of involuntary movement disorder in the subject, determining whether or not there is a sensory disorder in the subject, determining the risk of sensory disorder in the subject. determining the degree of sensory impairment in a subject, determining the presence or absence of a visual impairment in a subject, determining the risk of a visual impairment in a subject, determining the degree of a visual impairment in a subject, determining the presence or absence of an olfactory impairment in a subject, determining the risk of an olfactory impairment in a subject, determining the degree of an olfactory impairment in a subject, determining the presence or absence of a hearing impairment in a subject, determining the risk of a hearing impairment in a subject, determining the degree of a hearing impairment in a subject, determining the presence or absence of a balance impairment in a subject, determining the risk of a balance impairment in a subject, determining the degree of a balance impairment in a subject, determining the presence or absence of a sleep disorder in a subject, determining the risk of a sleep disorder in a subject, determining the degree of a sleep disorder in a subject, determining the presence or absence of a speech disorder in a subject, determining the risk of a speech disorder in a subject, determining the degree of a speech disorder in a subject, determining the presence or absence of an emotional dysregulation disorder in a subject, determining the risk of an emotional dysregulation disorder in a subject,These include determining the level of emotional dysregulation in a subject, determining the presence or absence of a headache in a subject, determining the risk of a headache in a subject, determining the level of a headache in a subject, determining the presence or absence of convulsions in a subject, determining the risk of convulsions in a subject, determining the level of convulsions in a subject, determining the presence or absence of tremors in a subject, determining the risk of tremors in a subject, determining the level of tremors in a subject, determining the presence or absence of hallucinations in a subject, determining the risk of hallucinations in a subject, determining the level of hallucinations in a subject, determining the presence or absence of delusions in a subject, determining the risk of delusions in a subject, determining the level of delusions in a subject, determining the presence or absence of illusions in a subject, determining the risk of illusions in a subject, determining the level of illusions in a subject, determining the presence or absence of abnormal behavior in a subject, determining the risk of abnormal behavior in a subject, determining the level of abnormal behavior in a subject, determining the presence or absence of depression in a subject, determining the risk of developing depression in a subject, or determining the severity of depression in a subject.

[0036] <1.4. Aging phenomenon> As used herein, the term "aging phenomenon" refers to physical and / or physiological changes that accompany aging. Examples of aging phenomena include skin changes such as age spots, wrinkles, sagging, and dullness, hair changes such as gray hair, thinning, and hair loss, brain morphological changes, brain tissue changes such as brain atrophy, peripheral blood changes, bone marrow cell changes, bone changes, and muscle changes.

[0037] As used herein, the term "degree of aging" refers to the degree of physical and / or physiological changes associated with aging. As used herein, the term "degree of aging" may be referred to as "degree of aging."

[0038] As used herein, "determination of the degree of aging" refers to determination of the degree of physical and physiological changes associated with aging. Determining the degree of aging includes, for example, determining the age of a subject, determining the degree of aging in brain tissue, determining the degree of aging in peripheral blood, determining the degree of aging in bone marrow cells, and the like.

[0039] <1.5. One or more proteins involved in energy metabolism reactions> As used herein, examples of the one or more proteins involved in an energy metabolic reaction include one or more proteins involved in glucose transport, lactate transport, energy metabolic control, mitochondrial biogenesis, glycolysis, the pentose phosphate pathway, the TCA cycle, the electron transport chain, fatty acid metabolism, gap junctions, or the sodium-potassium pump.

[0040] Examples of proteins involved in glucose transport include proteins constituting membrane proteins (glucose transporters) involved in glucose transport, which transport glucose used in cellular energy metabolism from outside the cell to inside the cell, and specifically, proteins belonging to the Glut (glucose transporter) family (Glut1 to 12) are included. Among the proteins belonging to the Glut family, gene expression or enzyme activity of Glut1, Glut2, Glut3 and Glut4, particularly Glut1 and Glut3, is preferred. The nucleotide sequence of mouse Glut1 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The nucleotide sequence of human Glut1 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4. The nucleotide sequence of mouse Glut3 is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6. The nucleotide sequence of human Glut3 is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8. The nucleotide sequences of SEQ ID NOs: 1, 3, 5 and 7 all include a nucleotide sequence (CDS) encoding the amino acid sequence of a protein, and nucleotide sequences of untranslated regions (UTR) located upstream and downstream of the CDS.

[0041] Examples of proteins involved in lactate transport include proteins constituting membrane proteins (lactate transporters) involved in lactate transport, which transport lactate produced by cellular energy metabolism from inside to outside the cells, and specifically include proteins belonging to the MCT (monocarboxylate transport protein) family. Among the proteins belonging to the MCT family, MCT1, MCT2, MCT3 and MCT4, particularly the gene expression or enzyme activity of MCT4, are preferred. The nucleotide sequence of mouse MCT4 is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10. The nucleotide sequence of human MCT4 is shown in SEQ ID NO: 11, and the amino acid sequence is shown in SEQ ID NO: 12. Both of the nucleotide sequences of SEQ ID NOs: 9 and 11 include a nucleotide sequence (CDS) that codes for the amino acid sequence of the protein, and the nucleotide sequences of the untranslated regions (UTR) located upstream and downstream of the CDS.

[0042] Proteins involved in energy metabolism control include proteins belonging to the PHD (prolyl hydroxylase domain-containing protein) family. Among the proteins belonging to the PHD family, PHD2 and PHD3, particularly the gene expression or enzyme activity of PHD3, are preferred. The nucleotide sequence of mouse PHD3 is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 14. The nucleotide sequence of human PHD3 is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16. Both the nucleotide sequences of SEQ ID NO: 13 and 15 include a nucleotide sequence (CDS) that codes for the amino acid sequence of the protein, and a nucleotide sequence of the untranslated region (UTR) located upstream and downstream of the CDS. In addition, examples of proteins involved in energy metabolism control include HIF1a (hypoxia-inducible factor 1 alpha), Sirt1 (sirtuin 1), PPARa (peroxisome proliferator-activated receptor alpha), and ERRa (estrogen-related receptor a).

[0043] An example of a protein involved in mitochondrial biogenesis is PGC1a (peroxisome proliferators-activated receptor-gamma co-activator-1 alpha).

[0044] Other examples of proteins involved in glycolysis include LDHa (lactate dehydrogenase A), LDHb (lactate dehydrogenase B), HK1 (hexokinase 1), PFK (phosphofructokinase), PKR (pyruvate kinase isozyme R), and Pkm1 ​​(pyruvate kinase isozymes M1).

[0045] Examples of proteins involved in the pentose phosphate pathway include glucose-6-phosphate dehydrogenase (G6PD), ribose-5-phosphate isomerase (RPI), ribulose-5-phosphate epimerase (RPE), 6-phosphogluconolactonase (6PGL), and transaldolase (TALDO).

[0046] Proteins involved in the TCA cycle include proteins belonging to the PDK (pyruvate dehydrogenase kinase) family. Among the proteins belonging to the PDK family, gene expression or enzyme activity of PDK1, PDK3 and PDK4 are preferred, and PDK1 is particularly preferred. The nucleotide sequence of mouse PDK1 is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 18. The nucleotide sequence of human PDK1 is shown in SEQ ID NO: 19, and the amino acid sequence is shown in SEQ ID NO: 20. Both the nucleotide sequences of SEQ ID NO: 17 and 19 include a nucleotide sequence (CDS) that codes for the amino acid sequence of the protein, and a nucleotide sequence of the untranslated region (UTR) located upstream and downstream of the CDS. In addition, other examples of proteins involved in the TCA cycle include IDH2 (isocitrate dehydrogenase 2), OGDH (2-oxoglutarate dehydrogenase), and CS (citrate synthase).

[0047] An example of a protein involved in the electron transport system is AMPK (AMP-activated protein kinase).

[0048] Examples of proteins involved in fatty acid metabolism include FABP1 (fatty acid binding protein 1), FABP4 (fatty acid binding protein 4), CD36 (fatty acid translocase: FAT), CPT1 (carnitine palmitoyltransferase 1), OXCT1 (3-oxo acid CoA-transferase 1), and ACC (acetyl-CoA carboxylase).

[0049] Examples of proteins involved in gap junctions include Cx37 (connexin 37) and Cx43 (connexin 43).

[0050] Examples of proteins involved in the sodium-potassium pump include ATP1A1 (sodium / potassium-transporting ATPase subunit alpha-1), ATP1A2 (sodium / potassium-transporting ATPase subunit alpha-2), and ATP1A3 (sodium / potassium-transporting ATPase subunit alpha-3).

[0051] <2. Method for obtaining an index for assessing dementia or brain function in a subject> One or more embodiments of the present invention include A method for obtaining an index for assessing dementia or brain function in a subject, comprising: The present invention relates to a method comprising measuring the gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample from a subject.

[0052] One or more embodiments of the present invention include A method for assessing dementia or brain function in a subject, comprising: Measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolism reaction in a sample from a subject; and Diagnosing dementia or brain function in the subject based on the measured values ​​of gene expression or enzyme activity of the one or more proteins. The present invention relates to a method comprising the steps of:

[0053] More preferably, each of said methods comprises: comparing the measured values ​​of gene expression or enzymatic activity of said one or more proteins with a reference value; Further includes:

[0054] In this embodiment, the "reference value" may be a measured value when the gene expression or enzyme activity of the one or more proteins in a sample derived from a normal specimen is measured under the same conditions as the gene expression or enzyme activity of the one or more proteins in a sample derived from a subject, or a standard value set from the measured value, or a measured value when the gene expression or enzyme activity of the one or more proteins in a sample derived from a patient whose presence or absence or severity of dementia is known is measured under the same conditions as the gene expression or enzyme activity of the one or more proteins in a sample derived from a subject, or a standard value set from the measured value.

[0055] One or more embodiments of the present invention are based on the unexpected finding that gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction is higher in body fluids such as peripheral blood, urine, saliva, etc. or bone marrow and lower in central nervous tissue such as brain tissue in subjects suffering from dementia, subjects who may develop dementia in the future, subjects with reduced brain function, or subjects whose brain function may be reduced in the future, compared to normal subjects. In addition, the gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction is higher in body fluids such as peripheral blood, urine, saliva, etc. or bone marrow and lower in central nervous tissue such as brain tissue, as the subject's dementia becomes more severe. In addition, the gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction is higher in body fluids such as peripheral blood, urine, saliva, etc. or bone marrow and lower in central nervous tissue such as brain tissue, as the subject's brain function becomes lower. Therefore, in the embodiment of the present invention, when the sample is a body fluid sample or a bone marrow sample, particularly a mononuclear cell-containing sample, a higher measured value than the reference value is an indicator that the subject suffers from dementia, that the subject is at risk of developing dementia, that the subject's dementia is more severe, that the subject's brain function is reduced, that the subject's brain function is at risk of reducing, or that the degree of reduction in the subject's brain function is more significant. Also, in the embodiment of the present invention, when the sample is a central nervous tissue sample, a lower measured value than the reference value is an indicator that the subject suffers from dementia, that the subject is at risk of developing dementia, that the subject's dementia is more severe, that the subject's brain function is reduced, that the subject's brain function is at risk of reducing, or that the degree of reduction in the subject's brain function is more significant.

[0056] In one or more embodiments of the invention, when the sample is a body fluid sample or a bone marrow sample, in particular a mononuclear cell-containing sample, the lower limit of the ratio of the measured value to the reference value is preferably 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2.0 or more, 2.1 or more, 2.2 or more, 2.3 or more, 2.4 or more, 2.5 or more, 2.6 or more, 2.7 or more, 2.8 or more, 2.9 or more, 3.0 or more, 3.2 or more, 3.4 or more, 3.6 or more, 3.8 or more, 4.0 or more, 4.2 or more, 4.4 or more, 4.6 or more, 4.8 or more, 5.0 or more, 5.2 or more, 5.4 or more, 5.6 or more, 5.8 or more, or 6.0 or more. Furthermore, when the sample is a body fluid sample or a bone marrow sample, particularly a mononuclear cell-containing sample, the upper limit of the ratio of the measured value to the reference value is not particularly limited, and can be, for example, 50.0 or less, 45.0 or less, 40.0 or less, 35.0 or less, 30.0 or less, 25.0 or less, 20.0 or less, 15.0 or less, 14.0 or less, 13.0 or less, 12.0 or less, 11.0 or less, 10.0 or less, 9.8 or less, 9.6 or less, 9.4 or less, 9.2 or less, or 9.0 or less. These ratios are particularly preferred when the reference value is a measured value when the gene expression or enzyme activity of the one or more proteins in a sample derived from a normal specimen is measured under the same conditions as the gene expression or enzyme activity of the one or more proteins in a sample derived from a subject, or a standard value set from the measured value.

[0057] In one or more embodiments of the present invention, it is preferable to measure gene expression or enzyme activity of one or more, two or more, three or more, four or more, or all proteins selected from Glut1, Glut3, MCT4, PHD3, and PDK1 as one or more proteins involved in the energy metabolic reaction. In an embodiment in which the sample is a body fluid sample or a bone marrow sample, particularly a mononuclear cell-containing sample, and gene expression or enzyme activity of two to five proteins out of the five proteins is measured, it is preferable to determine that the subject suffers from dementia, that the subject is at risk of developing dementia, that the subject's dementia is more severe, that the subject's brain function is reduced, that the subject is at risk of reducing brain function, or that the degree of reduction in brain function of the subject is more significant, when the measured values ​​of gene expression or enzyme activity of more than half (e.g., more than 1 / 2, more than 2 / 3, more than 2 / 4, more than 3 / 5) of the measured proteins are higher than the reference value.

[0058] In the embodiment of the present invention, either the gene expression or the enzyme activity of the one or more proteins may be measured, or both may be measured, more preferably the gene expression of the one or more proteins is measured.

[0059] The gene expression of the one or more proteins may be measured by detecting the mRNA (including the coding region and the untranslated region) of the gene of the one or more proteins in a sample, or by detecting the protein amount of the one or more proteins in a sample.

[0060] The measured value of gene expression of the one or more proteins may be the relative expression level of the gene expression of the one or more proteins to the expression level of one or more endogenous controls in the sample. As the endogenous controls, TBP (TATA-binding protein), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), β-actin, β2M (β2 microglobuline), HPRT1 (hypoxanthine phosphoribosyltransferase 1), 18S ribosomal RNA, particularly a housekeeping gene represented by 18S ribosomal RNA, can be used. The nucleotide sequence of mouse 18S ribosomal RNA is shown in SEQ ID NO: 21. The nucleotide sequence of human 18S ribosomal RNA is shown in SEQ ID NO: 22.

[0061] The detection of mRNA of a gene encoding the amino acid sequence of one or more proteins can be performed by Northern blotting, RT-PCR, real-time RT-PCR, DNA microarray (a method using a DNA chip), dot blot, RNase protection assay, etc. These methods can be performed by known methods.

