WO2021177447A1

WO2021177447A1 - Kit and method for determining dementia or brain function

Info

Publication number: WO2021177447A1
Application number: PCT/JP2021/008722
Authority: WO
Inventors: 明彦田口; 由紀子竹内; 由佳沖中; 友野　潤
Original assignee: 株式会社カネカ; 公益財団法人神戸医療産業都市推進機構
Priority date: 2020-03-06
Filing date: 2021-03-05
Publication date: 2021-09-10
Also published as: JPWO2021177447A1

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

Kits and methods for determining dementia or brain function

The present invention relates to a method for obtaining an index for determining dementia or determining brain function in a subject.
The present invention also relates to a kit for determining dementia or determining 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.

The number of dementia cases is increasing with the progress of aging, and research and development of therapeutic agents are being actively carried out. Dementia can be classified into Alzheimer's type, vascular type, Lewy body type, and frontotemporal type. Of these, Alzheimer's disease has the largest number of patients, and it is reported that it accounts for more than 60% of the total. In 1999, donepezil hydrochloride (trade name: Aricept, etc.) for Alzheimer-type dementia (improved memory and cognitive impairment and suppressed the progression of the disease), and in 2011, galantamine (the same Reminyl) and rivastigmine (the same Ixeron patch, rivastigmine patch). ) And Memantine (the same Memary) are on the market. These drugs slow the progression of dementia. However, no therapeutic agent for dementia is currently available and development is still difficult. Therefore, it is now being attempted to treat dementia by early detection and lifestyle-related improvement.

PET (positron emission tomography) and SPECT (single photon emission tomography) are known as imaging methods for diagnosing dementia. However, these imaging methods are not suitable for early determination of dementia because they are expensive and can be discriminated only when the symptoms are progressing.

In addition, cognitive function tests such as the Hasegawa simple intelligence evaluation scale for discriminating dementia are known, but the cognitive function test is a test used after the patient himself / herself recognizes the progression of symptoms, and is used in the early stage of onset. It cannot be used to judge dementia and brain function. There is also the problem that the results of cognitive function tests are ambiguous.

In addition, a method for determining dementia by a protein test in cerebrospinal fluid is also known, but there is a problem that it is highly invasive and cannot be used for early diagnosis.

A method for comprehensive analysis of blood microRNAs in subjects has also been developed for predicting the onset of dementia. There are problems that the operation of collecting blood microRNA as a pretreatment of the test sample is complicated and that an expensive measurement system is required.

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 increasing. For example, as a method for determining the degree of various aging phenomena such as skin spots and wrinkles, visual determination of appearance has been conventionally performed. However, since it is difficult to obtain an objective and quantitative judgment result by visual judgment, a search for a new biomarker capable of appropriately evaluating the degree of the aging phenomenon is underway.

Patent Documents 1 to 3 can be exemplified as patent documents that disclose a method for determining brain dysfunction based on the gene expression level (biomarker) of a specific protein.

Patent Document 1 examines 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 a subject-derived central nervous system tissue. The method is described.

Patent Document 2 detects phosphorylation of at least one substrate protein such as MARCKS in a subject, and when the degree of phosphorylation is higher than that of a normal sample, the subject suffers from or is at risk of developing Alzheimer's disease. A method for determining that the substance has sex is described.

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

Further, Patent Documents 4 to 6 can be exemplified as patent documents that disclose a method for determining the degree of aging phenomenon based on the gene expression level (biomarker) of a specific protein.

Patent Document 4 is a method for determining the degree of aging of the skin, which comprises measuring the expression of secreted proteins and / or intracellular proteins in skin cells and / or skin tissues and / or the gene expression thereof. The above-mentioned method is described in which the expression of a protein and / or an intracellular protein changes depending on the degree of skin aging. The biomarkers described in Patent Document 4 are described as Kallikrein 7, Keratin 7, and the like.

In Patent Document 5, the degree of aging of cells and / or individuals is evaluated using the expression level of the GREM1 protein and / or the GREM2 protein, or the gene encoding the GREM1 protein and / or the gene encoding the GREM2 protein in a biological sample as an index. How to do it is described.

Patent Document 6 describes a method for evaluating the cell senescence state of fibroblasts, which comprises a step of determining the expression level e of the gene product of OLFML2A and / or CRLF1 in a biological sample. ..

Special Table 2008-522199 International release WO2015 / 099094 International release WO 2019/012671 Japanese Unexamined Patent Publication No. 2012-189602 JP-A-2019-007759 Japanese Unexamined Patent Publication No. 2013-116088

One or more embodiments of the present invention provide a method and a kit for early determination of dementia by a simple operation, a method and a kit for determining brain function by a simple operation. The purpose is.
Another one or more embodiments of the present invention are aimed at providing a method capable of determining the degree of an aging phenomenon at an early stage by a simple operation and a kit for the same.

As a result of diligent studies, the present inventors have completed the following inventions.
(1) A method for obtaining an index for determining dementia or brain function in a subject.
A method comprising measuring the gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
(2) The method according to (1), wherein the determination of the dementia is a determination of the presence or absence of dementia, a determination of the risk of developing dementia, or a determination of the severity of dementia.
(3) The sample is a body fluid sample or a bone marrow sample.
It further comprises comparing the measured values of gene expression or enzyme activity of one or more proteins with reference values.
A measurement value of gene expression or enzyme activity of one or more proteins, which is higher than the reference value, indicates that the subject suffers from dementia and that the subject has a risk of developing dementia. Or, the method according to (2), which indicates 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 one or more proteins to the reference value is 1.1 or more and 50.0 or less.
(5) The sample is a central nervous system tissue sample.
It further comprises comparing the measured values of gene expression or enzyme activity of one or more proteins with reference values.
A measurement of gene expression or enzyme activity of one or more proteins, which is lower than the reference value, indicates that the subject suffers from dementia and that the subject has a risk of developing dementia. Or, the method according to (2), which indicates the severity of dementia in the subject.
(6) The determination of the brain function is as follows: determination of the presence or absence of deterioration of brain function, determination of the risk of deterioration of brain function, determination of the degree of deterioration of brain function, determination of presence or absence of disorder of the autonomic nervous system, autonomic nerve Judgment of risk of system disorder, judgment of degree of disorder of autonomic nervous system, judgment of presence or absence of memory disorder, judgment of risk of memory disorder, judgment of degree of memory disorder, judgment of presence or absence of motor disorder, judgment of movement disorder Judgment of risk of , Judgment of the risk of involuntary movement disorder, judgment of the degree of involuntary movement disorder, judgment of the presence or absence of sensory disorder, judgment of the risk of sensory disorder, judgment of the degree of sensory disorder, presence or absence of visual disorder Judgment, judgment of the risk of visual impairment, judgment of the degree of visual impairment, judgment of the presence or absence of olfactory disorder, judgment of the risk of olfactory disorder, judgment of the degree of olfactory disorder, presence or absence of hearing disorder Judgment, judgment of the risk of hearing disorder, judgment of the degree of hearing disorder, judgment of the presence or absence of balance disorder, judgment of the risk of balance disorder, judgment of the degree of balance disorder, sleep disorder Judgment of presence / absence, judgment of risk of sleep disorder, judgment of degree of sleep disorder, judgment of presence / absence of language disorder, judgment of risk of language disorder, judgment of degree of speech disorder, judgment of presence / absence of emotional dysregulation, emotion Judgment of the risk of dysregulation, judgment of the degree of emotional dysregulation, judgment of the presence or absence of headache, judgment of the risk of headache, judgment of the degree of headache, judgment of the presence or absence of convulsions, judgment of the risk of convulsions, judgment of convulsions Degree judgment, tremor presence / absence judgment, tremor risk judgment, tremor degree judgment, illusion presence / absence judgment, illusion risk judgment, illusion degree judgment, delusion presence / absence judgment, delusion Judgment of danger, judgment of degree of delusion, judgment of presence or absence of illusion, judgment of danger of illusion, judgment of degree of illusion, judgment of presence or absence of abnormal behavior, judgment of danger of abnormal behavior, judgment of degree of abnormal behavior The method according to (1), which is a determination, a determination of the presence or absence of depression, a determination of the risk of developing depression, or a determination of 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 determining dementia or brain function.
A kit containing a reagent for measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
(9) The kit according to (8), wherein the determination of the dementia is a determination of the presence or absence of dementia, a determination of the risk of developing dementia, or a determination of the severity of dementia.
(10) The determination of the brain function includes determination of the presence or absence of deterioration of brain function, determination of the risk of deterioration of brain function, determination of the degree of deterioration of brain function, determination of presence or absence of disorder of the autonomic nervous system, and autonomic nerve. Judgment of risk of system disorder, judgment of degree of disorder of autonomic nervous system, judgment of presence or absence of memory disorder, judgment of risk of memory disorder, judgment of degree of memory disorder, judgment of presence or absence of motor disorder, judgment of movement disorder Judgment of risk of , Judgment of the risk of involuntary movement disorder, judgment of the degree of involuntary movement disorder, judgment of the presence or absence of sensory disorder, judgment of the risk of sensory disorder, judgment of the degree of sensory disorder, presence or absence of visual disorder Judgment, judgment of the risk of visual impairment, judgment of the degree of visual impairment, judgment of the presence or absence of olfactory disorder, judgment of the risk of olfactory disorder, judgment of the degree of olfactory disorder, presence or absence of hearing disorder Judgment, judgment of the risk of hearing disorder, judgment of the degree of hearing disorder, judgment of the presence or absence of balance disorder, judgment of the risk of balance disorder, judgment of the degree of balance disorder, sleep disorder Judgment of presence / absence, judgment of risk of sleep disorder, judgment of degree of sleep disorder, judgment of presence / absence of language disorder, judgment of risk of language disorder, judgment of degree of speech disorder, judgment of presence / absence of emotional dysregulation, emotion Judgment of the risk of dysregulation, judgment of the degree of emotional dysregulation, judgment of the presence or absence of headache, judgment of the risk of headache, judgment of the degree of headache, judgment of the presence or absence of convulsions, judgment of the risk of convulsions, judgment of convulsions Degree judgment, tremor presence / absence judgment, tremor risk judgment, tremor degree judgment, illusion presence / absence judgment, illusion risk judgment, illusion degree judgment, delusion presence / absence judgment, delusion Judgment of danger, judgment of degree of delusion, judgment of presence or absence of illusion, judgment of danger of illusion, judgment of degree of illusion, judgment of presence or absence of abnormal behavior, judgment of danger of abnormal behavior, judgment of degree of abnormal behavior The kit according to (8), which is a determination, a determination of the presence or absence of depression, a determination of the risk of developing depression, or a determination of 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 reagent is specific to a primer pair for amplifying a nucleic acid containing a gene of one or more proteins, a probe that hybridizes with a nucleic acid containing a gene of one or more proteins, or the protein of one or more. The kit according to any one of (8) to (11), which is an antibody that binds specifically.
(13) A method for obtaining an index for determining the degree of aging phenomenon in a subject.
A method comprising measuring the gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
(14) The determination of the degree of the aging phenomenon is a determination of age, a determination of the degree of aging phenomenon of brain tissue, a determination of the degree of aging phenomenon of peripheral blood, or a determination of the degree of aging phenomenon of bone marrow cells. 13).
(15) The method according to (13) or (14), wherein the sample is a body fluid sample, a bone marrow sample, or a central nervous system 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.
A kit containing a reagent for measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
(18) The determination of the degree of the aging phenomenon is a determination of age, a determination of the degree of aging phenomenon of brain tissue, a determination of the degree of aging phenomenon of peripheral blood, or a determination of the degree of aging phenomenon of bone marrow cells. 17) The kit according to.
(19) The kit according to (17) or (18), wherein the sample is a body fluid sample, a bone marrow sample, or a central nervous system 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 reagent is specific to a primer pair for amplifying a nucleic acid containing a gene of one or more proteins, a probe that hybridizes with a nucleic acid containing a gene of one or more proteins, or the protein of one or more. The kit according to any one of (17) to (20), which is an antibody that binds specifically.

(22) Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in the sample derived from the subject includes (a) and (b) shown below, (1). The method according to any one of (7) and (13) to (16):
(A) Obtaining a mononuclear cell-containing sample by separating mononuclear cells from the sample.
(B) To measure the gene expression or enzyme activity of one or more proteins in the mononuclear cell-containing sample.
(23) The kit according to any one of (8) to (12) and (17) to (21), wherein the kit further contains a reagent and / or a column for separating mononuclear cells from the sample. ..

(24) A method for determining dementia or brain function in a subject.
To measure the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in a sample derived from a subject, and
A method comprising determining dementia or brain function in a subject based on measurements of gene expression or enzyme activity of one or more proteins.
Here, the subject is preferably a human or non-human animal.
Here, the sample is preferably a sample isolated from the subject.
(25) The method according to (24), wherein the determination of the dementia is a determination of the presence or absence of dementia, a determination of the risk of developing dementia, or a determination of the severity of dementia.
(26) The sample is a body fluid sample or a bone marrow sample.
It further comprises comparing the measured values of gene expression or enzyme activity of one or more proteins with reference values.
A measurement value of gene expression or enzyme activity of one or more proteins, which is higher than the reference value, indicates that the subject suffers from dementia and that the subject has a risk of developing dementia. Or, the method according to (25), which indicates the severity of dementia in the subject.
(27) The method according to (26), wherein the ratio of the measured value of gene expression or enzyme activity of one or more proteins to the reference value is 1.1 or more and 50.0 or less.
(28) The sample is a central nervous system tissue sample.
It further comprises comparing the measured values of gene expression or enzyme activity of one or more proteins with reference values.
A measurement of gene expression or enzyme activity of one or more proteins, which is lower than the reference value, indicates that the subject suffers from dementia and that the subject has a risk of developing dementia. Or, the method according to (25), which indicates the severity of dementia in the subject.
(29) The method according to (24), wherein the determination of the brain function is the determination according to (6).
(30) A method for determining the degree of aging phenomenon in a subject.
To measure the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in a sample derived from a subject, and
A method comprising determining the degree of aging in the subject based on measurements of gene expression or enzyme activity of one or more proteins.
Here, the subject is preferably a human or non-human animal.
Here, the sample is preferably a sample isolated from the subject.
(31) The method according to (30), wherein the determination of the degree of the aging phenomenon is the determination 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 system 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) Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in the sample derived from the subject includes (a) and (b) shown below (24). The method according to any one of (33):
(A) Obtaining a mononuclear cell-containing sample by separating mononuclear cells from the sample.
(B) To measure the gene expression or enzyme activity of one or more proteins in the mononuclear cell-containing sample.

(35) A biomarker for determining dementia or brain function, which comprises one or more proteins involved in an energy metabolism reaction or a nucleic acid containing a base sequence encoding the amino acid sequence of the one or more proteins.
(36) A biomarker for determining the degree of aging phenomenon, which comprises one or more proteins involved in an energy metabolism reaction or a nucleic acid containing 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.

(39) In the manufacture of a kit for determining dementia or determining brain function.
Use of reagents 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 reagent is specific to a primer pair for amplifying a nucleic acid containing a gene of one or more proteins, a probe that hybridizes with a nucleic acid containing a gene of one or more proteins, or the protein of one or more. The use according to (39) or (40), which is an antibody that binds specifically.
(42) The use according to any one of (39) to (41), wherein the kit has the characteristics of the kit according to any one of (8) to (12) and (23).

(43) In the manufacture of a kit for determining the degree of aging phenomenon,
Use of reagents 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 reagent is specific to a primer pair for amplifying a nucleic acid containing a gene of one or more proteins, a probe that hybridizes with a nucleic acid containing a gene of one or more proteins, or the protein of one or more. The use according to (43) or (44), which is an antibody that binds specifically.
(46) The use according to any one of (43) to (45), wherein the kit has the characteristics of the kit according to any one of (17) to (21) and (23).

(47) A method for measuring gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a subject.
A method comprising measuring the gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
Here, the subject is preferably a human or a non-human animal, and more preferably a human.
Here, the subject preferably desires or needs to determine a subject suspected of having dementia, a subject suffering from dementia, a subject treated for dementia, and a risk of developing dementia. Subjects to be treated, subjects with suspected deterioration of brain function, subjects with decreased brain function, subjects who have been treated for treatment of deterioration of brain function, subjects who desire or need to determine brain function , A subject suspected of having an aging phenomenon, a subject having an aging phenomenon, a subject having been treated to recover from the aging phenomenon, or a subject who desires or needs to determine the degree of the 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 system tissue sample.
(49) The method according to (47) or (48), further comprising comparing a measured value of gene expression or enzyme activity of 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) Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in the sample derived from the subject includes (a) and (b) shown below, (47). The method according to any one of (50):
(A) Obtaining a mononuclear cell-containing sample by separating mononuclear cells from the sample.
(B) To measure the gene expression or enzyme activity of one or more proteins in the mononuclear cell-containing sample.

(52) A method for determining dementia or brain function in a subject and improving or treating dementia or brain function in the subject.
To measure the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in a sample derived from a subject.
Dementia or brain function in the subject is determined based on the measured values of gene expression or enzyme activity of one or more proteins, and
A method comprising performing a treatment for improving or treating dementia or brain function with respect to the subject whose result of the determination indicates that it is necessary to improve or treat dementia or brain function.
Here, the subject is preferably a human or non-human animal.
Here, the sample is preferably a sample isolated from the subject.
(53) The method according to (52), wherein the determination of dementia is a determination of the presence or absence of dementia, a determination of the risk of developing dementia, or a determination of the severity of dementia.
(54) The sample is a body fluid sample or a bone marrow sample.
It further comprises comparing the measured values of gene expression or enzyme activity of one or more proteins with reference values.
A measurement value of gene expression or enzyme activity of one or more proteins, which is higher than the reference value, indicates that the subject suffers from dementia and that the subject has a risk of developing dementia. Or, the method according to (53), which indicates the severity of dementia in the subject.
(55) The method according to (54), wherein the ratio of the measured value of gene expression or enzyme activity of one or more proteins to the reference value is 1.1 or more and 50.0 or less.
(56) The sample is a central nervous system tissue sample.
It further comprises comparing the measured values of gene expression or enzyme activity of one or more proteins with reference values.
A measurement of gene expression or enzyme activity of one or more proteins, which is lower than the reference value, indicates that the subject suffers from dementia and that the subject has a risk of developing dementia. Or, the method according to (53), which indicates the severity of dementia in the subject.
(57) The method according to (52), wherein the determination of the brain function is the determination according to (6).
(58) A method for determining the degree of aging phenomenon in a subject and improving or treating the aging phenomenon in the subject.
To measure the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in a sample derived from a subject.
Based on the measured values of gene expression or enzyme activity of one or more proteins, the degree of aging phenomenon in the subject is determined, and
A method comprising performing a treatment for improving or treating the aging phenomenon with respect to the subject whose result of the determination indicates that it is necessary to improve or treat the aging phenomenon.
Here, the subject is preferably a human or non-human animal.
Here, the sample is preferably a sample isolated from the subject.
(59) The method according to (58), wherein the determination of the degree of the aging phenomenon is the determination 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 system 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) Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in the sample derived from the subject includes (a) and (b) shown below, (52). The method according to any one of (61):
(A) Obtaining a mononuclear cell-containing sample by separating mononuclear cells from the sample.
(B) To measure the gene expression or enzyme activity of one or more proteins in the mononuclear cell-containing sample.

This specification includes the disclosure content of Japanese Patent Application No. 2020-038991, which is the basis of the priority of the present application.

According to one or more embodiments of the present invention, dementia can be determined by a simple operation. Early determination of dementia is possible.
According to one or more embodiments of the present invention, brain function can be determined by a simple operation.
According to one or more embodiments of the present invention, the degree of the aging phenomenon can be determined by a simple operation.