[0062] The detection of the amount of the one or more proteins can be performed by an immunoassay using an antibody that specifically recognizes and binds to the one or more proteins. The antibody can be prepared by a known method. Examples of the immunoassay include a method using a solid phase carrier to which an antibody that specifically binds to the one or more proteins to be detected is immobilized, flow cytometry, Western blotting, etc. Examples of the method using a solid phase carrier include, but are not limited to, an enzyme-linked immunosorbent assay (ELISA) using an immobilized microtiter plate, and an agglutination method (immunoprecipitation) using immobilized particles, and the like. The amount of the one or more proteins in a sample can be detected by adopting a known immunoassay method. In addition, the detection of the amount of the one or more proteins can also be performed by a method using LC-MS / MS MRM, which is a protein mass spectrometry technique that does not use an antibody. These detection methods can also be performed according to a conventional protocol.

[0063] The enzymatic activity of the one or more proteins in a sample can be measured by a method depending on the enzymatic activity of the protein to be measured.

[0064] The ratio of mononuclear cells in the sample is preferably 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%. As used herein, the "ratio of mononuclear cells in a sample" refers to the ratio of the number of mononuclear cells to the number of all white blood cells contained in the sample. The ratio of mononuclear cells in a sample can be measured, for example, by a hematology analyzer, a hemocytometer, flow cytometry, etc., but is not limited thereto.

[0065] In one or more embodiments of the present invention, measuring gene expression or enzyme activity of one or more proteins involved in energy metabolic reactions in a sample derived from the subject preferably includes the following (a) and (b). (a) obtaining a mononuclear cell-containing sample by isolating mononuclear cells from said sample; (b) measuring the gene expression or enzymatic activity of said one or more proteins in said mononuclear cell-containing sample.

[0066] A sample derived from a subject may contain granulocytes, and the granulocytes in the sample may be a factor that reduces the measurement accuracy of the measured values ​​of the gene expression or enzyme activity of the one or more proteins. Therefore, by isolating mononuclear cells from the sample or removing granulocytes to obtain a mononuclear cell-containing sample with an increased ratio of mononuclear cells, and subjecting the obtained mononuclear cell-containing sample to measurement, a highly accurate measurement value can be obtained.

[0067] The ratio of mononuclear cells in the mononuclear cell-containing sample is preferably 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%. The ratio of granulocytes in the mononuclear cell-containing sample is preferably less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, or 0%. In this specification, the "ratio of mononuclear cells or granulocytes in the mononuclear cell-containing sample" refers to the ratio of the number of mononuclear cells or granulocytes to the number of all cells contained in the mononuclear cell-containing sample. The ratio of mononuclear cells or granulocytes in the mononuclear cell-containing sample can be measured, for example, by a hematology analyzer, a hemocytometer, flow cytometry, etc., but is not limited thereto.

[0068] A method for obtaining a mononuclear cell-containing sample from a sample derived from a subject is preferably a method using a reagent and / or column for separating mononuclear cells. Examples of the reagent and / or column include Ficoll-Paque PLUS (GE Healthcare), Lymphoprep (Abbott Diagnostics Technologies), Human Peripheral Blood Mononuclear Cell Isolation and Viability Kit (BioVision), BD Vacutainer (registered trademark) CPT blood collection tube for mononuclear cell separation (Becton Dickinson), pluriMate (pluriSelect), SepMate (STEMCELL Technologies), etc. By using the reagent and / or column, mononuclear cells and granulocytes in the sample can be separated, and a mononuclear cell-containing sample for measurement can be efficiently obtained.

[0069] <3. Method for obtaining an index for determining the degree of aging phenomenon in a subject> One or more embodiments of the present invention include A method for obtaining an index for determining the degree of aging in a subject, comprising: The present invention relates to a method comprising measuring the gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample from a subject.

[0070] One or more embodiments of the present invention include A method for determining the degree of aging in a subject, comprising: Measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolism reaction in a sample from a subject; and determining the degree of aging in said subject based on the measured values ​​of gene expression or enzymatic activity of said one or more proteins; The present invention relates to a method comprising the steps of:

[0071] More preferably, each of said methods comprises: comparing the measured values ​​of gene expression or enzymatic activity of said one or more proteins with a reference value; Further includes:

[0072] One or more embodiments of the present invention are based on the unexpected finding that the gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction is higher in body fluids such as peripheral blood, urine, saliva, etc. or bone marrow, and lower in central nervous tissue such as brain tissue, in an elderly subject, compared to a young subject. Also, one or more embodiments of the present invention show that the gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction is higher in body fluids such as peripheral blood, urine, saliva, etc. or bone marrow, and lower in central nervous tissue such as brain tissue, in subjects with aging of brain tissue, subjects with aging of peripheral blood, subjects with aging of bone marrow cells, or subjects with aging of bone marrow cells, compared to a normal subject. In one or more embodiments of the present invention, the gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction is higher in body fluids such as peripheral blood, urine, saliva, etc. or bone marrow, and lower in central nervous tissue such as brain tissue, in subjects with a high degree of aging in brain tissue, subjects with a high degree of aging in peripheral blood, or subjects with a high degree of aging in bone marrow cells, compared to normal subjects. In addition, the gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction is higher in body fluids such as peripheral blood, urine, saliva, etc. or bone marrow, and lower in central nervous tissue such as brain tissue, as the degree of aging in the subject's peripheral blood is higher. In addition, the gene expression or enzyme activity of one or more proteins involved in an energy metabolic reaction is higher in body fluids such as peripheral blood, urine, saliva, etc. or bone marrow, and lower in central nervous tissue such as brain tissue, as the degree of aging in the subject's peripheral blood is higher. In addition, the greater the degree of aging of the subject's bone marrow cells, the higher the gene expression or enzyme activity of one or more proteins involved in energy metabolic reactions in body fluids such as peripheral blood, urine, and saliva, or in bone marrow, and the lower the gene expression or enzyme activity in central nervous tissues such as brain tissue.Therefore, in the embodiment of the present invention, when the sample is a body fluid sample, a bone marrow sample, or a central nervous tissue sample, the measurement value serves as an indicator of the age of the subject, the possibility of aging of the subject's brain tissue progressing, the degree of aging of the subject's brain tissue, the possibility of aging of the subject's peripheral blood progressing, the degree of aging of the subject's peripheral blood, or the degree of aging of the subject's bone marrow cells.

[0073] In one or more embodiments of the present invention, it is preferable to measure gene expression or enzyme activity of one or more, two or more, three or more, four or more, or all proteins selected from Glut1, Glut3, MCT4, PHD3, and PDK1 as one or more proteins involved in the energy metabolic reaction. In an embodiment in which the sample is a body fluid sample or a bone marrow sample, particularly a mononuclear cell-containing sample, and gene expression or enzyme activity of two to five proteins out of the five proteins is measured, it is preferable to determine that the aging phenomenon of the subject is more advanced when the measured values ​​of gene expression or enzyme activity of more than half of the measured proteins (e.g., 1 / 2 or more, 2 / 3 or more, 2 / 4 or more, 3 / 5 or more) are higher than the reference value.

[0074] In the embodiment of the present invention, either the gene expression or the enzyme activity of the one or more proteins may be measured, or both may be measured, more preferably the gene expression of the one or more proteins is measured.

[0075] The gene expression of the one or more proteins may be measured by detecting the mRNA (including the coding region and the untranslated region) of the gene of the one or more proteins in a sample, or by detecting the protein amount of the one or more proteins in a sample.

[0076] The detection of mRNA of the gene for one or more proteins can be performed by Northern blotting, RT-PCR, real-time RT-PCR, DNA microarray (a method using a DNA chip), dot blot, RNase protection assay, etc. These methods can be performed by known methods.

[0077] The detection of the amount of the one or more proteins can be performed by an immunoassay using an antibody that specifically recognizes and binds to the one or more proteins. The antibody can be prepared by a known method. Examples of the immunoassay include a method using a solid phase carrier to which an antibody that specifically binds to the one or more proteins to be detected is immobilized, flow cytometry, Western blotting, etc. Examples of the method using a solid phase carrier include, but are not limited to, an enzyme-linked immunosorbent assay (ELISA) using an immobilized microtiter plate, and an agglutination method (immunoprecipitation) using immobilized particles, and the like. The amount of the one or more proteins in a sample can be detected by adopting a known immunoassay method. In addition, the detection of the amount of the one or more proteins can also be performed by a method using LC-MS / MS MRM, which is a protein mass spectrometry technique that does not use an antibody. These detection methods can also be performed according to a conventional protocol. The enzymatic activity of the one or more proteins in a sample can be measured by a method depending on the enzymatic activity of the protein to be measured.

[0078] <4. Kits for assessing dementia or brain function> One or more further embodiments of the present invention include A kit for determining dementia or brain function, comprising: The present invention relates to a kit comprising a reagent for measuring gene expression or enzyme activity of one or more proteins involved in energy metabolic reactions in a sample derived from a subject. The reagent is preferably a reagent that can be used in the above-mentioned method for measuring gene expression or enzyme activity of one or more proteins.

[0079] Specifically, examples of the probe include a primer pair for amplifying a nucleic acid (genomic DNA, mRNA, or cDNA prepared based on mRNA) containing the gene of the one or more proteins to be measured, a probe that hybridizes with a nucleic acid (genomic DNA, mRNA, or cDNA prepared based on mRNA) containing the gene of the one or more proteins to be measured, an antibody that specifically binds to the one or more proteins, and a reaction substrate for measuring the enzyme activity of the one or more proteins. The nucleic acid may also include a non-translated region, an intron region, a signal sequence region, and the like. Preferably, the nucleic acid is mRNA or cDNA. The mRNA or cDNA may include at least a part of the base sequence (CDS) encoding the amino acid sequence of the one or more proteins and the base sequence of the untranslated region (UTR) located upstream and downstream thereof. An example of the primer pair is a primer pair that can amplify at least a part of the base sequence of the CDS and UTR. An example of the probe is a probe that can hybridize to at least a part of the base sequence of the CDS and UTR.

[0080] An example of the primer pair is a primer pair for amplifying a nucleic acid containing a Glut1 gene, the primer pair including a Glut1 forward primer having at its 3' end a base sequence Af identical or homologous to any of the base sequences shown in (A1) to (A4) below, and a Glut1 reverse primer having at its 3' end a base sequence Ar identical or homologous to the complementary base sequence of any of the base sequences shown in (A5) to (A8) below. (A1) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 101 to 180 of SEQ ID NO: 1 (A2) A base sequence of SEQ ID NO: 3 that corresponds to the base sequence of (A1) when the base sequence of SEQ ID NO: 3 is aligned with the base sequence of SEQ ID NO: 1. (A3) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 256 to 336 of SEQ ID NO: 3 (A4) A base sequence of SEQ ID NO: 1 that corresponds to the base sequence of (A3) when the base sequence of SEQ ID NO: 1 is aligned with the base sequence of SEQ ID NO: 3. (A5) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 213 to 292 of SEQ ID NO: 1 (A6) A base sequence of SEQ ID NO: 3 that corresponds to the base sequence of (A5) when the base sequence of SEQ ID NO: 3 is aligned with the base sequence of SEQ ID NO: 1. (A7) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 339 to 418 of SEQ ID NO: 3 (A8) A base sequence of SEQ ID NO: 1, which corresponds to the base sequence of (A7) when the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 3 are aligned.

[0081] In the above (A1), the "base sequence from positions 101 to 180" is preferably the "base sequence from positions 111 to 170", more preferably the "base sequence from positions 121 to 160", more preferably the "base sequence from positions 121 to 155", and more preferably the "base sequence from positions 126 to 150".

[0082] In the above (A3), the "base sequence from positions 256 to 336" is preferably the "base sequence from positions 266 to 326", more preferably the "base sequence from positions 276 to 316", more preferably the "base sequence from positions 276 to 311", and more preferably the "base sequence from positions 281 to 306".

[0083] In the above (A1) and (A3), "10 bases or more" is preferably "15 bases or more," more preferably "17 bases or more," and more preferably "19 bases or more." In the above (A1) and (A3), "consecutive" preferably means "consecutive from the 3' end."

[0084] The base sequence Af may be identical or homologous to the base sequence of (A1), (A2), (A3), or (A4), but preferably, from the 3' end, a continuous base sequence of 2 or more bases, more preferably 3 or more bases, more preferably 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 19 or more bases is identical to the base sequence of (A1), (A2), (A3), or (A4), and the remainder of the base sequence Af is identical or homologous to the base sequence of (A1), (A2), (A3), or (A4). The range of "homology" is as described below. Particularly preferably, the base sequence Af is identical to the base sequence of (A1), (A2), (A3), or (A4).

[0085] The Glut1 forward primer may contain a polynucleotide containing the nucleotide sequence Af at the 3'-terminus, and may further contain another nucleotide sequence on the 5'-terminus side of the nucleotide sequence Af. The number of nucleotides in the polynucleotide contained in the Glut1 forward primer is not particularly limited, but is preferably 50 nucleotides or less, more preferably 40 nucleotides or less, and more preferably 30 nucleotides or less.

[0086] A specific example of the base sequence of (A1) is the base sequence of SEQ ID NO: 25. A specific example of the base sequence Af is the base sequence of SEQ ID NO: 25.

[0087] A specific example of the base sequence of (A3) is the base sequence of SEQ ID NO: 37. A specific example of the base sequence Af is the base sequence of SEQ ID NO: 37.

[0088] In the above (A5), the "base sequence from positions 213 to 292" is preferably the "base sequence from positions 223 to 282", more preferably the "base sequence from positions 233 to 272", more preferably the "base sequence from positions 238 to 272", and more preferably the "base sequence from positions 243 to 267".

[0089] In the above (A7), the "base sequence from positions 339 to 418" is preferably the "base sequence from positions 349 to 408", more preferably the "base sequence from positions 359 to 398", more preferably the "base sequence from positions 364 to 398", and more preferably the "base sequence from positions 369 to 393".

[0090] In the above (A5) and (A7), "10 bases or more" is preferably "15 bases or more," more preferably "17 bases or more," and more preferably "19 bases or more." In the above (A5) and (A7), "consecutive" preferably means "consecutive from the 5' end."

[0091] The base sequence Ar may be identical or homologous to the complementary base sequence of the base sequence of (A5), (A6), (A7), or (A8), but preferably 2 or more bases, more preferably 3 or more bases, more preferably 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 19 or more bases from the 3' end of the base sequence Ar are identical to the complementary base sequence of the base sequence of (A5), (A6), (A7), or (A8), and the remainder of the base sequence Ar is identical or homologous to the complementary base sequence of the base sequence of (A5), (A6), (A7), or (A8). The range of "homology" is as described below. Particularly preferably, the base sequence Ar is identical to the complementary base sequence of the base sequence of (A5), (A6), (A7) or (A8).