FIG. 1 shows the average relative expression level of each target gene with respect to the expression level of the 18S ribosomal RNA gene in the body fluid samples of the mice of Example 1 and Comparative Example 1. FIG. 2 shows the average relative expression level of each target gene with respect to the expression level of the 18S ribosomal RNA gene in the body fluid sample of each mouse of Example 2. FIG. 3 shows the relationship between the average relative expression level of each target gene in the body fluid sample of the mouse of Example 2 and the age of the mouse. FIG. 3A shows the relationship between the average relative expression level of the Glut1 gene in the body fluid sample of the mouse of Example 2 and the age of the mouse. FIG. 3B shows the relationship between the average relative expression level of the Glut3 gene in the body fluid sample of the mouse of Example 2 and the age of the mouse. FIG. 3C shows the relationship between the average relative expression level of the MCT4 gene in the body fluid sample of the mouse of Example 2 and the age of the mouse. FIG. 3D shows the relationship between the average relative expression level of the PHD3 gene in the body fluid sample of the mouse of Example 2 and the age of the mouse. FIG. 3E shows the relationship between the average relative expression level of the PDK1 gene in the body fluid sample of the mouse of Example 2 and the age of the mouse.

<1. Term>
<1.1. Sample derived from the subject>
As used herein, the term "subject" refers to a human or non-human animal that is the subject of determination of the degree of dementia, brain function, or aging phenomenon. 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.

As the sample derived from the subject, body fluid samples such as peripheral blood, saliva, urine, sputum, sweat, pharyngeal swab, and nasal swab, bone marrow sample, and central nervous tissue sample such as brain tissue are used. be able to. In particular, from the viewpoint of non-invasiveness at the time of sampling, a body fluid sample or a bone marrow sample is preferable, and a body fluid sample, particularly a peripheral blood sample, is most preferable. As the peripheral blood sample, various forms of peripheral blood samples such as serum, plasma, and whole blood can be used. A bone marrow fluid sample can be used as the bone marrow sample. The sample derived from the subject is preferably a sample isolated from the subject. As shown in the examples, the gene expression level or enzyme activity of the protein described later determines the subject suffering from dementia, the subject who may develop dementia in the future, the subject whose brain function is deteriorated, or the subject. , It has been confirmed that in subjects whose brain function may decline in the future, it is higher in body fluids such as peripheral blood or bone marrow than in normal specimens, and although not shown in Examples, brain tissue It has been confirmed that it is low in central nervous tissue such as. Further, as shown in 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 older subjects than in younger specimens. Although not shown in the examples, it has been confirmed to be low in central nervous system tissues such as brain tissue. Further, as shown in Examples, the gene expression level or enzyme activity of the protein described later is a subject in which the aging phenomenon of brain tissue is observed, a subject in which the aging phenomenon of brain tissue may progress, and aging of peripheral blood. In a subject in which the phenomenon is observed, a subject in which the aging phenomenon of peripheral blood may progress, a subject in which the aging phenomenon of bone marrow cells is observed, or a subject in which the aging phenomenon of bone marrow cells may progress. , It has been confirmed that it is higher in body fluids such as peripheral blood or bone marrow as compared with normal samples, and it is confirmed that it is lower in central nervous tissue such as brain tissue, although not shown in Examples.

Further, as the body fluid sample or bone marrow sample derived from the subject, a mononuclear cell-containing sample containing mononuclear cells (Mononuclear Cells; MNC) separated from the body fluid or bone marrow of the subject can be used. As used herein, the term "mononuclear cell" refers to lymphocytes and / or monocytes. Further, in the present specification, "white blood cell" refers to a general term for neutrophils, basophils, eosinophils, lymphocytes and monocytes.

<1.2. Dementia>
As used herein, the term "dementia" may be any of Alzheimer's disease, vascular dementia, Lewy body dementia, and frontotemporal dementia, but Alzheimer's disease is particularly preferable. be.

To determine dementia, determine the presence or absence of dementia in the subject, determine the risk of developing dementia in the subject, and determine the severity of dementia in the subject. Etc. are included.

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

To determine brain function, determine whether or not there is a decrease in brain function in the subject, determine the risk of decrease in brain function in the subject, and determine the degree of decrease in brain function in the subject. To determine the presence or absence of autonomic dysfunction in the subject, to determine the risk of autonomic dysfunction in the subject, to determine the degree of autonomic dysfunction in the subject, to determine the degree of autonomic dysfunction in the subject To determine the presence or absence of memory impairment in the subject, to determine the risk of memory impairment in the subject, to determine the degree of memory impairment in the subject, to determine the presence or absence of movement disorder in the subject, in the subject To determine the risk of movement disorder, to determine the degree of motor disorder in the subject, to determine the presence or absence of coordinated movement disorder in the subject, to determine the risk of coordinated movement disorder in the subject. , Determining the degree of impaired cooperative movement in the subject, determining the presence or absence of impaired involuntary movement in the subject, determining the risk of impaired involuntary movement in the subject, non-compliance in the subject Judging the degree of voluntary movement disorder, determining the presence or absence of sensory impairment in the subject, determining the risk of sensory impairment in the subject, determining the degree of sensory impairment in the subject, determining the subject To determine the presence or absence of visual impairment in the subject, to determine the risk of visual impairment in the subject, to determine the degree of visual impairment in the subject, to determine the presence or absence of olfactory impairment in the subject. , Judging the risk of olfactory dysfunction in the subject, determining the degree of olfactory dysfunction in the subject, determining the presence or absence of hearing deficits in the subject, risk of hearing deficits in the subject To determine the degree of hearing impairment in the subject, to determine the presence or absence of equilibrium impairment in the subject, to determine the risk of equilibrium impairment in the subject, in the subject Judging the degree of equilibrium disorder, determining the presence or absence of sleep disorder in the subject, determining the risk of sleep disorder in the subject, determining the degree of sleep disorder in the subject, determining the subject To determine the presence or absence of a language disorder in a subject, to determine the risk of a language disorder in a subject, to determine the degree of a language disorder in a subject, to determine the presence or absence of an emotional dysregulation in a subject, to determine the subject Judging the risk of emotional dysregulation in the subject, emotional dysregulation in the subject Determining the degree of harm, determining the presence or absence of headache in the subject, determining the risk of headache in the subject, determining the degree of headache in the subject, determining the presence or absence of convulsions in the subject To determine the risk of convulsions in the subject, to determine the degree of convulsions in the subject, to determine the presence or absence of tremors in the subject, to determine the risk of tremors in the subject, to determine Determining the degree of tremor in a sample, determining the presence or absence of hallucinations in a subject, determining the risk of hallucinations in a subject, determining the degree of hallucinations in a subject, presence or absence of delusions in a subject Judgment, judgment of the risk of delusion in the subject, judgment of the degree of delusion in the subject, judgment of the presence or absence of hallucination in the subject, judgment of the risk of hallucination in the subject, judgment in the subject Determining the degree of hallucination, determining the presence or absence of abnormal behavior in the subject, determining the risk of abnormal behavior in the subject, determining the degree of abnormal behavior in the subject, depression in the subject It includes determining the presence or absence of hallucinations, determining the risk of developing depression in a subject, determining the severity of depression in a subject, and the like.

<1.4. Aging phenomenon>
As used herein, the term "aging phenomenon" refers to physical and / or physiological changes associated with aging. Examples of aging phenomena include skin changes such as spots, wrinkles, sagging, and dullness, hair changes such as white hair, thinning hair, and hair loss, changes in brain morphology, changes in brain tissue such as brain atrophy, and changes in peripheral blood. , Changes in bone marrow cells, changes in bone, changes in muscle, etc. can be exemplified.

As used herein, the term "degree of aging phenomenon" refers to the degree of physical and / or physiological changes associated with aging. In the present specification, "degree of aging phenomenon" may be described as "degree of aging".

In the present specification, "determination of the degree of aging phenomenon" refers to determination of the degree of physical and physiological changes associated with aging. To determine the degree of aging phenomenon, for example, to determine the age of the subject, to determine the degree of aging phenomenon of brain tissue, to determine the degree of aging phenomenon of peripheral blood, and to determine the degree of aging phenomenon of bone marrow cells. It includes determining the degree of the phenomenon.

<1.5. One or more proteins involved in energy metabolism reactions>
In the present specification, one or more proteins involved in the energy metabolism reaction include glucose transport, lactic acid transport, energy metabolism regulation, mitochondrial biosynthesis, glycolysis, pentose phosphate pathway, TCA cycle, electron transport chain, fatty acid metabolism, and the like. One or more proteins involved in gap binding or sodium-potassium pumps can be exemplified.

Examples of proteins involved in glucose transport include proteins that constitute membrane proteins (glucose transporters) involved in glucose transport that transport glucose used for energy metabolism in cells from outside the cell to the inside of the cell. Examples of proteins belonging to the Glucose transporter family (Glucose 1 to 12). Among the proteins belonging to the Glut family, gene expression or enzymatic activity of Glut1, Glut2, Glut3 and Glut4, particularly Glut1 and Glut3 is preferable. 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 base 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 base 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 have a nucleotide sequence encoding the amino acid sequence of a protein (CDS) and a nucleotide sequence of an untranslated region (UTR) located upstream and downstream of the CDS. include.

As the protein involved in lactic acid transport, a protein constituting a membrane protein (lactic acid transporter) involved in lactic acid transport that transports lactic acid produced by energy metabolism in the cell from the inside of the cell to the outside of the cell can be exemplified. Examples include proteins belonging to the MCT (monocarboxylate transport protein) family. Among the proteins belonging to the MCT family, gene expression or enzymatic activity of MCT1, MCT2, MCT3 and MCT4, particularly MCT4, is preferable. 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. The nucleotide sequences of SEQ ID NOs: 9 and 11 both include the nucleotide sequence encoding the amino acid sequence of the protein (CDS) and the nucleotide sequence of the untranslated region (UTR) located upstream and downstream of the CDS.

Examples of proteins involved in the regulation of energy metabolism include proteins belonging to the PHD (procollagen-contining protein) family. Among the proteins belonging to the PHD family, gene expression or enzymatic activity of PHD2 and PHD3, particularly PHD3, is preferable. 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. The nucleotide sequences of SEQ ID NOs: 13 and 15 both include the nucleotide sequence encoding the amino acid sequence of the protein (CDS) and the nucleotide sequence of the untranslated region (UTR) located upstream and downstream of the CDS. In addition, as proteins involved in the regulation of energy metabolism, HIF1a (hypoxia-inducible factoror 1 alpha), Sirtuin 1 (sirtuin 1), PPARa (peroxisome proliferator-activated receptor) and exemplified receptor).

As a protein involved in mitochondrial biosynthesis, PGC1a (peroxisome proliferators-active receptor-ganma co-activator-1 alpha) can be exemplified.