[0092] The Glut1 reverse primer may contain a polynucleotide containing the base sequence Ar at the 3'-end, and may further contain another base sequence on the 5'-end side of the base sequence Ar. The number of bases of the polynucleotide contained in the Glut1 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.

[0093] A specific example of the base sequence of (A5) is the complementary base sequence of the base sequence of SEQ ID NO: 26. A specific example of the base sequence Ar is the base sequence of SEQ ID NO: 26.

[0094] A specific example of the base sequence of (A7) is the complementary base sequence of the base sequence of SEQ ID NO: 38. A specific example of the base sequence Ar is the base sequence of SEQ ID NO: 38.

[0095] An example of the primer pair is a primer pair for amplifying a nucleic acid containing a Glut3 gene, the primer pair including a Glut3 forward primer having at its 3' end a base sequence Bf identical or homologous to any of the base sequences shown in the following (B1) to (B4), and a Glut3 reverse primer having at its 3' end a base sequence Br identical or homologous to a complementary base sequence of any of the base sequences shown in the following (B5) to (B8). (B1) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 250 to 329 of SEQ ID NO: 5 (B2) Among the base sequences of SEQ ID NO: 7, a base sequence corresponding to the base sequence of (B1) when the base sequence of SEQ ID NO: 7 and the base sequence of SEQ ID NO: 5 are aligned. (B3) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 224 to 303 of SEQ ID NO: 7 (B4) A base sequence of SEQ ID NO:5 that corresponds to the base sequence of (B3) when the base sequence of SEQ ID NO:5 is aligned with the base sequence of SEQ ID NO:7. (B5) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 375 to 454 of SEQ ID NO: 5 (B6) A base sequence of SEQ ID NO: 7 that corresponds to the base sequence of (B5) when the base sequence of SEQ ID NO: 7 is aligned with the base sequence of SEQ ID NO: 5. (B7) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 288 to 367 of SEQ ID NO: 7 (B8) A base sequence of SEQ ID NO:5, which corresponds to the base sequence of (B7) when the base sequence of SEQ ID NO:5 is aligned with the base sequence of SEQ ID NO:7.

[0096] In the above (B1), the "base sequence from positions 250 to 329" is preferably the "base sequence from positions 260 to 319", more preferably the "base sequence from positions 270 to 309", more preferably the "base sequence from positions 270 to 304", and more preferably the "base sequence from positions 275 to 299".

[0097] In the above (B3), the "base sequence from positions 224 to 303" is preferably the "base sequence from positions 234 to 293", more preferably the "base sequence from positions 244 to 283", more preferably the "base sequence from positions 244 to 278", and more preferably the "base sequence from positions 249 to 273".

[0098] In the above (B1) and (B3), "10 bases or more" is preferably "15 bases or more," more preferably "17 bases or more," and more preferably "19 bases or more." In the above (B1) and (B3), "consecutive" preferably means "consecutive from the 3' end."

[0099] The base sequence Bf may be identical or homologous to the base sequence of (B1), (B2), (B3), or (B4), but preferably, from the 3' end, a continuous base sequence of 2 or more bases, more preferably 3 or more bases, more preferably 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 19 or more bases is identical to the base sequence of (B1), (B2), (B3), or (B4), and the remainder of the base sequence Bf is identical or homologous to the base sequence of (B1), (B2), (B3), or (B4). The range of "homology" is as described below. Particularly preferably, the base sequence Bf is identical to the base sequence of (B1), (B2), (B3), or (B4).

[0100] The Glut3 forward primer may contain a polynucleotide containing the nucleotide sequence Bf at the 3'-end, and may further contain another nucleotide sequence on the 5'-end side of the nucleotide sequence Bf. The number of nucleotides in the polynucleotide contained in the Glut3 forward primer is not particularly limited, but is preferably 50 nucleotides or less, more preferably 40 nucleotides or less, and more preferably 30 nucleotides or less.

[0101] A specific example of the base sequence of (B1) is the base sequence of SEQ ID NO: 27. A specific example of the base sequence Bf is the base sequence of SEQ ID NO: 27.

[0102] A specific example of the base sequence of (B3) is the base sequence of SEQ ID NO: 39. A specific example of the base sequence Bf is the base sequence of SEQ ID NO: 39.

[0103] In the above (B5), the "base sequence from positions 375 to 454" is preferably the "base sequence from positions 385 to 444", more preferably the "base sequence from positions 395 to 434", more preferably the "base sequence from positions 400 to 434", and more preferably the "base sequence from positions 405 to 429".

[0104] In the above (B7), the "base sequence from positions 288 to 367" is preferably the "base sequence from positions 298 to 357", more preferably the "base sequence from positions 308 to 347", more preferably the "base sequence from positions 313 to 347", and more preferably the "base sequence from positions 318 to 342".

[0105] In the above (B5) and (B7), "10 bases or more" is preferably "15 bases or more," more preferably "17 bases or more," and more preferably "19 bases or more." In the above (B5) and (B7), "consecutive" preferably means "consecutive from the 5' end."

[0106] The base sequence Br may be identical or homologous to the complementary base sequence of the base sequence of (B5), (B6), (B7), or (B8), but preferably a continuous base sequence of 2 or more bases, more preferably 3 or more bases, more preferably 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 19 or more bases from the 3' end of the base sequence Br is identical to the complementary base sequence of the base sequence of (B5), (B6), (B7), or (B8), and the remainder of the base sequence Br is identical or homologous to the complementary base sequence of the base sequence of (B5), (B6), (B7), or (B8). The range of "homology" is as described below. Particularly preferably, the base sequence Br is identical to the complementary base sequence of the base sequence (B5), the base sequence (B6), the base sequence (B7) or the base sequence (B8).

[0107] The Glut3 reverse primer may contain a polynucleotide containing the base sequence Br at the 3'-terminus, and may further contain another base sequence on the 5'-terminus side of the base sequence Br. The number of bases of the polynucleotide contained in the Glut3 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.

[0108] A specific example of the base sequence of (B5) is the complementary base sequence of the base sequence of SEQ ID NO: 28. A specific example of the base sequence Br is the base sequence of SEQ ID NO: 28.

[0109] A specific example of the base sequence of (B7) is the complementary base sequence of the base sequence of SEQ ID NO: 40. A specific example of the base sequence Br is the base sequence of SEQ ID NO: 40.

[0110] An example of the primer pair is a primer pair for amplifying a nucleic acid containing an MCT4 gene, which includes an MCT4 forward primer having a base sequence Cf at its 3' end that is identical or homologous to any of the base sequences shown in (C1) to (C4) below, and an MCT4 reverse primer having a base sequence Cr at its 3' end that is identical or homologous to the complementary base sequence of any of the base sequences shown in (C5) to (C8) below. (C1) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 40 to 118 of SEQ ID NO: 9 (C2) A base sequence of SEQ ID NO: 11 that corresponds to the base sequence of (C1) when the base sequence of SEQ ID NO: 11 is aligned with the base sequence of SEQ ID NO: 9. (C3) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 146 to 225 of SEQ ID NO: 11 (C4) A base sequence of SEQ ID NO: 9 that corresponds to the base sequence of (C3) when the base sequence of SEQ ID NO: 9 is aligned with the base sequence of SEQ ID NO: 11. (C5) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 107 to 186 of SEQ ID NO: 9 (C6) A base sequence of SEQ ID NO: 11 that corresponds to the base sequence of (C5) when the base sequence of SEQ ID NO: 11 is aligned with the base sequence of SEQ ID NO: 9. (C7) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 225 to 304 of SEQ ID NO: 11 (C8) A base sequence of SEQ ID NO: 9, which corresponds to the base sequence of (C7) when the base sequence of SEQ ID NO: 9 is aligned with the base sequence of SEQ ID NO: 11.

[0111] In the above (C1), the "base sequence from positions 40 to 118" is preferably the "base sequence from positions 50 to 108", more preferably the "base sequence from positions 60 to 98", more preferably the "base sequence from positions 60 to 93", and more preferably the "base sequence from positions 65 to 88".

[0112] In the above (C3), the "base sequence from positions 146 to 225" is preferably the "base sequence from positions 156 to 215", more preferably the "base sequence from positions 166 to 205", more preferably the "base sequence from positions 166 to 200", and more preferably the "base sequence from positions 171 to 195".

[0113] In the above (C1) and (C3), "10 bases or more" is preferably "15 bases or more," more preferably "17 bases or more," and more preferably "19 bases or more." In the above (C1) and (C3), "consecutive" preferably means "consecutive from the 3' end."

[0114] The base sequence Cf may be identical or homologous to the base sequence of (C1), (C2), (C3), or (C4), but preferably, from the 3' end, a continuous base sequence of 2 or more bases, more preferably 3 or more bases, more preferably 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 19 or more bases is identical to the base sequence of (C1), (C2), (C3), or (C4), and the remainder of the base sequence Cf is identical or homologous to the base sequence of (C1), (C2), (C3), or (C4). The range of "homology" is as described below. Particularly preferably, the base sequence Cf is identical to the base sequence of (C1), (C2), (C3), or (C4).

[0115] The MCT4 forward primer may contain a polynucleotide containing the base sequence Cf at the 3' end, and may further contain another base sequence on the 5' end side of the base sequence Cf. The number of bases of the polynucleotide contained in the MCT4 forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.

[0116] A specific example of the base sequence of (C1) is the base sequence of SEQ ID NO: 29. A specific example of the base sequence Cf is the base sequence of SEQ ID NO: 29.

[0117] A specific example of the base sequence of (C3) is the base sequence of SEQ ID NO: 41. A specific example of the base sequence Cf is the base sequence of SEQ ID NO: 41.

[0118] In the above (C5), the "base sequence from positions 107 to 186" is preferably the "base sequence from positions 117 to 176", more preferably the "base sequence from positions 127 to 166", more preferably the "base sequence from positions 132 to 166", and more preferably the "base sequence from positions 137 to 161".

[0119] In the above (C7), the "base sequence from positions 225 to 304" is preferably the "base sequence from positions 235 to 294", more preferably the "base sequence from positions 245 to 284", more preferably the "base sequence from positions 250 to 284", and more preferably the "base sequence from positions 255 to 279".

[0120] In the above (C5) and (C7), "10 bases or more" is preferably "15 bases or more," more preferably "17 bases or more," and more preferably "19 bases or more." In the above (C5) and (C7), "consecutive" preferably means "consecutive from the 5' end."

[0121] The base sequence Cr may be identical or homologous to the complementary base sequence of the base sequence of (C5), (C6), (C7), or (C8), but a continuous base sequence of preferably 2 or more bases, more preferably 3 or more bases, more preferably 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 19 or more bases from the 3' end of the base sequence Cr is identical to the complementary base sequence of the base sequence of (C5), (C6), (C7), or (C8), and the remainder of the base sequence Cr is identical or homologous to the complementary base sequence of the base sequence of (C5), (C6), (C7), or (C8). The range of "homology" is as described below. Particularly preferably, the base sequence Cr is identical to the complementary base sequence of the base sequence (C5), the base sequence (C6), the base sequence (C7) or the base sequence (C8).

[0122] The MCT4 reverse primer may contain a polynucleotide containing the base sequence Cr at the 3' end, and may further contain another base sequence on the 5' end side of the base sequence Cr. The number of bases of the polynucleotide contained in the MCT4 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.

[0123] A specific example of the base sequence of (C5) is the complementary base sequence of the base sequence of SEQ ID NO: 30. A specific example of the base sequence Cr is the base sequence of SEQ ID NO: 30.

[0124] A specific example of the base sequence of (C7) is the complementary base sequence of the base sequence of SEQ ID NO: 42. A specific example of the base sequence Cr is the base sequence of SEQ ID NO: 42.

[0125] An example of the primer pair is a primer pair for amplifying a nucleic acid containing a PHD3 gene, which includes a PHD3 forward primer having at its 3' end a base sequence Df that is identical or homologous to any of the base sequences shown in (D1) to (D4) below, and a PHD3 reverse primer having at its 3' end a base sequence Dr that is identical or homologous to the complementary base sequence of any of the base sequences shown in (D5) to (D8) below. (D1) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 864 to 943 of SEQ ID NO: 13 (D2) A base sequence of SEQ ID NO: 15 that corresponds to the base sequence of (D1) when the base sequence of SEQ ID NO: 15 is aligned with the base sequence of SEQ ID NO: 13. (D3) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 863 to 942 of SEQ ID NO: 15 (D4) A base sequence of SEQ ID NO: 13 that corresponds to the base sequence of (D3) when the base sequence of SEQ ID NO: 13 is aligned with the base sequence of SEQ ID NO: 15. (D5) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 991 to 1072 of SEQ ID NO: 13 (D6) A base sequence of SEQ ID NO: 15 that corresponds to the base sequence of (D5) when the base sequence of SEQ ID NO: 15 is aligned with the base sequence of SEQ ID NO: 13. (D7) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 1004 to 1083 of SEQ ID NO: 15. (D8) A base sequence of SEQ ID NO: 13, which corresponds to the base sequence of (D7) when the base sequence of SEQ ID NO: 13 is aligned with the base sequence of SEQ ID NO: 15.

[0126] In the above (D1), the "base sequence from positions 864 to 943" is preferably the "base sequence from positions 874 to 933", more preferably the "base sequence from positions 884 to 923", more preferably the "base sequence from positions 884 to 918", and more preferably the "base sequence from positions 889 to 913".

[0127] In (D3), the "base sequence from positions 863 to 942" is preferably the "base sequence from positions 873 to 932", more preferably the "base sequence from positions 883 to 922", more preferably the "base sequence from positions 883 to 917", and more preferably the "base sequence from positions 888 to 912".

[0128] In the above (D1) and (D3), "10 bases or more" is preferably "15 bases or more," more preferably "17 bases or more," and more preferably "19 bases or more." In the above (D1) and (D3), "consecutive" preferably means "consecutive from the 3' end."

[0129] The base sequence Df may be identical or homologous to the base sequence of (D1), (D2), (D3), or (D4), but preferably, from the 3' end, a continuous base sequence of 2 or more bases, more preferably 3 or more bases, more preferably 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 19 or more bases is identical to the base sequence of (D1), (D2), (D3), or (D4), and the remainder of the base sequence Df is identical or homologous to the base sequence of (D1), (D2), (D3), or (D4). The range of "homology" is as described below. Particularly preferably, the base sequence Df is identical to the base sequence of (D1), (D2), (D3), or (D4).

[0130] The PHD3 forward primer may contain a polynucleotide containing the base sequence Df at the 3' end, and may further contain another base sequence on the 5' end side of the base sequence Df. The number of bases of the polynucleotide contained in the PHD3 forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.

[0131] A specific example of the base sequence of (D1) is the base sequence of SEQ ID NO: 31. A specific example of the base sequence Df is the base sequence of SEQ ID NO: 31.

[0132] A specific example of the base sequence of (D3) is the base sequence of SEQ ID NO: 43. A specific example of the base sequence Df is the base sequence of SEQ ID NO: 43.

[0133] In (D5), the "base sequence from positions 991 to 1072" is preferably the "base sequence from positions 1001 to 1062", more preferably the "base sequence from positions 1011 to 1052", more preferably the "base sequence from positions 1016 to 1047", and more preferably the "base sequence from positions 1021 to 1042".

[0134] In (D7), the "base sequence from positions 1004 to 1083" is preferably the "base sequence from positions 1014 to 1073", more preferably the "base sequence from positions 1024 to 1063", more preferably the "base sequence from positions 1029 to 1063", and more preferably the "base sequence from positions 1034 to 1058".

[0135] In the above (D5) and (D7), "10 bases or more" is preferably "15 bases or more," more preferably "17 bases or more," and more preferably "19 bases or more." In the above (D5) and (D7), "consecutive" preferably means "consecutive from the 5' end."

[0136] The base sequence Dr may be identical or homologous to the complementary base sequence of the base sequence of (D5), (D6), (D7), or (D8), but preferably 2 or more bases, more preferably 3 or more bases, more preferably 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 19 or more bases from the 3' end of the base sequence Dr are identical to the complementary base sequence of the base sequence of (D5), (D6), (D7), or (D8), and the remainder of the base sequence Dr is identical or homologous to the complementary base sequence of the base sequence of (D5), (D6), (D7), or (D8). The range of "homology" is as described below. Particularly preferably, the base sequence Dr is identical to the complementary base sequence of the base sequence (D5), the base sequence (D6), the base sequence (D7) or the base sequence (D8).

[0137] The PHD3 reverse primer may contain a polynucleotide containing the base sequence Dr at the 3' end, and may further contain another base sequence at the 5' end side of the base sequence Dr. The number of bases of the polynucleotide contained in the PHD3 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.

[0138] A specific example of the base sequence of (D5) is the complementary base sequence of the base sequence of SEQ ID NO: 32. A specific example of the base sequence Dr is the base sequence of SEQ ID NO: 32.

[0139] A specific example of the base sequence of (D7) is the complementary base sequence of the base sequence of SEQ ID NO: 44. A specific example of the base sequence Dr is the base sequence of SEQ ID NO: 44.

[0140] An example of the primer pair is a primer pair for amplifying a nucleic acid containing the PDK1 gene, which includes a PDK1 forward primer having at its 3' end a base sequence Ef that is identical or homologous to any of the base sequences shown in (E1) to (E4) below, and a PDK1 reverse primer having at its 3' end a base sequence Er that is identical or homologous to the complementary base sequence of any of the base sequences shown in (E5) to (E8) below. (E1) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 445 to 528 of SEQ ID NO: 17 (E2) A base sequence of SEQ ID NO: 19 that corresponds to the base sequence of (E1) when the base sequence of SEQ ID NO: 19 is aligned with the base sequence of SEQ ID NO: 17. (E3) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 981 to 1060 of SEQ ID NO: 19 (E4) A base sequence of SEQ ID NO: 17 that corresponds to the base sequence of (E3) when the base sequence of SEQ ID NO: 17 is aligned with the base sequence of SEQ ID NO: 19. (E5) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 545 to 624 of SEQ ID NO: 17. (E6) A base sequence of SEQ ID NO: 19 that corresponds to the base sequence of (E5) when the base sequence of SEQ ID NO: 19 is aligned with the base sequence of SEQ ID NO: 17. (E7) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 1078 to 1157 of SEQ ID NO: 19 (E8) A base sequence of SEQ ID NO: 17, which corresponds to the base sequence of (E7) when the base sequence of SEQ ID NO: 17 is aligned with the base sequence of SEQ ID NO: 19.

[0141] In the above (E1), the "base sequence from positions 445 to 528" is preferably the "base sequence from positions 455 to 518", more preferably the "base sequence from positions 465 to 508", more preferably the "base sequence from positions 465 to 503", and more preferably the "base sequence from positions 470 to 498".

[0142] In the above (E3), the "base sequence from positions 981 to 1060" is preferably the "base sequence from positions 991 to 1050", more preferably the "base sequence from positions 1001 to 1040", more preferably the "base sequence from positions 1001 to 1035", and more preferably the "base sequence from positions 1006 to 1030".

[0143] In the above (E1) and (E3), "10 bases or more" is preferably "15 bases or more," more preferably "17 bases or more," and more preferably "19 bases or more." In the above (E1) and (E3), "consecutive" preferably means "consecutive from the 3' end."

[0144] The base sequence Ef may be identical or homologous to the base sequence of (E1), (E2), (E3) or (E4), but preferably, from the 3'-end, a continuous base sequence of 2 or more bases, more preferably 3 or more bases, more preferably 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 19 or more bases is identical to the base sequence of (E1), (E2), (E3) or (E4), and the remainder of the base sequence Ef is identical or homologous to the base sequence of (E1), (E2), (E3) or (E4). The range of "homology" is as described below. Particularly preferably, the base sequence Ef is identical to the base sequence of (E1), (E2), (E3) or (E4).

[0145] The PDK1 forward primer may contain a polynucleotide containing the nucleotide sequence Ef at the 3'-end, and may further contain another nucleotide sequence on the 5'-end side of the nucleotide sequence Ef. The number of nucleotides in the polynucleotide contained in the PDK1 forward primer is not particularly limited, but is preferably 50 nucleotides or less, more preferably 40 nucleotides or less, and more preferably 30 nucleotides or less.

[0146] A specific example of the base sequence of (E1) is the base sequence of SEQ ID NO: 33. A specific example of the base sequence Ef is the base sequence of SEQ ID NO: 33.

[0147] A specific example of the base sequence of (E3) is the base sequence of SEQ ID NO: 45. A specific example of the base sequence Ef is the base sequence of SEQ ID NO: 45.

[0148] In the above (E5), the "base sequence from positions 545 to 624" is preferably the "base sequence from positions 555 to 614", more preferably the "base sequence from positions 565 to 604", more preferably the "base sequence from positions 570 to 604", and more preferably the "base sequence from positions 575 to 599".

[0149] In the above (E7), the "base sequence from positions 1078 to 1157" is preferably the "base sequence from positions 1088 to 1147", more preferably the "base sequence from positions 1098 to 1137", more preferably the "base sequence from positions 1103 to 1137", and more preferably the "base sequence from positions 1108 to 1132".

[0150] In the above (E5) and (E7), "10 bases or more" is preferably "15 bases or more," more preferably "17 bases or more," and more preferably "19 bases or more." In the above (E5) and (E7), "consecutive" preferably means "consecutive from the 5' end."

[0151] The base sequence Er may be identical or homologous to the complementary base sequence of the base sequence of (E5), (E6), (E7), or (E8), but preferably a continuous base sequence of 2 or more bases, more preferably 3 or more bases, more preferably 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 19 or more bases from the 3' end of the base sequence Er is identical to the complementary base sequence of the base sequence of (E5), (E6), (E7), or (E8), and the remainder of the base sequence Er is identical or homologous to the complementary base sequence of the base sequence of (E5), (E6), (E7), or (E8). The range of "homology" is as described below. Particularly preferably, the base sequence Er is identical to the complementary base sequence of the base sequence (E5), the base sequence (E6), the base sequence (E7) or the base sequence (E8).

[0152] The PDK1 reverse primer may contain a polynucleotide containing the base sequence Er at the 3'-terminus, and may further contain another base sequence on the 5'-terminus side of the base sequence Er. The number of bases of the polynucleotide contained in the PDK1 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.

[0153] A specific example of the base sequence of (E5) is the complementary base sequence of the base sequence of SEQ ID NO: 34. A specific example of the base sequence Er is the base sequence of SEQ ID NO: 34.

[0154] A specific example of the base sequence of (E7) is the complementary base sequence of the base sequence of SEQ ID NO: 46. A specific example of the base sequence Er is the base sequence of SEQ ID NO: 46.

[0155] The kit may include a primer pair for amplifying a nucleic acid of an endogenous control gene as the primer pair. An example of such a primer pair is an 18S ribosomal RNA primer pair for amplifying a nucleic acid encoding 18S ribosomal RNA, the primer pair including an 18S ribosomal RNA forward primer having a nucleotide sequence Ff identical or homologous to any of the nucleotide sequences shown in (F1) to (F4) below at its 3' end, and an 18S ribosomal RNA reverse primer having a nucleotide sequence Fr identical or homologous to a complementary nucleotide sequence of any of the nucleotide sequences shown in (F5) to (F8) below at its 3' end. (F1) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 1216 to 1297 of SEQ ID NO: 21 (F2) A base sequence of SEQ ID NO: 22 that corresponds to the base sequence of (F1) when the base sequence of SEQ ID NO: 22 is aligned with the base sequence of SEQ ID NO: 21. (F3) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 1216 to 1297 of SEQ ID NO: 22 (F4) A base sequence of SEQ ID NO: 21 that corresponds to the base sequence of (F3) when the base sequence of SEQ ID NO: 21 is aligned with the base sequence of SEQ ID NO: 22. (F5) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 1317 to 1396 of SEQ ID NO: 21 (F6) A base sequence of SEQ ID NO: 22 that corresponds to the base sequence of (F5) when the base sequence of SEQ ID NO: 22 is aligned with the base sequence of SEQ ID NO: 21. (F7) A base sequence of 10 or more consecutive bases contained in the base sequence of positions 1317 to 1396 of SEQ ID NO: 21 (F8) A base sequence of SEQ ID NO: 21 that corresponds to the base sequence of (F7) when the base sequence of SEQ ID NO: 21 is aligned with the base sequence of SEQ ID NO: 22.

[0156] In (F1), the "base sequence from positions 1216 to 1297" is preferably the "base sequence from positions 1226 to 1287", more preferably the "base sequence from positions 1236 to 1277", more preferably the "base sequence from positions 1241 to 1272", and more preferably the "base sequence from positions 1241 to 1267".

[0157] In the above (F3), the "base sequence from positions 1216 to 1297" is preferably the "base sequence from positions 1226 to 1287", more preferably the "base sequence from positions 1236 to 1277", more preferably the "base sequence from positions 1236 to 1272", and more preferably the "base sequence from positions 1241 to 1267".

[0158] In the above (F1) and (F3), "10 bases or more" is preferably "15 bases or more," more preferably "17 bases or more," and more preferably "19 bases or more." In the above (F1) and (F3), "consecutive" preferably means "consecutive from the 3' end."

[0159] The base sequence Ff may be identical or homologous to the base sequence of (F1), (F2), (F3), or (F4), but preferably, from the 3' end, a continuous base sequence of 2 or more bases, more preferably 3 or more bases, more preferably 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 19 or more bases is identical to the base sequence of (F1), (F2), (F3), or (F4), and the remainder of the base sequence Ff is identical or homologous to the base sequence of (F1), (F2), (F3), or (F4). The range of "homology" is as described below. Particularly preferably, the base sequence Ff is identical to the base sequence of (F1), (F2), (F3), or (F4).

[0160] The 18S ribosomal RNA forward primer may contain a polynucleotide containing the base sequence Ff at the 3' end, and may further contain another base sequence on the 5' end side of the base sequence Ff. The number of bases of the polynucleotide contained in the 18S ribosomal RNA forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.

[0161] A specific example of the base sequence of (F1) is the base sequence of SEQ ID NO: 23. A specific example of the base sequence Ff is the base sequence of SEQ ID NO: 23.

[0162] A specific example of the base sequence of (F3) is the base sequence of SEQ ID NO: 35. A specific example of the base sequence Ff is the base sequence of SEQ ID NO: 35.

[0163] In the above (F5), the "base sequence from positions 1317 to 1396" is preferably the "base sequence from positions 1327 to 1386", more preferably the "base sequence from positions 1337 to 1376", more preferably the "base sequence from positions 1342 to 1376", and more preferably the "base sequence from positions 1347 to 1371".

[0164] In the above (F7), the "base sequence from positions 1317 to 1396" is preferably the "base sequence from positions 1327 to 1386", more preferably the "base sequence from positions 1337 to 1376", more preferably the "base sequence from positions 1342 to 1376", and more preferably the "base sequence from positions 1347 to 1371".

[0165] In the above (F5) and (F7), "10 bases or more" is preferably "15 bases or more," more preferably "17 bases or more," and more preferably "19 bases or more." In the above (F5) and (F7), "consecutive" preferably means "consecutive from the 5' end."

[0166] The base sequence Fr may be identical or homologous to the complementary base sequence of the base sequence of (F5), (F6), (F7), or (F8), but preferably 2 or more bases, more preferably 3 or more bases, more preferably 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, more preferably 19 or more bases from the 3' end of the base sequence Fr are identical to the complementary base sequence of the base sequence of (F5), (F6), (F7), or (F8), and the remainder of the base sequence Fr is identical or homologous to the complementary base sequence of the base sequence of (F5), (F6), (F7), or (F8). The range of "homology" is as described below. Particularly preferably, the base sequence Fr is identical to the complementary base sequence of the base sequence (F5), the base sequence (F6), the base sequence (F7) or the base sequence (F8).

[0167] The 18S ribosomal RNA reverse primer may contain a polynucleotide containing the base sequence Fr at the 3' end, and may further contain another base sequence on the 5' end side of the base sequence Fr. The number of bases of the polynucleotide contained in the 18S ribosomal RNA reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.

[0168] A specific example of the base sequence of (F5) is the complementary base sequence of the base sequence of SEQ ID NO: 24. A specific example of the base sequence Fr is the base sequence of SEQ ID NO: 24.

[0169] A specific example of the base sequence of (F7) is the complementary base sequence of the base sequence of SEQ ID NO: 36. A specific example of the base sequence Fr is the base sequence of SEQ ID NO: 36.

[0170] In the present specification, when "base sequence Y is homologous to base sequence X" or "base sequence X and base sequence Y are homologous", the base sequences X and Y may be partially different as long as the polynucleotide consisting of the complementary sequence of base sequence X and the polynucleotide consisting of base sequence Y can form sufficient hydrogen bonds to hybridize and form a stable double strand under the annealing conditions of the nucleic acid amplification reaction. For example, the polynucleotide consisting of the complementary sequence of base sequence X and the polynucleotide consisting of base sequence Y may have several mismatches, such as one mismatch in 10 nucleotides, one mismatch in 20 nucleotides, or one mismatch in 30 nucleotides. Typically, when the base sequence Y is "homologous" to the base sequence X, it means that the base sequences X and Y satisfy any of the following relationships.