Examples of proteins involved in glycolysis include LDHa (lactate dehydrogenase A), LDHb (lactate dehydrogenase B), HK1 (hexokinase 1), PFK (phosphofructase kinase), and PKR. Can also be exemplified.

Proteins involved in the pentose phosphate pathway include G6PD (glucose-6-phosphate dehydrange), RPI (ribose-5-phosphate isomerase), RPE (ribose-5-phosphate epimerase), and 6PGLose (6-PGLose. ) Can be exemplified.

Examples of proteins involved in the TCA cycle include proteins belonging to the PDK (pyruvate dehydogenesis kinase) family. Among the proteins belonging to the PDK family, PDK1, PDK3 and PDK4, particularly PDK1 gene expression or enzymatic activity is preferable. 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. The nucleotide sequences of SEQ ID NOs: 17 and 19 both include the nucleotide sequence encoding the amino acid sequence of the protein (CDS) and the nucleotide sequence of the untranslated region (UTR) located upstream and downstream of the CDS. Further, as proteins involved in the TCA cycle, IDH2 (isocitrate dehydrogenase 2), OGDH (2-oxoglute dehydrogenase) and CS (citrate synthase) can also be exemplified.

As a protein involved in the electron transport chain, AMPK (AMP-activated protein kinase) can be exemplified.

Examples of proteins involved in fatty acid metabolism include FABP1 (fatty acid binding protein 1), FABP4 (fatty acid binding protein 4), CD36 (fatty acid transit protein: FAT), CPT1 (carnitine palmitoyl), and CPT1 (carnitine palmitoyl). -Transphase 1) and ACC (protein-CoA fatty acid) can be exemplified.

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

Proteins involved in the sodium-potassium pump include ATP1A1 (sodium / potassium-transporting TAPase subunit alpha-1), ATP1A2 (sodium / potassium-transporting TAPase subunitAtropa 3) can be exemplified.

<2. How to obtain an index for determining dementia or brain function in a subject>
One or more embodiments of the present invention
It is a method of acquiring an index for determining dementia or brain function in a subject.
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 derived from a subject.

One or more embodiments of the present invention
A method for determining dementia or brain function in a subject.
To measure the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in a sample derived from a subject, and
The present invention relates to a method comprising determining dementia or brain function in the subject based on the measured values of gene expression or enzyme activity of one or more proteins.

More preferably, each of the above methods
Comparing the measured values of gene expression or enzyme activity of one or more proteins with the reference values,
Is further included.

In the present embodiment, the "reference value" refers to the gene expression or enzyme activity of the one or more proteins in a sample derived from a normal sample, and the gene expression or enzyme activity of the one or more proteins in a sample derived from a subject. It may be a measured value when measured under the same conditions or a reference value set from the measured value, or the above-mentioned one or more proteins in a sample derived from a patient whose presence or absence or severity of dementia is known. Even if the gene expression or enzyme activity is measured under the same conditions as the gene expression or enzyme activity of the one or more proteins in the sample derived from the subject, or even if it is a reference value set from the measured value. good.

In one or more embodiments of the present invention, gene expression or enzymatic activity of one or more proteins involved in an energy metabolism response is a subject suffering from dementia, a subject who may develop dementia in the future. , Subjects with reduced brain function, or subjects with a possibility of decreased brain function in the future, are higher in body fluids such as peripheral blood, urine, saliva, or bone marrow than normal samples, and brain tissue Based on the unexpected finding that it is low in central nervous tissue such as. In addition, the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction is higher in body fluids such as peripheral blood, urine, saliva or bone marrow as the severity of dementia in the subject is higher, and in brain tissues and the like. Low in central nervous tissue. In addition, the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction is higher in body fluids such as peripheral blood, urine, saliva or bone marrow as the brain function in the subject is lower, and the central nervous tissue such as brain tissue. Low in. 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, the subject recognizes that the measured value is higher than the reference value. The subject has a disease, the subject has a risk of developing dementia, the subject has a higher degree of dementia, and the subject's brain function is impaired. It is an index showing that there is a risk that the brain function of the subject is deteriorated, or that the degree of deterioration of the brain function of the subject is more remarkable. Further, in the embodiment of the present invention, when the sample is a central nervous system sample, the measured value lower than the reference value means that the subject suffers from dementia. The sample has a risk of developing dementia, the severity of dementia of the subject is higher, the brain function of the subject is deteriorated, and the brain function of the subject is deteriorated. It is an index showing that the subject has sex or that the degree of deterioration of the brain function of the subject is more remarkable.

In one or more embodiments of the present invention, when the sample is a body fluid sample or a bone marrow sample, particularly a mononuclear cell-containing sample, the lower limit of the ratio of the measured value to the reference value is 1.1 or more and 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 4.4 or higher, 3.6 or higher, 3.8 or higher, 4.0 or higher, 4.2 or higher, 4.4 or higher, 4.6 or higher, 4.8 or higher, 5.0 or higher, 5.2 or higher, 5 It is preferably .4 or more, 5.6 or more, 5.8 or more, or 6.0 or more. 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, but is, for example, 50.0 or less, 45.0. 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 Below, it can be 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 the same as the gene expression or enzyme activity of the one or more proteins in the sample derived from the normal sample, and the gene expression or enzyme activity of the one or more proteins in the sample derived from the subject. It is particularly preferable that it is a measured value when measured under the conditions of (1) or a reference value set from the measured value.

In one or more embodiments of the present invention, one or more, two or more, three or more, four or more or all selected from Glut1, Glut3, MCT4, PHD3 and PDK1 as one or more proteins involved in the energy metabolism reaction. It is preferable to measure the gene expression or enzyme activity of the protein. In the embodiment in which the sample is a body fluid sample or a bone marrow sample, particularly a mononuclear cell-containing sample, and the gene expression or enzyme activity of 2 or more and 5 or less of the five proteins is measured, half of the measured proteins are used. When the measured value of gene expression or enzyme activity of the above (for example, 1/2 or more, 2/3 or more, 2/4 or more, 3/5 or more) is higher than the reference value, the subject has dementia. The subject has a risk of developing dementia, the subject has a higher severity of dementia, the subject's brain function is impaired, the subject's brain It is preferable to determine that there is a risk of functional deterioration, or that the degree of deterioration of the brain function of the subject is more remarkable.

In the embodiment of the present invention, only one of the gene expression and the enzyme activity of the one or more proteins may be measured, or both may be measured. More preferably, the gene expression of one or more proteins is measured.

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 the sample, or the gene expression in the sample may be measured. It may be measured by detecting the protein amount of one or more proteins.

The measured value of the gene expression of the one or more proteins may be the relative expression level of the gene expression of the one or more proteins with respect to the gene expression level of the one or more endogenous controls in the sample. As endogenous controls, TBP (TATA-binding protein), GAPDH (glyceraldehyde-3-phosphate dehydogenase), β-actin, β2M (β2 microglobuline), HPRT1 (hypoxanthine), HPRT1 (hypoxanthine), HPRT1 (hypoxanthine) A representative housekeeping gene 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.

Northern blotting, RT-PCR method, real-time RT-PCR method, DNA microarray method (method using DNA chip), dot blotting method, RN for detection of mRNA of the gene encoding the amino acid sequence of one or more proteins. An ase protection assay method or the like can be used. These methods can be performed by known methods.

The protein amount of the one or more proteins can be detected by an immunoassay method 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 method include a method using a solid-phase carrier on which an antibody that specifically binds to the one or more proteins to be detected is immobilized, flow cytometry, Western blotting, and the like. 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 method) using immobilized particles. A known immunoassay can be used to detect the protein content of one or more of the above proteins in a sample. Further, the detection of the protein amount of the above-mentioned one or more proteins can also be performed by a method using LC-MS / MSMRM or the like, which is a protein mass spectrometry technique that does not use an antibody. These detection methods can also be carried out by a conventional protocol.

The enzymatic activity of the one or more proteins in the sample can be measured by a method according to the enzymatic activity of the protein to be measured.

The ratio of mononuclear cells in the sample is 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% is preferable. As used herein, the term "ratio of mononuclear cells in a sample" refers to the ratio of the number of mononuclear cells to the total number of leukocytes contained in the sample. The ratio of mononuclear cells in the sample can be measured by, for example, a blood cell analyzer, a hemocytometer, flow cytometry, or the like, but is not limited thereto.

In one or more embodiments of the present invention, measuring the gene expression or enzyme activity of one or more proteins involved in an energy metabolism reaction in a sample derived from the subject is shown in (a) and (b) below. ) Is preferably included.
(A) Obtaining a mononuclear cell-containing sample by separating mononuclear cells from the sample.
(B) To measure the gene expression or enzyme activity of one or more proteins in the mononuclear cell-containing sample.

The sample derived from the subject may contain granulocytes, and the granulocytes in the sample become a factor that lowers the measurement accuracy of the measured values of gene expression or enzyme activity of one or more proteins. Sometimes. Therefore, a mononuclear cell-containing sample in which the proportion of mononuclear cells is increased by separating mononuclear cells or removing granulocytes from the sample is obtained, and the obtained mononuclear cell-containing sample is used for measurement. Therefore, a highly accurate measured value can be obtained.

The ratio of mononuclear cells in the mononuclear cell-containing sample is 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%. It is preferable to have. Further, the ratio of granulocytes in the mononuclear cell-containing sample may be less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2% or 0%. preferable. In the present specification, the "ratio of mononuclear cells or granulocytes in the mononuclear cell-containing sample" is defined by the number of mononuclear cells or granulocytes with respect to the total number of cells contained in the mononuclear cell-containing sample. Refers to the ratio. The ratio of mononuclear cells or granulocytes in a mononuclear cell-containing sample can be measured by, for example, a blood cell analyzer, a hemocytometer, flow cytometry, or the like, but is not limited thereto.

As a method for obtaining a mononuclear cell-containing sample from a sample derived from a subject, a method using a reagent and / or a column for separating mononuclear cells is preferable. Examples of the reagent and / or column include Ficoll-Paque PLUS (manufactured by GE Healthcare), Lymphoprep (manufactured by Abbott Laboratories Technologies), and Human Peripheral Blood Monologic Laboratory (manufactured by Human Biological Blood Mononulus Technology). Registered trademark) CPT mononuclear cell separation blood collection tube (manufactured by Becton Dickinson), purriMate (manufactured by purriSelect), SepMate (manufactured by STEMCELL Technologies) and the like can be mentioned. By using the reagent and / or the column, the mononuclear cell and the granulocyte in the sample can be separated, and a mononuclear cell-containing sample for measurement can be efficiently obtained.

<3. How to obtain an index for determining the degree of aging in a subject>
One or more embodiments of the present invention
It is a method of obtaining an index for determining the degree of aging phenomenon in a subject.
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 derived from a subject.

One or more embodiments of the present invention
It is a method of determining the degree of aging phenomenon in a subject.
To measure the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in a sample derived from a subject, and
The present invention relates to a method comprising determining the degree of aging phenomenon in the subject based on the measured values of gene expression or enzyme activity of one or more proteins.

In one or more embodiments of the invention, the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism response is such that in older subjects, in peripheral blood, urine, saliva, etc., as compared to younger specimens. It is based on the unexpected finding that it is high in body fluids or bone marrow and low in central nervous tissue such as brain tissue. Further, in one or more embodiments of the present invention, the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction may cause the aging phenomenon of the brain tissue to progress in the subject in which the aging phenomenon of the brain tissue is observed. A subject with sex, a subject with peripheral blood aging, a subject with potential for peripheral blood aging, a subject with bone marrow cell aging, or a bone marrow cell aging phenomenon In the subject in which the disease may progress, it is higher in body fluids such as peripheral blood, urine, saliva or bone marrow, and lower in central nervous tissue such as brain tissue, as compared with normal specimens. Further, in one or more embodiments of the present invention, the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction is such that the degree of aging phenomenon of brain tissue is large and the degree of aging phenomenon of peripheral blood is high. In a large subject or a subject having a large degree of aging phenomenon of bone marrow cells, it is higher in body fluids such as peripheral blood, urine, saliva or bone marrow, and lower in central nervous tissue such as brain tissue, as compared with normal samples. In addition, the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction is higher in body fluids such as peripheral blood, urine, saliva, or bone marrow as the degree of aging phenomenon of the brain tissue of the subject increases. Low in central nervous tissue such as. In addition, the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction increases in body fluids such as peripheral blood, urine, saliva, or bone marrow as the degree of aging phenomenon of peripheral blood of the subject increases, and brain tissue. Low in central nervous tissue such as. In addition, the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction is higher in body fluids such as peripheral blood, urine, saliva, or bone marrow as the degree of aging phenomenon of the bone marrow cells of the subject is greater, and the brain tissue. Low in central nervous tissue such as. 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 measured value is used to determine the age of the subject and the aging of the brain tissue of the subject. The possibility of the phenomenon progressing, the degree of the aging phenomenon of the brain tissue of the subject, the possibility of the aging phenomenon of the peripheral blood of the subject progressing, the degree of the aging phenomenon of the peripheral blood of the subject, or the subject It is an index showing the degree of aging phenomenon of the bone marrow cells of the sample.

In one or more embodiments of the present invention, one or more, two or more, three or more, four or more or all selected from Glut1, Glut3, MCT4, PHD3 and PDK1 as one or more proteins involved in the energy metabolism reaction. It is preferable to measure the gene expression or enzyme activity of the protein. In the embodiment in which the sample is a body fluid sample or a bone marrow sample, particularly a mononuclear cell-containing sample, and the gene expression or enzyme activity of 2 or more and 5 or less of the five proteins is measured, half of the measured proteins. When the measured value of gene expression or enzyme activity of the above (for example, 1/2 or more, 2/3 or more, 2/4 or more, 3/5 or more) is higher than the reference value, the aging phenomenon of the subject. It is preferable to determine that is more advanced.

Northern blotting, RT-PCR method, real-time RT-PCR method, DNA microarray method (method using DNA chip), dot blotting method, RNase protection assay method, etc. Can be used. These methods can be performed by known methods.

The protein amount of the one or more proteins can be detected by an immunoassay method 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 method include a method using a solid-phase carrier on which an antibody that specifically binds to the one or more proteins to be detected is immobilized, flow cytometry, Western blotting, and the like. 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 method) using immobilized particles. A known immunoassay can be used to detect the protein content of one or more of the above proteins in a sample. Further, the detection of the protein amount of the above-mentioned one or more proteins can also be performed by a method using LC-MS / MS MRM or the like, which is a protein mass spectrometry technique that does not use an antibody. These detection methods can also be carried out by a conventional protocol.
The enzymatic activity of the one or more proteins in the sample can be measured by a method according to the enzymatic activity of the protein to be measured.

<4. Kit for determining dementia or brain function>
Another embodiment of the present invention
A kit for determining dementia or brain function.
The present invention relates to a kit containing a reagent for measuring gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
As the reagent, a reagent that can be used in the above-mentioned method for measuring gene expression or enzyme activity of one or more proteins is preferable.

Specifically, a primer pair for amplifying a nucleic acid (genome DNA, mRNA or cDNA prepared based on mRNA) containing a gene of the one or more proteins to be measured, and the one or more proteins to be measured. To measure the enzymatic activity of a probe that hybridizes with a nucleic acid containing the gene (genomic DNA, mRNA or cDNA prepared based on the mRNA), an antibody that specifically binds to the one or more proteins, and the enzyme activity of the one or more proteins. The reaction substrate of the above can be exemplified. The nucleic acid may also include an untranslated region, an intron region, a signal sequence region, and the like. Preferably, the nucleic acid is mRNA or cDNA. The mRNA or cDNA may contain 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 capable of amplifying at least a part of the base sequences of CDS and UTR. An example of the probe is a probe that can hybridize to at least a part of the base sequences of CDS and UTR.

As an example of the primer pair, 3 is a primer pair for amplifying a nucleic acid containing the Glut1 gene, which has the same or homologous base sequence Af as any of the base sequences shown in (A1) to (A4) below. 'The Glut1 forward primer contained at the end and the Glut1 reverse primer containing the same or homologous base sequence Ar as the complementary base sequence of any of the base sequences shown in (A5) to (A8) below at the 3'end. An example includes a Glut1 primer pair containing.
(A1) Consecutive 10 or more base sequences contained in the base sequences of positions 101 to 180 among the base sequences of SEQ ID NO: 1 (A2) Bases of SEQ ID NO: 3 among the base sequences of SEQ ID NO: 3. When the sequence and the base sequence of SEQ ID NO: 1 are aligned, the base sequence corresponding to the base sequence of (A1) is changed to the base sequence of positions 256 to 336 among the base sequences of (A3) SEQ ID NO: 3. Consecutive base sequence of 10 or more bases (A4) Of the base sequences of SEQ ID NO: 1, when the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 3 are aligned, the base sequence of (A3) is obtained. Corresponding base sequence (A5) Of the base sequences of SEQ ID NO: 1, consecutive base sequences of 10 or more bases included in the base sequences of positions 213 to 292 (A6) Of the base sequences of SEQ ID NO: 3, the sequence When the base sequence of No. 3 and the base sequence of SEQ ID NO: 1 are aligned, the base sequence corresponding to the base sequence of (A5) (A7) Of the base sequences of SEQ ID NO: 3, positions 339 to 418 (A8) Of the nucleotide sequences of SEQ ID NO: 1, when the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 3 are aligned, the above (A7) The base sequence corresponding to the base sequence of.

In the above (A1), the "base sequence at positions 101 to 180" is preferably the "base sequence at positions 111 to 170", and more preferably "base sequence at positions 121 to 160". It is a "base sequence", more preferably "base sequence at positions 121 to 155", and more preferably "base sequence at positions 126 to 150".

In the above (A3), the "base sequence at positions 256 to 336" is preferably the "base sequence at positions 266 to 326", and more preferably "base sequence at positions 276 to 316". It is a "base sequence", more preferably "base sequence at positions 276 to 311", and more preferably "base sequence at positions 281 to 306".

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), "continuous" preferably means "continuous from the 3'end".

The base sequence Af may be the same as or homologous to the base sequence of (A1), the base sequence of (A2), the base sequence of (A3), or the base sequence of (A4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably The continuous base sequence of 19 bases or more is the same as the base sequence of (A1), (A2), (A3) or (A4), and the rest of the base sequence Af is It is the same as or homologous to the base sequence of (A1), the base sequence of (A2), the base sequence of (A3), or the base sequence of (A4). The range of "homology" is as described later. Particularly preferably, the base sequence Af is the same as the base sequence of (A1), the base sequence of (A2), the base sequence of (A3), or the base sequence of (A4).

The Glut1 forward primer may be one containing a polynucleotide containing the base sequence Af at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Af. The number of bases of the polynucleotide contained in the Glut1 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.

As a specific example of the base sequence of (A1), the base sequence of SEQ ID NO: 25 can be exemplified. As a specific example of the base sequence Af, the base sequence of SEQ ID NO: 25 can be exemplified.

As a specific example of the base sequence of (A3) above, the base sequence of SEQ ID NO: 37 can be exemplified. As a specific example of the base sequence Af, the base sequence of SEQ ID NO: 37 can be exemplified.

In the above (A5), the "base sequence at positions 213 to 292" is preferably the "base sequence at positions 223 to 282", and more preferably "base sequence at positions 233 to 272". It is a "base sequence", more preferably "base sequence at positions 238 to 272", and more preferably "base sequence at positions 243 to 267".

In the above (A7), the "base sequence at positions 339 to 418" is preferably the "base sequence at positions 349 to 408", and more preferably "base sequence at positions 359 to 398". It is a "base sequence", more preferably "base sequence at positions 364 to 398", and more preferably "base sequence at positions 369 to 393".

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), "continuous" preferably means "continuous from the 5'end".

The base sequence Ar may be the same as or homologous to the base sequence of (A5), the base sequence of (A6), the base sequence of (A7), or the complementary base sequence of the base sequence of (A8), but the base From the 3'end of the sequence Ar, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (A5), the base sequence of (A6), the base sequence of (A7), or the complementary base sequence of the base sequence of (A8). The rest of the base sequence Ar is the same as or homologous to the base sequence of (A5), the base sequence of (A6), the base sequence of (A7), or the complementary base sequence of the base sequence of (A8). The range of "homology" is as described later. Particularly preferably, the base sequence Ar is the same as the base sequence of (A5), the base sequence of (A6), the base sequence of (A7), or the complementary base sequence of the base sequence of (A8).