[0171] (A) The base sequence Y is a base sequence in which one or several bases have been deleted, substituted, added and / or inserted in the base sequence X. (B) The base sequence Y has an identity of 70% or more with the base sequence X. (C) A polynucleotide consisting of base sequence Y can hybridize under stringent conditions to a polynucleotide consisting of a base sequence complementary to SEQ ID NO:X. (D) A thymine (T) at any position in one of base sequences X and Y is replaced with a uracil (U) in the other.

[0172] In the above (A), "one or several" preferably refers to 1 to 5, more preferably 1 to 4, more preferably 1 to 3, particularly preferably 1 or 2, and most preferably 1. In the above (A), "one or several" refers to the total number of deleted, substituted, added, and / or inserted bases.

[0173] In (B), the identity value indicates a value calculated by using software (e.g., FASTA, DNASIS, and BLAST) that calculates the identity between multiple base sequences with default settings. The identity value of base sequences is calculated by calculating the number of matching bases when a pair of base sequences are aligned to maximize the degree of identity, and is calculated as a ratio of the number of matching bases to the total number of bases of the compared base sequences. Here, when there is a gap, the total number of bases is the number of bases counted with one gap as one base. For details of the method of determining identity, see, for example, Altschul et al., Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215, 403-410, 1990.

[0174] In (B) above, the identity is more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more.

[0175] In the above (C), the term "stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed, and can be appropriately determined by referring to, for example, Green and Sambrook, Molecular Cloning, 4th Ed (2012), Cold Spring Harbor Laboratory Press. Specifically, stringent conditions can be set by the temperature during Southern hybridization, the salt concentration contained in the solution, and the temperature during the washing step of Southern hybridization, and the salt concentration contained in the solution. More specifically, stringent conditions include, for example, a sodium concentration of 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 40 to 68°C, preferably 40 to 65°C, in the hybridization step. More specifically, hybridization can be performed in 1 to 7×SSC, 0.02 to 3% SDS, and at a temperature of 40°C to 60°C. In addition, a washing step may be carried out after hybridization. The washing step can be carried out, for example, in 0.1 to 2×SSC, 0.1 to 0.3% SDS, and at a temperature of 50 to 65° C. One or both of the primers in each pair may be modified with other substances such as a labeling substance.

[0176] The probe is a nucleic acid containing a gene for one or more proteins, for example, the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3 of the Glut1 gene (particularly, the base sequence of positions 131 to 262 of the base sequence of SEQ ID NO: 1; the base sequence of SEQ ID NO: 3 corresponding to the base sequence of positions 131 to 262 of the base sequence of SEQ ID NO: 1 when the base sequence of SEQ ID NO: 3 is aligned with the base sequence of SEQ ID NO: 1; the base sequence of positions 286 to 388 of the base sequence of SEQ ID NO: 3; or the base sequence of SEQ ID NO: 1). a base sequence corresponding to the 286th to 388th bases in the base sequence of SEQ ID NO: 3 when the base sequence of SEQ ID NO: 1 is aligned with the base sequence of SEQ ID NO: 3; a base sequence of SEQ ID NO: 5 or SEQ ID NO: 7 of the Glut3 gene (particularly, a base sequence of the 280th to 424th bases in the base sequence of SEQ ID NO: 5 when the base sequence of SEQ ID NO: 7 is aligned with the base sequence of SEQ ID NO: 5; a base sequence of SEQ ID NO: 7 of the Glut3 gene (particularly, a base sequence of the 280th to 424th bases in the base sequence of SEQ ID NO: 5 when the base sequence of SEQ ID NO: 7 is aligned with the base sequence of SEQ ID NO: 5); the nucleotide sequence of positions 254 to 337 of the nucleotide sequence of SEQ ID NO: 7; or, among the nucleotide sequences of SEQ ID NO: 5, a nucleotide sequence corresponding to the nucleotide sequence of positions 254 to 337 of the nucleotide sequence of SEQ ID NO: 7 when the nucleotide sequence of SEQ ID NO: 5 is aligned with the nucleotide sequence of SEQ ID NO: 7), the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 11 of the MCT4 gene (particularly, the nucleotide sequence of positions 70 to 156 of the nucleotide sequence of SEQ ID NO: 9; among the nucleotide sequences of SEQ ID NO: 11, a nucleotide sequence corresponding to the nucleotide sequence of positions 254 to 337 of the nucleotide sequence of SEQ ID NO: 7 when the nucleotide sequence of SEQ ID NO: 11 is aligned with the nucleotide sequence of SEQ ID NO: 9) a base sequence corresponding to the base sequence of positions 70 to 156 in the base sequence of SEQ ID NO: 9; a base sequence of positions 176 to 274 in the base sequence of SEQ ID NO: 11; or, when the base sequence of SEQ ID NO: 9 is aligned with the base sequence of SEQ ID NO: 11, a base sequence corresponding to the base sequence of positions 176 to 274 in the base sequence of SEQ ID NO: 11), the base sequence of SEQ ID NO: 13 or SEQ ID NO: 15 of the PHD3 gene (particularly, the base sequence of positions 894 to 1042 in the base sequence of SEQ ID NO: 13;Among the base sequences of SEQ ID NO:15, a base sequence corresponding to the base sequence of positions 894 to 1042 in the base sequence of SEQ ID NO:3 when the base sequence of SEQ ID NO:15 is aligned with the base sequence of SEQ ID NO:13; a base sequence of positions 893 to 1053 in the base sequence of SEQ ID NO:15; or among the base sequences of SEQ ID NO:13, a base sequence corresponding to the base sequence of positions 893 to 1053 in the base sequence of SEQ ID NO:15 when the base sequence of SEQ ID NO:13 is aligned with the base sequence of SEQ ID NO:15), or the base sequence of SEQ ID NO:17 or SEQ ID NO:19 of the PDK1 gene (particularly, the base sequence of positions 475 to 594 in the base sequence of SEQ ID NO:17; The nucleic acid fragment may include a nucleic acid fragment containing a continuous partial base sequence of 10 or more bases, preferably 15 or more bases, more preferably 17 or more bases, and more preferably 20 or more bases contained in a base sequence corresponding to the base sequence of positions 475 to 594 of the base sequence of SEQ ID NO: 17 when the base sequence of SEQ ID NO: 19 is aligned with the base sequence of SEQ ID NO: 17; a base sequence corresponding to the base sequence of positions 1011 to 1127 of the base sequence of SEQ ID NO: 19 when the base sequence of SEQ ID NO: 17 is aligned with the base sequence of SEQ ID NO: 19, or a nucleic acid fragment containing a continuous partial base sequence of 10 or more bases, preferably 15 or more bases, more preferably 17 or more bases, and more preferably 20 or more bases, or a base sequence homologous to the complementary base sequence thereof. The upper limit of the length of the partial base sequence is not particularly limited, but may be, for example, 50 or less bases, 40 or less bases, or 30 or less bases. The probe may be a nucleic acid fragment modified with another substance such as a labeling substance.

[0177] The antibody that specifically binds to one or more proteins may be a polyclonal antibody or a monoclonal antibody. Furthermore, the antibody may be used as a fragment as long as it can specifically bind to the protein to be measured. Examples of antibody fragments include Fab fragments, F(ab')2 fragments, and single-chain antibodies (scFv). The antibody may be immobilized on a solid phase carrier such as a microtiter plate or particles.

[0178] The reaction substrate for measuring the enzymatic activity of the one or more proteins can be appropriately selected depending on the activity of the protein to be measured.

[0179] The kit according to the embodiment may further include a dilution or reaction buffer solution containing components necessary for the measurement, a washing solution, a color-developing reagent, a reaction vessel, and the like.

[0180] The kit according to the embodiment preferably further includes a reagent and / or a column for separating mononuclear cells from the sample. Examples of the reagent and / or column include Ficoll-Paque PLUS (GE Healthcare), Lymphoprep (Abbott Diagnostics Technologies), Human Peripheral Blood Mononuclear Cell Isolation and Viability Kit (BioVision), BD Vacutainer CPT blood collection tube for mononuclear cell separation (Becton Dickinson), pluriMate (pluriSelect), and SepMate (STEMCELL Technologies). The kit can separate mononuclear cells and granulocytes in the sample, and efficiently obtain a mononuclear cell-containing sample for measurement.

[0181] <5. Kit for determining the degree of aging phenomenon in a subject> One or more further embodiments of the present invention include A kit for determining the degree of aging in a subject, comprising: The present invention relates to a kit comprising a reagent for measuring gene expression or enzyme activity of one or more proteins involved in energy metabolic reactions in a sample derived from a subject.

[0182] The assessment of the degree of aging is preferably assessment of age, assessment of the degree of aging in brain tissue, assessment of the degree of aging in peripheral blood, or assessment of the degree of aging in bone marrow cells.

[0183] The bone marrow cells are preferably hematopoietic stem cells, mesenchymal stem cells, or immature cells.

[0184] The reagent is preferably a reagent that can be used in the above-mentioned method for measuring gene expression or enzyme activity of one or more proteins.

[0185] Specifically, examples of the probe include a primer pair for amplifying a nucleic acid (genomic DNA, mRNA, or cDNA prepared based on mRNA) containing the gene of the one or more proteins to be measured, a probe that hybridizes with a nucleic acid (mRNA or cDNA prepared based on mRNA) containing the gene of the one or more proteins to be measured, an antibody that specifically binds to the one or more proteins, and a reaction substrate for measuring the enzyme activity of the one or more proteins. The genomic DNA may also include a non-translated region, an intron region, a signal sequence region, and the like. Preferably, the nucleic acid is mRNA or cDNA. The mRNA or cDNA may include at least a part of the base sequence (CDS) encoding the amino acid sequence of the one or more proteins and the base sequence of the untranslated region (UTR) located upstream and downstream thereof. An example of the primer pair is a primer pair that can amplify at least a part of the base sequence of the CDS and UTR. An example of the probe is a probe that can hybridize to at least a part of the base sequence of the CDS and UTR.

[0186] The primer pair can be selected from the same range as the suitable examples of the primer pair described above in relation to "4. Kit for assessing dementia or brain function."

[0187] The probe can be selected from the same range as the suitable examples of the probe described above in relation to "4. Kit for assessing dementia or brain function."

[0188] The antibody that specifically binds to one or more proteins may be a polyclonal antibody or a monoclonal antibody. Furthermore, the antibody may be used as a fragment as long as it can specifically bind to the protein to be measured. Examples of antibody fragments include Fab fragments, F(ab')2 fragments, and single-chain antibodies (scFv). The antibody may be immobilized on a solid phase carrier such as a microtiter plate or particles.

[0189] The reaction substrate for measuring the enzymatic activity of the one or more proteins can be appropriately selected depending on the activity of the protein to be measured.

[0190] The kit according to the embodiment may further include a dilution or reaction buffer solution containing components necessary for the measurement, a washing solution, a color-developing reagent, a reaction vessel, and the like. EXAMPLES

[0191] The present invention will be specifically described in the following examples, but the present invention is not limited to these examples.

[0192] All animal experiments described below were approved by the Animal Experiment Review Committee of the Kobe Biomedical Research Institute, a public interest incorporated foundation, and were conducted in accordance with the basic guidelines for the conduct of animal experiments in research institutions, etc., established by the Ministry of Education, Culture, Sports, Science and Technology. Experiments and results will be reported in accordance with the ARRIVE guidelines prepared by the National Centre for 3Rs (NC3Rs).

[0193] 1. Comparative Example 1 <Method> In Comparative Example 1, indicators for assessing dementia and brain function in subjects were examined using body fluid samples derived from healthy mice.

[0194] 1-1. Preparation of experimental animals Male CB-17 mice (Oriental Yeast Co., Ltd.) aged 5 weeks or less were used in the following experiment.

[0195] 1-2.Behavioral tests Five behavioral tests (open field test, wire hang test, passive avoidance test, open space swimming test and rotarod test) were performed to evaluate dementia and / or brain function in mice.

[0196] 1-2-1.Open field test The open field test is a behavioral test to evaluate the responsiveness of mice to changes in light and darkness using the average number of movements (average of the total number of horizontal and vertical movements) of the mice under light and dark conditions as an index. The open field test was performed as follows.

[0197] A mouse was placed in a transparent acrylic box (bottom: 30 cm long x 30 cm wide). With the white light on (bright state), the mouse was allowed to freely explore the acrylic box for 30 minutes. The light was then turned off, and the box was kept dark for 30 minutes. The number of horizontal movements of the mouse was evaluated by counting the number of times the mouse interrupted infrared beams set at 10 cm intervals along the vertical and horizontal axes at a height of 2 cm from the floor. The number of vertical movements (number of times the mouse stood up) of the mouse was evaluated by counting the number of times the mouse interrupted infrared beams set at 3 cm intervals along the vertical axis at a height of 5 cm from the floor. The test was performed 9 times or more, and the average number of movements was calculated.

[0198] 1-2-2. Wire hang test The wire hang test is a behavioral test to evaluate the muscle strength of mice by using the average time it takes for a mouse suspended from a wire mesh to fall (average fall time) as an index. The wire hang test was performed as follows.

[0199] A mouse was placed on a wire mesh with a gap of 12 mm. Next, the mouse was turned upside down with the wire mesh and hung at a height of 20 cm. The time it took for the mouse to fall from the wire mesh (fall time) was recorded. The test was performed eight or more times, and the average fall time (average fall time) was calculated.

[0200] 1-2-3. Passive avoidance test The passive avoidance test is a behavioral test to evaluate the learning and memory functions of mice by using the average reaction latency (mean reaction latency) of mice that have been previously exposed to an aversive stimulus as an index. The passive avoidance test was performed as follows.

[0201] The passive avoidance test was conducted using a box consisting of a light room and a dark room separated by a board with a hole through which the mouse could pass, and an electric stimulator was installed on the floor of the dark room. In the preliminary stage, the mouse was placed in the light room with white light on and allowed to freely explore the box. Since the mouse prefers dark places, it gradually enters the dark room, but when it enters the dark room, it is given a light electric stimulus. The time that the mouse stayed in the light room (i.e., the time until it entered the dark room) was measured and recorded. 24 hours later, for the main test, the mouse was placed in the light room again and the time that the mouse stayed in the light room (i.e., the time until it entered the dark room) was measured again. The time that the mouse stayed in the light room (the time until it entered the dark room) was measured up to 180 seconds. The reaction latency was calculated as the absolute value of the difference between the time that the mouse stayed in the light room in the main test and the time that the mouse stayed in the light room in the preliminary stage. The test was conducted five or more times, and the average reaction latency was calculated.