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.

As a specific example of the base sequence of (A5) above, a complementary base sequence of the base sequence of SEQ ID NO: 26 can be exemplified. As a specific example of the base sequence Ar, the base sequence of SEQ ID NO: 26 can be exemplified.

As a specific example of the base sequence of (A7), the complementary base sequence of the base sequence of SEQ ID NO: 38 can be exemplified. As a specific example of the base sequence Ar, the base sequence of SEQ ID NO: 38 can be exemplified.

As an example of the primer pair, 3 is a primer pair for amplifying a nucleic acid containing the Glut3 gene, which has the same or homologous base sequence Bf as any of the base sequences shown in (B1) to (B4) below. 'The Glut3 forward primer contained at the end and the Glut3 reverse primer containing the same or homologous base sequence Br as the complementary base sequence of any of the base sequences shown in (B5) to (B8) below at the 3'end. An example includes a Glut3 primer pair containing.
(B1) Consecutive 10 or more base sequences contained in the base sequences of positions 250 to 329 of the base sequence of SEQ ID NO: 5 (B2) Base of SEQ ID NO: 7 among the base sequences of SEQ ID NO: 7. When the sequence and the base sequence of SEQ ID NO: 5 are aligned, the base sequence corresponding to the base sequence of (B1) (B3) is the base sequence of positions 224 to 303 among the base sequences of SEQ ID NO: 7. Consecutive base sequence of 10 or more bases (B4) Among the base sequences of SEQ ID NO: 5, when the base sequence of SEQ ID NO: 5 and the base sequence of SEQ ID NO: 7 are aligned, the base sequence of (B3) is obtained. Corresponding base sequence (B5) Of the base sequences of SEQ ID NO: 5, consecutive base sequences of 10 or more bases contained in the base sequences of positions 375 to 454 (B6) Of the base sequences of SEQ ID NO: 7, the sequence When the base sequence of No. 7 and the base sequence of SEQ ID NO: 5 are aligned, the base sequence corresponding to the base sequence of (B5) (B7) Of the base sequences of SEQ ID NO: 7, positions 288 to 367 (B8) Of the base sequences of SEQ ID NO: 5, when the base sequence of SEQ ID NO: 5 and the base sequence of SEQ ID NO: 7 are aligned, the above (B7) The base sequence corresponding to the base sequence of.

In the above (B1), the "base sequence at positions 250 to 329" is preferably the "base sequence at positions 260 to 319", and more preferably "base sequence at positions 270 to 309". It is a "base sequence", more preferably "base sequence at positions 270 to 304", and more preferably "base sequence at positions 275 to 299".

In the above (B3), the "base sequence at positions 224 to 303" is preferably the "base sequence at positions 234 to 293", and more preferably "base sequence at positions 244 to 283". It is a "base sequence", more preferably "base sequence at positions 244 to 278", and more preferably "base sequence at positions 249 to 273".

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), "continuous" preferably means "continuous from the 3'end".

The base sequence Bf may be the same as or homologous to the base sequence of (B1), the base sequence of (B2), the base sequence of (B3), or the base sequence of (B4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably The continuous base sequence of 19 bases or more is the same as the base sequence of (B1), (B2), (B3) or (B4), and the rest of the base sequence Bf is It is the same as or homologous to the base sequence of (B1), the base sequence of (B2), the base sequence of (B3), or the base sequence of (B4). The range of "homology" is as described later. Particularly preferably, the base sequence Bf is the same as the base sequence of (B1), the base sequence of (B2), the base sequence of (B3), or the base sequence of (B4).

The Glut3 forward primer may be one containing a polynucleotide containing the base sequence Bf at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Bf. The number of bases of the polynucleotide contained in the Glut3 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.

As a specific example of the base sequence of (B1), the base sequence of SEQ ID NO: 27 can be exemplified. As a specific example of the base sequence Bf, the base sequence of SEQ ID NO: 27 can be exemplified.

As a specific example of the base sequence of (B3), the base sequence of SEQ ID NO: 39 can be exemplified. As a specific example of the base sequence Bf, the base sequence of SEQ ID NO: 39 can be exemplified.

In the above (B5), the "base sequence at positions 375 to 454" is preferably the "base sequence at positions 385 to 444", and more preferably "base sequence at positions 395 to 434". It is a "base sequence", more preferably "base sequence at positions 400 to 434", and more preferably "base sequence at positions 405 to 429".

In the above (B7), the "base sequence at positions 288 to 367" is preferably the "base sequence at positions 298 to 357", and more preferably "base sequence at positions 308 to 347". It is a "base sequence", more preferably "base sequence at positions 313 to 347", and more preferably "base sequence at positions 318 to 342".

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), "continuous" preferably means "continuous from the 5'end".

The base sequence Br may be the same as or homologous to the base sequence of (B5), the base sequence of (B6), the base sequence of (B7), or the complementary base sequence of the base sequence of (B8), but the base From the 3'end of the sequence Br, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (B5), the base sequence of (B6), the base sequence of (B7), or the complementary base sequence of the base sequence of (B8). The rest of the base sequence Br is the same as or homologous to the base sequence of (B5), the base sequence of (B6), the base sequence of (B7), or the complementary base sequence of the base sequence of (B8). The range of "homology" is as described later. Particularly preferably, the base sequence Br is the same as the base sequence of (B5), the base sequence of (B6), the base sequence of (B7), or the complementary base sequence of the base sequence of (B8).

The Glut3 reverse primer may be one containing a polynucleotide containing the base sequence Br at the 3'end, and may further contain another base sequence on the 5'end 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.

As a specific example of the base sequence of (B5), the complementary base sequence of the base sequence of SEQ ID NO: 28 can be exemplified. As a specific example of the base sequence Br, the base sequence of SEQ ID NO: 28 can be exemplified.

As a specific example of the base sequence of (B7), the complementary base sequence of the base sequence of SEQ ID NO: 40 can be exemplified. As a specific example of the base sequence Br, the base sequence of SEQ ID NO: 40 can be exemplified.

As an example of the primer pair, 3 is a primer pair for amplifying a nucleic acid containing the MCT4 gene, which has the same or homologous base sequence Cf as any of the base sequences shown in (C1) to (C4) below. 'The MCT4 forward primer contained at the end and the MCT4 reverse primer containing the same or homologous base sequence Cr as the complementary base sequence of any of the base sequences shown in (C5) to (C8) below at the 3'end. An example includes an MCT4 primer pair containing.
(C1) Consecutive base sequence of 10 or more bases contained in the base sequence of positions 40 to 118 of the base sequence of SEQ ID NO: 9 (C2) Base of SEQ ID NO: 11 among the base sequences of SEQ ID NO: 11. When the sequence and the base sequence of SEQ ID NO: 9 are aligned, the base sequence corresponding to the base sequence of (C1) is changed to the base sequence of positions 146 to 225 among the base sequences of (C3) SEQ ID NO: 11. Consecutive base sequence of 10 or more bases (C4) Of the base sequences of SEQ ID NO: 9, when the base sequence of SEQ ID NO: 9 and the base sequence of SEQ ID NO: 11 are aligned, the base sequence of (C3) is obtained. Corresponding base sequence (C5) Of the base sequences of SEQ ID NO: 9, consecutive base sequences of 10 or more bases included in the base sequences of positions 107 to 186 (C6) Of the base sequences of SEQ ID NO: 11, the sequence When the base sequence of No. 11 and the base sequence of SEQ ID NO: 9 are aligned, the base sequence corresponding to the base sequence of (C5) (C7) Of the base sequences of SEQ ID NO: 11, positions 225 to 304 (C8) Of the base sequences of SEQ ID NO: 9, when the base sequence of SEQ ID NO: 9 and the base sequence of SEQ ID NO: 11 are aligned, the above (C7) The base sequence corresponding to the base sequence of.

In the above (C1), the "base sequence at positions 40 to 118" is preferably the "base sequence at positions 50 to 108", and more preferably "base sequence at positions 60 to 98". It is a "base sequence", more preferably a "base sequence at positions 60 to 93", and more preferably a "base sequence at positions 65 to 88".

In the above (C3), the "base sequence at positions 146 to 225" is preferably the "base sequence at positions 156 to 215", and more preferably "base sequence at positions 166 to 205". It is a "base sequence", more preferably "base sequence at positions 166 to 200", and more preferably "base sequence at positions 171 to 195".

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), "continuous" preferably means "continuous from the 3'end".

The base sequence Cf may be the same as or homologous to the base sequence of (C1), the base sequence of (C2), the base sequence of (C3), or the base sequence of (C4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably The continuous base sequence of 19 bases or more is the same as the base sequence of (C1), (C2), (C3) or (C4), and the rest of the base sequence Cf is It is the same as or homologous to the base sequence of (C1), the base sequence of (C2), the base sequence of (C3), or the base sequence of (C4). The range of "homology" is as described later. Particularly preferably, the base sequence Cf is the same as the base sequence of (C1), the base sequence of (C2), the base sequence of (C3), or the base sequence of (C4).

The MCT4 forward primer may be one containing 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.

As a specific example of the base sequence of (C1), the base sequence of SEQ ID NO: 29 can be exemplified. As a specific example of the base sequence Cf, the base sequence of SEQ ID NO: 29 can be exemplified.

As a specific example of the base sequence of (C3), the base sequence of SEQ ID NO: 41 can be exemplified. As a specific example of the base sequence Cf, the base sequence of SEQ ID NO: 41 can be exemplified.

In the above (C5), the "base sequence at positions 107 to 186" is preferably the "base sequence at positions 117 to 176", and more preferably "base sequence at positions 127 to 166". It is a "base sequence", more preferably "base sequence at positions 132 to 166", and more preferably "base sequence at positions 137 to 161".

In the above (C7), the "base sequence at positions 225 to 304" is preferably the "base sequence at positions 235 to 294", and more preferably "base sequence at positions 245 to 284". It is a "base sequence", more preferably "a base sequence at positions 250 to 284", and more preferably "a base sequence at positions 255 to 279".

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), "continuous" preferably means "continuous from the 5'end".