[0202] 1-2-4.Open space swimming test The open space swimming test is a behavioral test to evaluate coping behavior and depressive tendency of mice by using the average swimming distance (average swimming distance) as an index when mice are placed in a large pool filled with water. The open space swimming test was performed as follows.

[0203] Mice were placed in a 120 cm diameter pool and allowed to swim freely. Their swimming behavior was recorded by a video camera, and the swimming distance for 60 seconds was analyzed continuously for 10 minutes using image analysis software.

[0204] 1-2-5.Rotarod test The Rotarod test is a behavioral test to evaluate the motor function and sense of balance of mice by using the average time it takes for a mouse to fall off a rotating rotarod (average fall time) as an index. The rotarod test was performed as follows.

[0205] A mouse was placed on the lane of a rotarod that rotated 20 times per minute, and the time it took for the mouse to fall off the rotating rotarod (fall time) was measured. The test was performed three times, and the average fall time (average fall time) was calculated.

[0206] 1-3.Quantitative RT-PCR Approximately 1 mL of peripheral blood was collected from the mice subjected to the behavioral test, and 0.4 mL of the blood was used as a body fluid sample to be analyzed in the following experiment. Total RNA was extracted from the body fluid sample using an RNA extraction kit (Mouse RiboPure Blood RNA Isolation Kit (Thermo Fisher Scientific / Model: AM1951)) according to the manufacturer's protocol. Using the obtained total RNA as a template, cDNA was synthesized (reverse transcribed) from 1 μg of total RNA using a cDNA synthesis kit (PrimeScript (registered trademark) II 1st strand cDNA Synthesis Kit (Takara Bio Inc. / Model: 6210B (A × 4))) according to the manufacturer's protocol. Using the obtained cDNA as a template, a real-time PCR reaction was performed using a nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / Model: A25777)). The real-time PCR system used was the Agilent AriaMx Real-Time PCR System (Agilent). The real-time PCR reaction conditions were 1 cycle of 50°C for 3 minutes, 1 cycle of 95°C for 3 minutes, 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, 1 cycle of 95°C for 30 seconds, 1 cycle of 65°C for 30 seconds, and 1 cycle of 95°C for 30 seconds. The 18S ribosomal RNA gene, which is a housekeeping gene, was used as an endogenous control, and the relative expression level of each target gene relative to the endogenous control was analyzed by the Pfaffl method. The Pfaffl method is a type of relative quantification method of the comparative Ct method that takes into account the PCR amplification efficiency of the housekeeping gene and the target gene, and the method described in Pfaffl MW. Nucleic Acids Res. 2001; 29(9): e45. can be used. The test was performed five or more times, and the average value of the relative expression levels (average relative expression level) was calculated. Table 1 shows the base sequences of the primers for each gene.

[0207] [Table 1]

[0208] In Table 1, "Fw" indicates a forward primer, and "Rv" indicates a reverse primer. In addition, in each base sequence in Table 1, the left end indicates the 5' end, and the right end indicates the 3' end.

[0209] 2. Example 1 <Method> In Example 1, an index for assessing dementia and brain function in a subject was investigated using a body fluid sample derived from a mouse that had developed dementia.

[0210] 2-1. Preparation of experimental animals Male CB-17 mice (Oriental Yeast Co., Ltd.) aged 80 weeks or older were used in the following experiment.

[0211] 2-2.Behavioral tests In the same manner as in "1-2. Behavioral Tests" of Comparative Example 1, five behavioral tests (open field test, wire hang test, passive avoidance test, open space swimming test, and rotarod test) were carried out.

[0212] 2-3.Quantitative RT-PCR In the same manner as in "1-3. Quantitative RT-PCR" of Comparative Example 1, the relative expression level of each target gene in the mouse body fluid sample was analyzed.

[0213] <Result> The results of the behavioral tests in Example 1 and Comparative Example 1 are shown below. In the open field test, the average number of movements in the light state of the mouse of Comparative Example 1 was 537 ± 106 times, and the average number of movements in the dark state was 747 ± 160 times. On the other hand, the average number of movements in the light state of the mouse of Example 1 was 358 ± 180 times, and the average number of movements in the dark state was 217 ± 188 times. Therefore, when the state was changed from the light state to the dark state, the average number of movements of the mouse of Comparative Example 1 showed an increasing tendency, whereas the average number of movements of the mouse of Example 1 showed a decreasing tendency. From the above, it was suggested that the reactivity of the mouse of Example 1 to the change in light and dark was reduced compared to the mouse of Comparative Example 1.

[0214] In the wire hanging test, the average fall time of the mice in Comparative Example 1 was 84.9±41.1 seconds, whereas the average fall time of the mice in Example 1 was 25.6±22.9 seconds. This suggests that the muscle strength of the mice in Example 1 was reduced compared to that of the mice in Comparative Example 1.

[0215] In the passive avoidance test, the average time that the mice of Comparative Example 1 stayed in the light room in the preliminary stage was 31.5 ± 22.9 seconds, whereas the average time that the mice of Example 1 stayed in the light room in the preliminary stage was 20.0 ± 14.1 seconds. In addition, the average time that the mice of Comparative Example 1 stayed in the light room in this test was 180.0 ± 0.0 seconds, whereas the average time that the mice of Example 1 stayed in the light room in the preliminary stage was 47.8 ± 67.4 seconds. Thus, the average reaction latency of the mice of Comparative Example 1 was calculated to be 148.5 ± 22.9 seconds, and the average reaction latency of the mice of Example 1 was calculated to be 32.8 ± 63.5 seconds. From the above, it was suggested that the mice of Comparative Example 1 were able to learn and remember by associating entry into the dark room with the fear of the aversive stimulus, whereas the mice of Example 1 had impaired learning and memory functions.

[0216] In the open space swimming test, the average swimming distance of the mice of Comparative Example 1 was 794±192cm, whereas the average swimming distance of the mice of Example 1 was 1193±154cm. Compared with the mice of Comparative Example 1, the mice of Example 1 tended to show a shorter time of immobility (i.e., floating in the water without struggling, making only the minimum movement necessary to get their heads out of the water surface) and tended to swim around more than necessary. From the above, it was suggested that the coping behavior of the mice of Example 1 was weakened compared to the mice of Comparative Example 1, and that they showed a tendency to be depressed.

[0217] In the rotarod test, the average fall time of the mice in Comparative Example 1 was 41.6±25.2 seconds, whereas the average fall time of the mice in Example 1 was 6.8±4.5 seconds. This suggests that the motor function and sense of balance of the mice in Example 1 are reduced compared to those in Comparative Example 1.

[0218] From the results of the above behavioral tests, it was determined that the mice of Comparative Example 1 did not develop dementia and did not have impaired brain function, whereas the mice of Example 1 were determined to have dementia and / or impaired brain function. The results of quantitative RT-PCR in Comparative Example 1 and Example 1 are shown below.

[0219] Figure 1 and Table 2 show the average relative expression levels (average relative expression levels) of each target gene to the expression level of the 18S ribosomal RNA gene in the body fluid samples of each mouse in Comparative Example 1 and Example 1, calculated by the Pfaffl method.

[0220] [Table 2]

[0221] As shown in FIG. 1 and Table 2, in the body fluid samples of the mice of Comparative Example 1, the average relative expression level of the Glut1 gene was 0.56, the average relative expression level of the Glut3 gene was 0.67, the average relative expression level of the MCT4 gene was 0.45, the average relative expression level of the PHD3 gene was 0.58, and the average relative expression level of the PDK1 gene was 0.43. On the other hand, in the body fluid samples of the mice of Example 1, the average relative expression level of the Glut1 gene was 3.39, the average relative expression level of the Glut3 gene was 1.70, the average relative expression level of the MCT4 gene was 3.17, the average relative expression level of the PHD3 gene was 2.33, and the average relative expression level of the PDK1 gene was 3.03. Compared with the mice of Comparative Example 1, the gene expressions of the Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene, and PDK1 gene in the body fluid samples of the mice of Example 1 were significantly increased. Therefore, the following genes were identified as ones whose expression levels change significantly with the onset of dementia and / or decline in brain function: genes for proteins involved in glucose transport (Glut1 gene and Glut3 gene), genes for proteins involved in lactate transport (MCT4 gene), genes for proteins involved in glycolysis (PHD3 gene), and genes for proteins involved in the TCA cycle (PDK1 gene).

[0222] Therefore, the possibility of using the above genes and / or proteins encoded by the above genes as indicators for determining the onset of dementia and / or decline in brain function in a subject has been demonstrated. In addition, the possibility of using genes of proteins involved in energy metabolism and / or proteins involved in energy metabolism as indicators for determining the onset of dementia and / or decline in brain function in a subject has been demonstrated.

[0223] Furthermore, it was found that the average relative expression levels of each target gene in the body fluid samples of the mice in Example 1 were higher than the respective standard values ​​shown in (a) to (e) below, and the average relative expression levels of each target gene in the mice in Comparative Example 1 were lower than the respective standard values ​​shown in (a) to (e) below. (a) Baseline gene expression of Glut1 gene: 1.30 (b) Baseline gene expression of Glut3 gene: 1.30 (c) Reference value of gene expression of MCT4 gene: 1.40 (d) Baseline gene expression of PHD3 gene: 1.20 (e) Baseline gene expression of the PDK1 gene: 1.30

[0224] In the body fluid samples of the mice of Example 1, the ratio of the average relative expression level of the Glut1 gene to the above-mentioned standard value was 2.6. In the body fluid samples of the mice of Example 1, the ratio of the average relative expression level of the Glut3 gene to the above-mentioned standard value was 1.3. In the body fluid samples of the mice of Example 1, the ratio of the average relative expression level of the MCT4 gene to the above-mentioned standard value was 2.3. In the body fluid samples of the mice of Example 1, the ratio of the average relative expression level of the PHD3 gene to the above-mentioned standard value was 1.9. In the body fluid samples of the mice of Example 1, the ratio of the average relative expression level of the PDK1 gene to the above-mentioned standard value was 2.3.

[0225] Therefore, by using the relative expression level of one or more of the above genes being higher than the above reference value as an index, an index for determining dementia or brain function in a subject can be obtained. That is, by measuring the relative expression level of one or more of the above genes in a body fluid sample derived from a subject and comparing the relative expression level with the above reference value, an index for determining dementia or brain function in a subject can be obtained.

[0226] In addition, by measuring the gene expression or enzyme activity of one or more proteins involved in energy metabolic reactions in a sample derived from a subject, and comparing the measured values ​​of the gene expression or enzyme activity of the one or more proteins with a reference value, an index for assessing dementia or brain function in a subject can be obtained.

[0227] 3. Example 2 <Method> In Example 2, an index for determining the degree of aging in a subject was investigated using body fluid samples derived from multiple mice with known degrees of aging.

[0228] 3-1. Preparation of experimental animals Male CB-17 strain mice (Oriental Yeast Co., Ltd.) aged 1, 5, 10, 52, and 104 weeks were used in the following experiment.

[0229] 3-2.Quantitative RT-PCR Approximately 1 mL of peripheral blood was collected from the mouse, and 0.4 mL of the blood was used as a body fluid sample to be analyzed in the following experiment. Total RNA was extracted from the body fluid sample using an RNA extraction kit (Mouse RiboPure Blood RNA Isolation Kit (Thermo Fisher Scientific / Model: AM1951)) according to the manufacturer's protocol. Using the obtained total RNA as a template, cDNA was synthesized (reverse transcribed) from 1 μg of total RNA using a cDNA synthesis kit (PrimeScript (registered trademark) II 1st strand cDNA Synthesis Kit (Takara Bio Inc. / Model: 6210B (A × 4))) according to the manufacturer's protocol. Using the obtained cDNA as a template, a real-time PCR reaction was performed using a nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / Model: A25777)). The real-time PCR system used was the Agilent AriaMx Real-Time PCR System (Agilent). The real-time PCR reaction conditions were 1 cycle of 50°C for 3 minutes, 1 cycle of 95°C for 3 minutes, 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, 1 cycle of 95°C for 30 seconds, 1 cycle of 65°C for 30 seconds, and 1 cycle of 95°C for 30 seconds. The 18S ribosomal RNA gene, which is a housekeeping gene, was used as an endogenous control, and the relative expression level of each target gene relative to the endogenous control was analyzed by the Pfaffl method. The Pfaffl method is a type of relative quantification method of the comparative Ct method that takes into account the PCR amplification efficiency of the housekeeping gene and the target gene, and the method described in Pfaffl MW. Nucleic Acids Res. 2001; 29(9): e45. can be used. The test was performed five or more times, and the average value of the relative expression levels (average relative expression level) was calculated. Table 3 shows the base sequences of the primers for each gene.

[0230] [Table 3]

[0231] In Table 3, "Fw" indicates a forward primer, and "Rv" indicates a reverse primer. In addition, in each base sequence in Table 3, the left end indicates the 5' end, and the right end indicates the 3' end.

[0232] <Result> The results of the quantitative RT-PCR in Example 2 are shown below.

[0233] FIG. 2 and Table 4 show the average relative expression level (average relative expression level) of each target gene to the expression level of the 18S ribosomal RNA gene in the body fluid samples of each mouse in Example 2, calculated by the Pfaffl method.

[0234] [Table 4]

[0235] As shown in FIG. 2 and Table 4, in the body fluid samples from 1-week-old mice, the average relative expression levels of the Glut1 gene were 0.42, the average relative expression levels of the Glut3 gene were 0.27, the average relative expression levels of the MCT4 gene were 0.20, the average relative expression levels of the PHD3 gene were 0.35, and the average relative expression levels of the PDK1 gene were 0.28. In the body fluid samples from 5-week-old mice, the average relative expression levels of the Glut1 gene were 0.68, the average relative expression levels of the Glut3 gene were 0.81, the average relative expression levels of the MCT4 gene were 0.54, the average relative expression levels of the PHD3 gene were 0.62, and the average relative expression levels of the PDK1 gene were 0.83. In the body fluid samples from 10-week-old mice, the average relative expression levels of the Glut1 gene were 1.16, the average relative expression levels of the Glut3 gene were 1.18, the average relative expression levels of the MCT4 gene were 1.22, the average relative expression levels of the PHD3 gene were 1.12, and the average relative expression levels of the PDK1 gene were 1.17. In the body fluid samples from 52-week-old mice, the mean relative expression levels of Glut1, Glut3, MCT4, PHD3, and PDK1 were 1.47, 1.83, 2.47, 1.74, and 1.71, respectively. In the body fluid samples from 104-week-old mice, the mean relative expression levels of Glut1, Glut3, MCT4, PHD3, and PDK1 were 1.99, 1.96, 2.82, 2.35, and 1.96, respectively. With increasing age, the gene expressions of Glut1, Glut3, MCT4, PHD3, and PDK1 in the body fluid samples from mice were significantly increased. Therefore, genes whose expression levels change significantly with increasing severity of aging were identified as follows: genes for proteins involved in glucose transport (Glut1 gene and Glut3 gene), gene for proteins involved in lactate transport (MCT4 gene), gene for proteins involved in glycolysis (PHD3 gene), and gene for proteins involved in the TCA cycle (PDK1 gene).

[0236] Therefore, the possibility of using the above genes and / or proteins encoded by the above genes as indicators for determining the degree of aging in a subject has been demonstrated. Also, the possibility of using genes of proteins involved in energy metabolism and / or proteins involved in energy metabolism as indicators for determining the degree of aging in a subject has been demonstrated.

[0237] Fig. 3 shows the relationship between the average relative expression level of each target gene in the body fluid samples of the mice in Example 2 and the age of the mice in weeks. As shown in Fig. 3, the relationship between the average relative expression level of each target gene in the body fluid samples of the mice in Example 2 and the age of the mice in weeks is represented by the following formulas (A) to (E). (A) Relationship between the average relative expression level of Glut1 gene and age in weeks: y = 0.3257 × ln(x) + 0.3316 (B) Relationship between the average relative expression level of Glut3 gene and age in weeks: y = 0.3788 × ln(x) + 0.2658 (C) Relationship between the average relative expression level of the MCT4 gene and age in weeks: y = 0.6078 × ln(x)-0.0663 (D) Relationship between the average relative expression level of the PHD3 gene and age in weeks: y = 0.4283 × ln(x) + 0.1677 (E) Relationship between the average relative expression level of the PDK1 gene and age in weeks: y = 0.3626 × ln(x) + 0.2848 Here, ln represents the natural logarithm, x represents the age of the mouse in weeks, and y represents the average relative expression level of each target gene in the mouse body fluid sample.

[0238] Therefore, the age of a subject can be determined based on the above relational expression formula using the relative expression levels of one or more of the above genes as an index. That is, the age of a subject can be determined by measuring the relative expression levels of one or more of the above genes in a body fluid sample derived from the subject and using the relative expression levels and the above relational expression formula.

[0239] Furthermore, by measuring the gene expression or enzyme activity of one or more proteins involved in energy metabolic reactions in a sample derived from a subject, an index for determining the degree of aging in the subject can be obtained.

[0240] 4. Example 3 <Manufacturing dementia / brain function assessment kits> A kit for assessing dementia or brain function (dementia / brain function assessment kit) is manufactured by the following procedure.

[0241] The primer pair for amplifying the Glut1 gene shown in Table 1 is dissolved in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) to prepare a primer pair solution for amplifying the Glut1 gene at a final concentration of 10 μM. The primer pair for amplifying the 18S ribosomal RNA gene shown in Table 1 is dissolved in TE buffer to prepare a primer pair solution for amplifying the 18S ribosomal RNA gene at a final concentration of 10 μM. The primer pair solution for amplifying the Glut1 gene is used as a reagent for measuring gene expression of Glut1, a protein involved in glucose transport. Furthermore, the primer pair solution for amplifying the Glut1 gene, the primer pair solution for amplifying the 18S ribosomal RNA gene, an RNA extraction reagent (Mouse RiboPure Blood RNA Isolation Kit (Thermo Fisher Scientific / model number: AM1951)), a cDNA synthesis reagent (PrimeScript (registered trademark) II 1st strand cDNA Synthesis Kit (Takara Bio Inc. / model number: 6210A), and a nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / model number: A25777)) are combined to form a dementia / brain function assessment kit.

[0242] <Use of Dementia / Brain Function Assessment Kit> The dementia / brain function assessment kit can be used in the following manner.

[0243] Peripheral blood is collected from a subject mouse, and a part of the blood is used as a body fluid sample to be analyzed. Total RNA is extracted from the body fluid sample using an RNA extraction reagent constituting the dementia / brain function assessment kit. Using the obtained total RNA as a template, cDNA is synthesized using a cDNA synthesis reagent constituting the dementia / brain function assessment kit. Using the obtained cDNA as a template, a real-time PCR reaction is performed using a primer pair solution for amplifying the Glut1 gene, a primer pair solution for amplifying the 18S ribosomal RNA gene, and a nucleic acid amplification reagent constituting the dementia / brain function assessment kit. The real-time PCR system used is an Agilent AriaMx Real-Time PCR System (Agilent). The real-time PCR reaction conditions are 1 cycle of 50°C for 3 minutes, 1 cycle of 95°C for 3 minutes, 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, 1 cycle of 95°C for 30 seconds, 1 cycle of 65°C for 30 seconds, and 1 cycle of 95°C for 30 seconds. The relative expression level of the Glut1 gene to the 18S ribosomal RNA gene is analyzed by the Pfaffl method. By comparing the relative expression level of the Glut1 gene to the 18S ribosomal RNA gene with the standard value of 1.30 for gene expression of the Glut1 gene, the presence or absence of dementia in mice can be determined.

[0244] 5. Example 4 <Manufacturing of aging assessment kits> A kit for determining the degree of aging phenomenon (aging degree determination kit) is manufactured according to the following procedure.

[0245] The primer pair for amplifying the Glut3 gene shown in Table 3 is dissolved in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) to prepare a primer pair solution for amplifying the Glut3 gene at a final concentration of 10 μM. The primer pair for amplifying the 18S ribosomal RNA gene shown in Table 3 is dissolved in TE buffer to prepare a primer pair solution for amplifying the 18S ribosomal RNA gene at a final concentration of 10 μM. The primer pair solution for amplifying the Glut3 gene is used as a reagent for measuring gene expression of Glut3, a protein involved in glucose transport. Furthermore, the primer pair solution for amplifying the Glut3 gene, the primer pair solution for amplifying the 18S ribosomal RNA gene, an RNA extraction reagent (Mouse RiboPure Blood RNA Isolation Kit (Thermo Fisher Scientific / model number: AM1951)), a cDNA synthesis reagent (PrimeScript (registered trademark) II 1st strand cDNA Synthesis Kit (Takara Bio Inc. / model number: 6210A), and a nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / model number: A25777)) are combined to form an aging degree determination kit.

[0246] <Using the Aging Assessment Kit> The aging degree determination kit can be used in the following manner. Peripheral blood is obtained from a number of mice whose ages are known in weeks, and a part of the blood is used as a body fluid sample for preparing a standard. Total RNA is extracted from the analysis sample using an RNA extraction reagent constituting the aging degree determination kit. Using the obtained total RNA as a template, cDNA is synthesized using a cDNA synthesis reagent constituting the aging degree determination kit. Using the obtained cDNA as a template, a real-time PCR reaction is performed using a primer pair solution for amplifying the Glut3 gene, a primer pair solution for amplifying the 18S ribosomal RNA gene, and a nucleic acid amplification reagent constituting the aging degree determination kit. The real-time PCR system used is an Agilent AriaMx Real-Time PCR System (Agilent). The real-time PCR reaction conditions are 1 cycle of 50°C for 3 minutes, 1 cycle of 95°C for 3 minutes, 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, 1 cycle of 95°C for 30 seconds, 1 cycle of 65°C for 30 seconds, and 1 cycle of 95°C for 30 seconds. The relative expression level of the Glut3 gene to the 18S ribosomal RNA gene is analyzed by the Pfaffl method. The relationship between the obtained relative expression level and age in weeks is calculated.

[0247] Next, peripheral blood is collected from the subject mouse, and a part of the blood is used as a body fluid sample to be analyzed. The body fluid sample to be analyzed is used to measure the relative expression level of the Glut3 gene to the 18S ribosomal RNA gene by the same method. The relative expression level of the Glut3 gene in the body fluid sample to be analyzed is used as an index to determine the age of the subject in weeks based on the above-mentioned relational formula.

[0248] 6. Comparative Example 2 <Method> In Comparative Example 2, indicators for assessing dementia and brain function in subjects were examined using body fluid samples derived from humans (volunteer donors).

[0249] 6-1. Recruitment of subjects The following experiment was carried out with the cooperation of five volunteer donors (volunteer donors A to E). The ages of volunteer donors A to E were 85, 59, 28, 27, and 21, respectively.

[0250] 6-2. Dementia screening test To evaluate dementia and / or brain function, the Mini-Mental State Examination (MMSE) was administered to volunteer donors. The MMSE is a cognitive function test consisting of a total of 11 questions, and can evaluate cognitive functions such as time orientation, place orientation, immediate recall, attention and arithmetic ability, delayed recall (short-term memory), linguistic ability, and graphic ability (spatial cognition). In the MMSE, the maximum score for the MMSE was 30 points, and a score of 27 or less was judged to be mild cognitive impairment (MCI), and a score of 23 or less was judged to be dementia.

[0251] 6-3. Blood cell analysis Peripheral blood was collected from volunteer donors, and the percentage of mononuclear cells in the peripheral blood samples was determined using an automated hemocytometer.

[0252] 6-4.Quantitative RT-PCR Peripheral blood was collected from volunteer donors using a blood collection tube for RNA isolation (PAXgene® RNA blood collection tube) (manufactured by Becton Dickinson). The blood collection tube was gently mixed by inversion and then left at room temperature to obtain total RNA derived from the peripheral blood (whole blood) of the volunteer donor. The total RNA was frozen and stored at -20 to -80°C. Using the obtained total RNA as a template, cDNA was synthesized (reverse transcribed) from 0.3 μg of total RNA using a cDNA synthesis kit (PrimeScript® II 1st strand cDNA Synthesis Kit (Takara Bio Inc. / model number: 6210A)) according to the manufacturer's protocol. Using the obtained cDNA as a template, real-time PCR reaction was performed using a nucleic acid amplification reagent (PowerUp SYBR® Green Master Mix (Thermo Fisher Scientific / model number: A25777)). The real-time PCR system used was the Agilent AriaMx Real-Time PCR System (Agilent). The real-time PCR reaction conditions were 1 cycle of 50°C for 3 minutes, 1 cycle of 95°C for 3 minutes, 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, 1 cycle of 95°C for 30 seconds, 1 cycle of 65°C for 30 seconds, and 1 cycle of 95°C for 30 seconds. The 18S ribosomal RNA gene, which is a housekeeping gene, was used as an endogenous control, and the relative expression level of each target gene relative to the endogenous control was analyzed by the Pfaffl method. The Pfaffl method is a type of relative quantification method of the comparative Ct method that takes into account the PCR amplification efficiency of the housekeeping gene and the target gene, and the method described in Pfaffl MW. Nucleic Acids Res. 2001; 29 (9): e45. can be used. Table 5 shows the base sequences of the primers for each gene.

[0253] [Table 5]

[0254] In Table 5, "Fw" indicates a forward primer, and "Rv" indicates a reverse primer. In addition, in each base sequence in Table 5, the left end indicates the 5' end, and the right end indicates the 3' end.

[0255] 7. Example 5 <Method> In Example 5, indicators for assessing dementia and brain function in subjects were investigated using body fluid samples derived from humans (volunteer donors).

[0256] 7-1.Recruitment of subjects The following experiment was conducted with the cooperation of four volunteer donors (volunteer donors F to I). The ages of volunteer donors F to I were 82, 81, 58, and 51, respectively.

[0257] 7-2. Dementia screening test A mini-mental state examination was conducted on the volunteer donors in the same manner as in "6-2. Dementia screening test" of Comparative Example 2.

[0258] 7-3. Blood cell analysis The ratio of mononuclear cells in peripheral blood samples of volunteer donors was measured in the same manner as in "6-3. Blood cell analysis" of Comparative Example 2.

[0259] 7-4.Quantitative RT-PCR In the same manner as in "6-4. Quantitative RT-PCR" of Comparative Example 2, the relative expression levels of each target gene in peripheral blood samples from volunteer donors were analyzed.

[0260] <Result> The results of the Mini-Mental State Examination (MMSE) for Comparative Example 2 and Example 5 are shown below. The MMSE scores of volunteer donors A to E in Comparative Example 2 were 29 or 30 points. On the other hand, the MMSE scores of volunteer donors F to I in Example 5 were 25 or 26 points. From the above, volunteer donors A to E in Comparative Example 2 were determined to have not developed dementia and had no decline in brain function, whereas volunteer donors F to I in Example 5 were determined to be suspected of mild cognitive impairment and suspected of decline in brain function.

[0261] The results of quantitative RT-PCR in Comparative Example 2 and Example 5 are shown below. Table 6 shows the relative expression level of each target gene to the expression level of the 18S ribosomal RNA gene in the peripheral blood samples of each volunteer donor in Comparative Example 2 and Example 5, calculated by the Pfaffl method.

[0262] [Table 6]

[0263] In Table 6, "Age" indicates the age of each volunteer donor, "Mononuclear cells" indicates the proportion of mononuclear cells in the peripheral blood sample of each volunteer donor (the proportion of mononuclear cells to the total number of white blood cells contained in the peripheral blood sample), and "MMSE" indicates the MMSE score.

[0264] As shown in Table 6, in the peripheral blood sample of volunteer donor A, the relative expression level of Glut1 gene was 0.33, the relative expression level of Glut3 gene was 0.25, the relative expression level of MCT4 gene was 0.09, the relative expression level of PHD3 gene was 0.50, and the relative expression level of PDK1 gene was 0.72. In the peripheral blood sample of volunteer donor B, the relative expression level of Glut1 gene was 0.45, the relative expression level of Glut3 gene was 0.26, the relative expression level of MCT4 gene was 0.19, the relative expression level of PHD3 gene was 0.24, and the relative expression level of PDK1 gene was 0.35. In the peripheral blood sample of volunteer donor C, the relative expression level of Glut1 gene was 0.12, the relative expression level of Glut3 gene was 0.17, the relative expression level of MCT4 gene was 0.72, the relative expression level of PHD3 gene was 0.24, and the relative expression level of PDK1 gene was 0.73. In the peripheral blood sample from volunteer donor D, the relative expression levels of Glut1, Glut3, MCT4, PHD3, and PDK1 were 0.55, 0.32, 1.00, 0.44, and 0.58, respectively. In the peripheral blood sample from volunteer donor E, the relative expression levels of Glut1, Glut3, MCT4, PHD3, and PDK1 were 0.92, 0.41, 0.75, 0.76, and 0.88, respectively.

[0265] As shown in Table 6, in the peripheral blood sample of volunteer donor F, the relative expression level of Glut1 gene was 1.14, the relative expression level of Glut3 gene was 1.15, the relative expression level of MCT4 gene was 1.35, the relative expression level of PHD3 gene was 1.11, and the relative expression level of PDK1 gene was 1.26. In the peripheral blood sample of volunteer donor G, the relative expression level of Glut1 gene was 2.96, the relative expression level of Glut3 gene was 0.97, the relative expression level of MCT4 gene was 1.44, the relative expression level of PHD3 gene was 2.65, and the relative expression level of PDK1 gene was 2.28. In the peripheral blood sample of volunteer donor H, the relative expression level of Glut1 gene was 1.52, the relative expression level of Glut3 gene was 1.56, the relative expression level of MCT4 gene was 1.38, the relative expression level of PHD3 gene was 1.59, and the relative expression level of PDK1 gene was 1.27. In the peripheral blood sample of volunteer donor I, the relative expression level of Glut1 gene was 1.76, the relative expression level of Glut3 gene was 0.62, the relative expression level of MCT4 gene was 2.08, the relative expression level of PHD3 gene was 2.18, and the relative expression level of PDK1 gene was 1.28.