The base sequence Cr may be the same as or homologous to the base sequence of (C5), the base sequence of (C6), the base sequence of (C7), or the complementary base sequence of the base sequence of (C8). From the 3'end of the sequence Cr, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (C5), the base sequence of (C6), the base sequence of (C7), or the complementary base sequence of the base sequence of (C8). The rest of the base sequence Cr is the same as or homologous to the base sequence of (C5), the base sequence of (C6), the base sequence of (C7), or the complementary base sequence of the base sequence of (C8). The range of "homology" is as described later. Particularly preferably, the base sequence Cr is the same as the base sequence of (C5), the base sequence of (C6), the base sequence of (C7), or the complementary base sequence of the base sequence of (C8).

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.

As a specific example of the base sequence of (C5), the complementary base sequence of the base sequence of SEQ ID NO: 30 can be exemplified. As a specific example of the base sequence Cr, the base sequence of SEQ ID NO: 30 can be exemplified.

As a specific example of the base sequence of (C7), the complementary base sequence of the base sequence of SEQ ID NO: 42 can be exemplified. As a specific example of the base sequence Cr, the base sequence of SEQ ID NO: 42 can be exemplified.

As an example of the primer pair, 3 is a primer pair for amplifying a nucleic acid containing a PHD3 gene, which has the same or homologous base sequence Df as any of the base sequences shown in (D1) to (D4) below. 'The PHD3 forward primer contained at the end and the PHD3 reverse primer containing the same or homologous base sequence Dr as the complementary base sequence of any of the base sequences shown in (D5) to (D8) below at the 3'end. An example includes a pair of PHD3 primers.
(D1) Consecutive 10 or more base sequences contained in the base sequences of positions 864 to 943 among the base sequences of SEQ ID NO: 13 (D2) Bases of SEQ ID NO: 15 among the base sequences of SEQ ID NO: 15. When the sequence and the base sequence of SEQ ID NO: 13 are aligned, the base sequence corresponding to the base sequence of (D1) is changed to the base sequence of positions 863 to 942 among the base sequences of (D3) SEQ ID NO: 15. Consecutive base sequence of 10 or more bases (D4) Among the base sequences of SEQ ID NO: 13, when the base sequence of SEQ ID NO: 13 and the base sequence of SEQ ID NO: 15 are aligned, the base sequence of (D3) is obtained. Corresponding base sequence (D5) Of the base sequences of SEQ ID NO: 13, consecutive base sequences of 10 or more bases included in the base sequences of positions 991 to 1072 (D6) Of the base sequences of SEQ ID NO: 15, the sequence When the base sequence of No. 15 and the base sequence of SEQ ID NO: 13 are aligned, the base sequence corresponding to the base sequence of (D5) (D7) Of the base sequences of SEQ ID NO: 15, positions 1004 to 1083 (D8) Of the base sequences of SEQ ID NO: 13, when the base sequence of SEQ ID NO: 13 and the base sequence of SEQ ID NO: 15 are aligned, the above (D7) The base sequence corresponding to the base sequence of.

In the above (D1), the "base sequence at positions 864 to 943" is preferably the "base sequence at positions 874 to 933", and more preferably "base sequence at positions 884 to 923". It is a "base sequence", more preferably "base sequence at positions 884 to 918", and more preferably "base sequence at positions 889 to 913".

In the above (D3), the "base sequence at positions 863 to 942" is preferably the "base sequence at positions 873 to 932", and more preferably "base sequence at positions 883 to 922". It is a "base sequence", more preferably "base sequence at positions 883 to 917", and more preferably "base sequence at positions 888 to 912".

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), "continuous" preferably means "continuous from the 3'end".

The base sequence Df may be the same as or homologous to the base sequence of (D1), the base sequence of (D2), the base sequence of (D3), or the base sequence of (D4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably The continuous base sequence of 19 bases or more is the same as the base sequence of (D1), (D2), (D3) or (D4), and the rest of the base sequence Df is It is the same as or homologous to the base sequence of (D1), the base sequence of (D2), the base sequence of (D3), or the base sequence of (D4). The range of "homology" is as described later. Particularly preferably, the base sequence Df is the same as the base sequence of (D1), the base sequence of (D2), the base sequence of (D3), or the base sequence of (D4).

The PHD3 forward primer may be one containing 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.

As a specific example of the base sequence of (D1), the base sequence of SEQ ID NO: 31 can be exemplified. As a specific example of the base sequence Df, the base sequence of SEQ ID NO: 31 can be exemplified.

As a specific example of the base sequence of (D3), the base sequence of SEQ ID NO: 43 can be exemplified. As a specific example of the base sequence Df, the base sequence of SEQ ID NO: 43 can be exemplified.

In the above (D5), the "base sequence at positions 991 to 1072" is preferably the "base sequence at positions 1001 to 1062", and more preferably "base sequence at positions 1011 to 1052". It is a "base sequence", more preferably "base sequence at positions 1016 to 1047", and more preferably "base sequence at positions 1021 to 1042".

In the above (D7), the "base sequence at positions 1004 to 1083" is preferably the "base sequence at positions 1014 to 1073", and more preferably "base sequence at positions 1024 to 1063". It is a "base sequence", more preferably "base sequence at positions 1029 to 1063", and more preferably "base sequence at positions 1034 to 1058".

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), "continuous" preferably means "continuous from the 5'end".

The base sequence Dr may be the same as or homologous to the base sequence of (D5), the base sequence of (D6), the base sequence of (D7), or the complementary base sequence of the base sequence of (D8). From the 3'end of the sequence Dr, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (D5), the base sequence of (D6), the base sequence of (D7), or the complementary base sequence of the base sequence of (D8). The rest of the base sequence Dr is the same as or homologous to the base sequence of (D5), the base sequence of (D6), the base sequence of (D7), or the complementary base sequence of the base sequence of (D8). The range of "homology" is as described later. Particularly preferably, the base sequence Dr is the same as the base sequence of (D5), the base sequence of (D6), the base sequence of (D7), or the complementary base sequence of the base sequence of (D8).

The PHD3 reverse primer may be one containing a polynucleotide containing the base sequence Dr at the 3'end, and may further contain another base sequence on 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.

As a specific example of the base sequence of (D5), the complementary base sequence of the base sequence of SEQ ID NO: 32 can be exemplified. As a specific example of the base sequence Dr, the base sequence of SEQ ID NO: 32 can be exemplified.

As a specific example of the base sequence of (D7), the complementary base sequence of the base sequence of SEQ ID NO: 44 can be exemplified. As a specific example of the base sequence Dr, the base sequence of SEQ ID NO: 44 can be exemplified.

As an example of the primer pair, 3 is a primer pair for amplifying a nucleic acid containing a PDK1 gene, which has the same or homologous base sequence Ef as any of the base sequences shown in (E1) to (E4) below. 'The PDK1 forward primer contained at the end and the PDK1 reverse primer containing the same or homologous base sequence Er as the complementary base sequence of any of the base sequences shown in (E5) to (E8) below at the 3'end. An example includes a PDK1 primer pair containing.
(E1) Consecutive 10 or more base sequences contained in the base sequences of positions 445 to 528 of the base sequence of SEQ ID NO: 17 (E2) Base of SEQ ID NO: 19 among the base sequences of SEQ ID NO: 19. When the sequence and the base sequence of SEQ ID NO: 17 are aligned, the base sequence corresponding to the base sequence of (E1) (E3) is the base sequence of positions 981 to 1060 among the base sequences of SEQ ID NO: 19. Consecutive base sequence of 10 or more bases (E4) Among the base sequences of SEQ ID NO: 17, when the base sequence of SEQ ID NO: 17 and the base sequence of SEQ ID NO: 19 are aligned, the base sequence of (E3) is obtained. Corresponding base sequence (E5) Of the base sequences of SEQ ID NO: 17, consecutive base sequences of 10 or more bases included in the base sequences of positions 545 to 624 (E6) Of the base sequences of SEQ ID NO: 19, the sequence When the base sequence of No. 19 and the base sequence of SEQ ID NO: 17 are aligned, the base sequence corresponding to the base sequence of (E5) (E7) Of the base sequences of SEQ ID NO: 19, positions 1078 to 1157 (E8) Of the base sequences of SEQ ID NO: 17, when the base sequence of SEQ ID NO: 17 and the base sequence of SEQ ID NO: 19 are aligned, the above (E7) The base sequence corresponding to the base sequence of.

In the above (E1), the "base sequence at positions 445 to 528" is preferably the "base sequence at positions 455 to 518", and more preferably "base sequence at positions 465 to 508". It is a "base sequence", more preferably "base sequence at positions 465 to 503", and more preferably "base sequence at positions 470 to 498".

In the above (E3), the "base sequence at positions 981 to 1060" is preferably the "base sequence at positions 991 to 1050", and more preferably "base sequence at positions 1001 to 1040". It is a "base sequence", more preferably "base sequence at positions 1001 to 1035", and more preferably "base sequence at positions 1006 to 1030".

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), "continuous" preferably means "continuous from the 3'end".

The base sequence Ef may be the same as or homologous to the base sequence of (E1), the base sequence of (E2), the base sequence of (E3), or the base sequence of (E4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably The continuous base sequence of 19 bases or more is the same as the base sequence of (E1), (E2), (E3) or (E4), and the rest of the base sequence Ef is It is the same as or homologous to the base sequence of (E1), the base sequence of (E2), the base sequence of (E3), or the base sequence of (E4). The range of "homology" is as described later. Particularly preferably, the base sequence Ef is the same as the base sequence of (E1), the base sequence of (E2), the base sequence of (E3), or the base sequence of (E4).

The PDK1 forward primer may be one containing a polynucleotide containing the base sequence Ef at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Ef. The number of bases of the polynucleotide contained in the PDK1 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.

As a specific example of the base sequence of (E1), the base sequence of SEQ ID NO: 33 can be exemplified. As a specific example of the base sequence Ef, the base sequence of SEQ ID NO: 33 can be exemplified.

As a specific example of the base sequence of (E3), the base sequence of SEQ ID NO: 45 can be exemplified. As a specific example of the base sequence Ef, the base sequence of SEQ ID NO: 45 can be exemplified.

In the above (E5), the "base sequence at positions 545 to 624" is preferably the "base sequence at positions 555 to 614", and more preferably "base sequence at positions 565 to 604". It is a "base sequence", more preferably "base sequence at positions 570 to 604", and more preferably "base sequence at positions 575 to 599".