[0266] As shown in Table 6, the gene expression of Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene, and PDK1 gene in the peripheral blood samples of volunteer donors F to I of Example 5 was significantly increased compared to volunteer donors A to E of Comparative Example 2. Thus, genes involved in glucose transport (Glut1 gene and Glut3 gene), genes involved in lactate transport (MCT4 gene), genes involved in glycolysis (PHD3 gene), and genes involved in the TCA cycle (PDK1 gene) were identified as genes whose expression levels change significantly with the onset of dementia and / or decline in brain function.

[0267] Therefore, the possibility of using the above genes and / or proteins encoded by the above genes as indicators for determining the onset of dementia and / or decline in brain function in a subject has been demonstrated. In addition, the possibility of using genes of proteins involved in energy metabolism and / or proteins involved in energy metabolism as indicators for determining the onset of dementia and / or decline in brain function in a subject has been demonstrated.

[0268] Furthermore, it was found that the relative expression levels of each target gene in the peripheral blood samples of volunteer donors F to I in Example 5 were higher than the respective standard values ​​shown in (a) to (e) below, and the relative expression levels of each target gene in volunteer donors A to E in Comparative Example 2 were lower than the respective standard values ​​shown in (a) to (e) below. (a) Baseline value of gene expression of Glut1 gene: 1.00 (b) Baseline value of gene expression of Glut3 gene: 0.50 (c) Reference value of gene expression of MCT4 gene: 1.10 (d) Reference value of gene expression of PHD3 gene: 0.90 (e) Baseline gene expression of the PDK1 gene: 1.00

[0269] In the peripheral blood sample of volunteer donor F, the ratios of the relative expression levels of Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene and PDK1 gene to the above-mentioned reference values ​​were 1.1, 2.3, 1.2, 1.2 and 1.3, respectively.

[0270] In the peripheral blood sample of volunteer donor G, the ratios of the relative expression levels of Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene and PDK1 gene to the above-mentioned reference values ​​were 3.0, 1.9, 1.3, 2.9 and 2.3, respectively.

[0271] In the peripheral blood sample of volunteer donor H, the ratios of the relative expression levels of Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene and PDK1 gene to the above-mentioned reference values ​​were 1.5, 3.1, 1.3, 1.8 and 1.3, respectively.

[0272] In the peripheral blood sample of volunteer donor I, the ratios of the relative expression levels of Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene, and PDK1 gene to the above-mentioned reference values ​​were 1.8, 1.2, 1.9, 2.4, and 1.3, respectively.

[0273] Therefore, by using the relative expression level of one or more of the above genes being higher than the above reference value as an index, an index for determining dementia or brain function in a subject can be obtained. That is, by measuring the relative expression level of one or more of the above genes in a body fluid sample derived from a subject and comparing the relative expression level with the above reference value, an index for determining dementia or brain function in a subject can be obtained.

[0274] In addition, by measuring the gene expression or enzyme activity of one or more proteins involved in energy metabolic reactions in a sample derived from a subject, and comparing the measured values ​​of the gene expression or enzyme activity of the one or more proteins with a reference value, an index for assessing dementia or brain function in a subject can be obtained.

[0275] 8. Example 6 <Method> In Example 6, dementia and brain function of a human (volunteer donor) were assessed from a body fluid sample derived from the human based on the indices for assessing dementia and brain function obtained in Comparative Example 2 and Example 5. In Example 6, the Glut1 gene and the PDK1 gene were used as indices for assessing dementia and brain function.

[0276] 8-1. Recruitment of subjects The following experiment was conducted with the cooperation of nine volunteer donors (volunteer donors J to R). The ages of volunteer donors J to R were 52, 81, 88, 81, 53, 50, 84, 53, and 22, respectively.

[0277] 8-2.Quantitative RT-PCR In the same manner as in "6-4. Quantitative RT-PCR" of Comparative Example 2, the relative expression levels of the Glut1 gene and the PDK1 gene in peripheral blood samples of volunteer donors were analyzed.

[0278] 8-3. Assessment of dementia and brain function The relative expression levels of the Glut1 gene and the PDK1 gene obtained in the above "8-2. Quantitative RT-PCR" were compared with the reference values ​​of the Glut1 gene and the PDK1 gene (both 1.00) to determine the risk of developing dementia and the presence or absence of decline in brain function in each volunteer donor.

[0279] 8-4. Dementia screening test A mini-mental state examination was conducted on the volunteer donors in the same manner as in "6-2. Dementia screening test" of Comparative Example 2.

[0280] <Result> The results of the quantitative RT-PCR in Example 6 are shown below. Table 7 shows the relative expression levels of the Glut1 gene and the PDK1 gene to the expression level of the 18S ribosomal RNA gene in the peripheral blood samples of each volunteer donor in Example 6, calculated by the Pfaffl method.

[0281] [Table 7]

[0282] As shown in Table 7, in the peripheral blood samples of volunteer donors K and M, the relative expression levels of the Glut1 gene and the PDK1 gene were higher than the reference values ​​(all 1.00) of the Glut1 gene and the PDK1 gene obtained in Comparative Example 2 and Example 5. On the other hand, in the peripheral blood samples of other volunteer donors (J, L, N to R), the relative expression levels of the Glut1 gene and the PDK1 gene were lower than the reference values ​​(all 1.00) of the Glut1 gene and the PDK1 gene. From the above, volunteer donors K and M were determined to be at risk of developing dementia and to be suspected of having a decline in brain function.

[0283] The results of the Mini-Mental State Examination (MMSE) in Example 6 are shown below. Volunteer donor K's MMSE score was 22 points, and volunteer donor M's MMSE score was 25 points. The other volunteer donors (J, L, N-R) had MMSE scores ranging from 28 to 30 points. Based on the above, volunteer donor K was judged to have suspected dementia and a decline in brain function. Volunteer donor M was judged to have suspected mild cognitive impairment and a decline in brain function. The other volunteer donors (J, L, N-R) were judged to have not developed dementia and no decline in brain function.

[0284] Therefore, by using as an indicator that the relative expression level of one or more of the above genes is higher than the above reference value, an indicator for assessing dementia or brain function in a subject can be obtained.

[0285] 9. Example 7 <Manufacturing dementia / brain function assessment kits> A kit for assessing dementia or brain function (dementia / brain function assessment kit) is manufactured by the following procedure.

[0286] The primer pair for amplifying the PDK1 gene shown in Table 5 is dissolved in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) to prepare a primer pair solution for amplifying the PDK1 gene at a final concentration of 10 μM. The primer pair for amplifying the 18S ribosomal RNA gene shown in Table 5 is dissolved in TE buffer to prepare a primer pair solution for amplifying the 18S ribosomal RNA gene at a final concentration of 10 μM. The primer pair solution for amplifying the PDK1 gene is used as a reagent for measuring gene expression of PDK1, a protein involved in the TCA cycle. Furthermore, the primer pair solution for amplifying the PDK1 gene, the primer pair solution for amplifying the 18S ribosomal RNA gene, a mononuclear cell separation reagent (Ficoll-Paque PLUS (manufactured by GE Healthcare / model number: 17144002)), an RNA extraction reagent (Kaneka Simple RNA Extraction Kit (for RT-PCR) (Kaneka Corporation / model number: KN-T110004)), a cDNA synthesis reagent (PrimeScript (registered trademark) II 1st strand cDNA Synthesis Kit (Takara Bio Inc. / model number: 6210A), and a nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / model number: A25777)) are combined to form a dementia / brain function assessment kit.

[0287] <Use of Dementia / Brain Function Assessment Kit> The dementia / brain function assessment kit can be used in the following manner.

[0288] Peripheral blood is collected from a human subject. Mononuclear cells are separated from the peripheral blood using the mononuclear cell separation reagent constituting the dementia / brain function assessment kit, and a portion of the obtained mononuclear cells is used as a body fluid sample to be analyzed. Total RNA is extracted from the body fluid sample using the RNA extraction reagent constituting the dementia / brain function assessment kit. Using the obtained total RNA as a template, cDNA is synthesized using the cDNA synthesis reagent constituting the dementia / brain function assessment kit. Using the obtained cDNA as a template, a real-time PCR reaction is performed using a primer pair solution for amplifying the PDK1 gene, a primer pair solution for amplifying the 18S ribosomal RNA gene, and a nucleic acid amplification reagent constituting the dementia / brain function assessment kit. The real-time PCR system used is the Agilent AriaMx Real-Time PCR System (Agilent). The real-time PCR reaction conditions are 1 cycle of 50°C for 3 minutes, 1 cycle of 95°C for 3 minutes, 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, 1 cycle of 95°C for 30 seconds, 1 cycle of 65°C for 30 seconds, and 1 cycle of 95°C for 30 seconds. The relative expression level of the PDK1 gene to the 18S ribosomal RNA gene is analyzed by the Pfaffl method. The relative expression level of the PDK1 gene to the expression level of the 18S ribosomal RNA gene is compared with the reference value of gene expression of the PDK1 gene, thereby determining whether or not a person suffers from dementia.

[0289] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A kit for determining dementia or brain function, comprising: A reagent for measuring the expression level of a gene encoding one or more proteins involved in an energy metabolic reaction in a sample derived from a subject, the one or more proteins are selected from human Glut1 consisting of the amino acid sequence shown in SEQ ID NO:4, human Glut3 consisting of the amino acid sequence shown in SEQ ID NO:8, human MCT4 consisting of the amino acid sequence shown in SEQ ID NO:12, human PHD3 consisting of the amino acid sequence shown in SEQ ID NO:16, and human PDK1 consisting of the amino acid sequence shown in SEQ ID NO:20; the sample is a peripheral blood sample, The assessment of dementia is assessment of the presence or absence of dementia, assessment of the risk of developing dementia, or assessment of the severity of dementia, The assessment of brain function is assessment of the presence or absence of brain function decline, assessment of the risk of brain function decline, or assessment of the degree of brain function decline, A kit in which a higher relative expression level of a gene encoding one or more proteins to the expression level of a housekeeping gene compared to a reference value indicates the presence of dementia, the risk of developing dementia, or the severity of dementia, or indicates the presence of a decline in brain function, the risk of a decline in brain function, or the degree of decline in brain function.

2. The kit described in claim 1, wherein the decline in brain function is memory impairment, movement disorder, vision impairment, balance disorder, or depression.

3. The kit according to claim 1 or 2, wherein the reagent is a primer pair for amplifying at least a portion of a nucleic acid comprising a gene encoding the one or more proteins, a probe that hybridizes with a nucleic acid comprising a gene encoding the one or more proteins, or an antibody that specifically binds to the one or more proteins.

4. The kit according to any one of claims 1 to 3, further comprising a reagent and / or a column for isolating mononuclear cells from the sample.

5. The reference value is A relative expression level of a measured value of an expression level of a gene encoding the one or more proteins in a sample derived from a normal specimen, measured under the same conditions as those for measuring the expression level of a gene encoding the one or more proteins in a sample derived from the subject, relative to the expression level of a housekeeping gene, or a reference value set from the relative expression level, or A relative expression level of a measured value of a gene encoding the one or more proteins in a sample derived from a patient known to have dementia or whose severity is known to be dementia, relative to the expression level of a housekeeping gene, when the expression level of the gene encoding the one or more proteins in a sample derived from the subject is measured under the same conditions as those for the measurement of the expression level of the gene encoding the one or more proteins in a sample derived from the subject, or a reference value set from the relative expression level The kit according to any one of claims 1 to 4,

6. A kit described in any one of claims 1 to 5, wherein the housekeeping gene is a human 18S ribosomal RNA gene having the base sequence shown in SEQ ID NO:

22.

7. A method for obtaining an index for assessing dementia or brain function in a subject, comprising: measuring the expression level of a gene encoding one or more proteins involved in an energy metabolic reaction in a sample derived from a subject; the one or more proteins are selected from human Glut1 consisting of the amino acid sequence shown in SEQ ID NO:4, human Glut3 consisting of the amino acid sequence shown in SEQ ID NO:8, human MCT4 consisting of the amino acid sequence shown in SEQ ID NO:12, human PHD3 consisting of the amino acid sequence shown in SEQ ID NO:16, and human PDK1 consisting of the amino acid sequence shown in SEQ ID NO:20; the sample is a peripheral blood sample, The assessment of dementia is assessment of the presence or absence of dementia, assessment of the risk of developing dementia, or assessment of the severity of dementia, The assessment of brain function is assessment of the presence or absence of brain function decline, assessment of the risk of brain function decline, or assessment of the degree of brain function decline, A method in which a higher relative expression level of a gene encoding one or more proteins to the expression level of a housekeeping gene compared to a reference value indicates the presence of dementia, the risk of developing dementia, or the severity of dementia, or indicates the presence of a decline in brain function, the risk of a decline in brain function, or the degree of a decline in brain function.

8. The method described in claim 7, wherein the decline in brain function is memory impairment, movement disorder, vision impairment, balance disorder, or depression.

9. The method according to claim 7 or 8, wherein measuring the expression levels of the genes encoding the one or more proteins in the sample derived from the subject comprises the following steps (a) and (b): (a) obtaining a mononuclear cell-containing sample by isolating mononuclear cells from said sample; (b) measuring the expression level of genes encoding said one or more proteins in said mononuclear cell-containing sample.

10. The reference value is A relative expression level of a measured value of an expression level of a gene encoding the one or more proteins in a sample derived from a normal specimen, measured under the same conditions as those for measuring the expression level of a gene encoding the one or more proteins in a sample derived from the subject, relative to the expression level of a housekeeping gene, or a reference value set from the relative expression level, or A relative expression level of a measured value of a gene encoding the one or more proteins in a sample derived from a patient known to have dementia or whose severity is known to be dementia, relative to the expression level of a housekeeping gene, when the expression level of the gene encoding the one or more proteins in a sample derived from the subject is measured under the same conditions as those for the measurement of the expression level of the gene encoding the one or more proteins in a sample derived from the subject, or a reference value set from the relative expression level The method according to any one of claims 7 to 9, wherein

11. A method according to any one of claims 7 to 10, wherein the housekeeping gene is a human 18S ribosomal RNA gene consisting of the base sequence shown in SEQ ID NO:22.

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