In the above (E7), the "base sequence at positions 1078 to 1157" is preferably the "base sequence at positions 1088 to 1147", and more preferably "base sequence at positions 1098 to 1137". It is a "base sequence", more preferably "base sequence at positions 1103 to 1137", and more preferably "base sequence at positions 1108 to 1132".

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), "continuous" preferably means "continuous from the 5'end".

The base sequence Er may be the same as or homologous to the base sequence of (E5), the base sequence of (E6), the base sequence of (E7), or the complementary base sequence of the base sequence of (E8), but the base From the 3'end of the sequence Er, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (E5), the base sequence of (E6), the base sequence of (E7), or the complementary base sequence of the base sequence of (E8). The rest of the base sequence Er is the same as or homologous to the base sequence of (E5), the base sequence of (E6), the base sequence of (E7), or the complementary base sequence of the base sequence of (E8). The range of "homology" is as described later. Particularly preferably, the base sequence Er is the same as the base sequence of (E5), the base sequence of (E6), the base sequence of (E7), or the complementary base sequence of the base sequence of (E8).

The PDK1 reverse primer may be one containing a polynucleotide containing the base sequence Er at the 3'end, and may further contain another base sequence on the 5'end 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.

As a specific example of the base sequence of (E5), the complementary base sequence of the base sequence of SEQ ID NO: 34 can be exemplified. As a specific example of the base sequence Er, the base sequence of SEQ ID NO: 34 can be exemplified.

As a specific example of the base sequence of (E7), the complementary base sequence of the base sequence of SEQ ID NO: 46 can be exemplified. As a specific example of the base sequence Er, the base sequence of SEQ ID NO: 46 can be exemplified.

The kit can include, as a primer pair, a primer pair for amplifying the nucleic acid of the endogenous control gene. As an example of such a primer pair, it is a primer pair for amplifying a nucleic acid encoding 18S ribosome RNA, and has the same or homologous base as any of the base sequences shown in (F1) to (F4) below. The 18S ribosome RNA forward primer containing the sequence Ff at the 3'end and the base sequence Fr which is the same as or homologous to the complementary base sequence of any of the base sequences shown in (F5) to (F8) below are contained at the 3'end. 18S ribosome RNA reverse primers and 18S ribosome RNA primer pairs including the 18S ribosome RNA primer pair can be exemplified.
(F1) Consecutive base sequence of 10 or more bases contained in the base sequences of positions 1216 to 1297 of the base sequence of SEQ ID NO: 21 (F2) Base of SEQ ID NO: 22 among the base sequences of SEQ ID NO: 22 When the sequence and the base sequence of SEQ ID NO: 21 are aligned, the base sequence corresponding to the base sequence of (F1) is changed to the base sequence of positions 1216 to 1297 among the base sequences of (F3) SEQ ID NO: 22. Consecutive base sequence of 10 or more bases (F4) Of the base sequences of SEQ ID NO: 21, when the base sequence of SEQ ID NO: 21 and the base sequence of SEQ ID NO: 22 are aligned, the base sequence of (F3) is obtained. Corresponding base sequence (F5) Of the base sequences of SEQ ID NO: 21, consecutive base sequences of 10 or more bases included in the base sequences of positions 1317 to 1396 (F6) Of the base sequences of SEQ ID NO: 22, the sequence When the base sequence of No. 22 and the base sequence of SEQ ID NO: 21 are aligned, the base sequence corresponding to the base sequence of (F5) (F7) Of the base sequences of SEQ ID NO: 21, positions 1317 to 1396 (F8) Of the base sequences of SEQ ID NO: 21, when the base sequence of SEQ ID NO: 21 and the base sequence of SEQ ID NO: 22 are aligned, the above (F7) The base sequence corresponding to the base sequence of.

In the above (F1), the "base sequence at positions 1216 to 1297" is preferably the "base sequence at positions 1226 to 1287", and more preferably "base sequence at positions 1236 to 1277". It is a "base sequence", more preferably "base sequence at positions 1241 to 1272", and more preferably "base sequence at positions 1241 to 1267".

In the above (F3), the "base sequence at positions 1216 to 1297" is preferably the "base sequence at positions 1226 to 1287", and more preferably "base sequence at positions 1236 to 1277". It is a "base sequence", more preferably "base sequence at positions 1236 to 1272", and more preferably "base sequence at positions 1241 to 1267".

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), "continuous" preferably means "continuous from the 3'end".

The base sequence Ff may be the same as or homologous to the base sequence of (F1), the base sequence of (F2), the base sequence of (F3), or the base sequence of (F4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably The continuous base sequence of 19 bases or more is the same as the base sequence of (F1), (F2), (F3) or (F4), and the rest of the base sequence Ff is It is the same as or homologous to the base sequence of (F1), the base sequence of (F2), the base sequence of (F3), or the base sequence of (F4). The range of "homology" is as described later. Particularly preferably, the base sequence Ff is the same as the base sequence of (F1), the base sequence of (F2), the base sequence of (F3), or the base sequence of (F4).

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.

As a specific example of the base sequence of (F1), the base sequence of SEQ ID NO: 23 can be exemplified. As a specific example of the base sequence Ff, the base sequence of SEQ ID NO: 23 can be exemplified.

As a specific example of the base sequence of (F3), the base sequence of SEQ ID NO: 35 can be exemplified. As a specific example of the base sequence Ff, the base sequence of SEQ ID NO: 35 can be exemplified.

In the above (F5), the "base sequence at positions 1317 to 1396" is preferably the "base sequence at positions 1327 to 1386", and more preferably "base sequence at positions 1337 to 1376". It is a "base sequence", more preferably "base sequence at positions 1342 to 1376", and more preferably "base sequence at positions 1347 to 1371".

In the above (F7), the "base sequence at positions 1317 to 1396" is preferably the "base sequence at positions 1327 to 1386", and more preferably "base sequence at positions 1337 to 1376". It is a "base sequence", more preferably "base sequence at positions 1342 to 1376", and more preferably "base sequence at positions 1347 to 1371".

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), "continuous" preferably means "continuous from the 5'end".

The base sequence Fr may be the same as or homologous to the base sequence of (F5), the base sequence of (F6), the base sequence of (F7), or the complementary base sequence of the base sequence of (F8), but the base From the 3'end of the sequence Fr, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (F5), the base sequence of (F6), the base sequence of (F7), or the complementary base sequence of the base sequence of (F8). The rest of the base sequence Fr is the same as or homologous to the base sequence of (F5), the base sequence of (F6), the base sequence of (F7), or the complementary base sequence of the base sequence of (F8). The range of "homology" is as described later. Particularly preferably, the base sequence Fr is the same as the base sequence of (F5), the base sequence of (F6), the base sequence of (F7), or the complementary base sequence of the base sequence of (F8).

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.

As a specific example of the base sequence of (F5), the complementary base sequence of the base sequence of SEQ ID NO: 24 can be exemplified. As a specific example of the base sequence Fr, the base sequence of SEQ ID NO: 24 can be exemplified.

As a specific example of the base sequence of (F7), the complementary base sequence of the base sequence of SEQ ID NO: 36 can be exemplified. As a specific example of the base sequence Fr, the base sequence of SEQ ID NO: 36 can be exemplified.

In the present specification, when "base sequence Y homologous to base sequence X" or "base sequence X and base sequence Y are homologous", a polynucleotide consisting of a complementary sequence of base sequence X and a base sequence As long as the polynucleotide consisting of Y is a combination capable of hybridizing under the annealing conditions of the nucleic acid amplification reaction and forming a hydrogen bond sufficient to form a stable double strand, the nucleotide sequences X and Y May be partially different. For example, a polynucleotide consisting of a complementary sequence of the base sequence X and a polynucleotide consisting of the base sequence Y have several mismatches such as 1 mismatch in 10 nucleotides, 1 mismatch in 20 nucleotides, or 1 mismatch in 30 nucleotides. There may be a mismatch. 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.

(A) The base sequence Y is a base sequence in which one or several bases are deleted, substituted, added and / or inserted in the base sequence X.
(B) The base sequence Y is a base sequence having 70% or more identity with the base sequence X.
(C) A polynucleotide having a base sequence Y can hybridize with a polynucleotide having a base sequence complementary to SEQ ID NO: X under stringent conditions.
(D) Thymine (T) at an arbitrary position in one of the base sequence X and the base sequence Y is replaced with uracil (U) in the other.

In the above (A), "1 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. It is one. In (A) above, "1 or several" refers to the total number of deleted, substituted, added and / or inserted bases.

In the above (B), the identity value indicates a value calculated with default settings using software (for example, FASTA, DNASIS, and BLAST) that calculates the identity between a plurality of base sequences. For the base sequence identity value, the number of matching bases when the pair of base sequences are aligned so as to maximize the degree of matching is calculated, and the total number of bases in the compared base sequence of the number of matching bases is calculated. Calculated as a percentage of the number. Here, when there is a gap, the total number of bases described above is the number of bases counted with one gap as one base. For details on the method of determining identity, see, for example, Altschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al, J. et al. Mol. Biol. See 215, 403-410, 1990.

In (B), 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. , More preferably 99% or more identity.

In the above (C), the “stringent condition” means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, Green and Sambrook, Molecular Cloning, 4th Ed (2012). , Cold Spring Harbor Laboratory Press can be appropriately determined. Specifically, stringent conditions can be set by the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution. More specifically, as stringent conditions, for example, in the hybridization step, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 40 to 68 ° C, preferably 40 to 65 ° C. More specifically, hybridization can be performed at 1-7 × SSC, 0.02-3% SDS, and a temperature of 40 ° C-60 ° C. Further, a washing step may be performed after hybridization, and the washing step can be performed, for example, at 0.1 to 2 × SSC, 0.1 to 0.3% SDS, and a temperature of 50 to 65 ° C.
One or both of the above primer pairs may be modified with another substance such as a labeling substance.

The probe is a nucleotide sequence of a nucleic acid containing the gene of one or more proteins, for example, the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 of the Glut1 gene (particularly, positions 131 to 262 of the nucleotide sequence of SEQ ID NO: 1). Nucleotide sequence at position; Of the nucleotide sequence of SEQ ID NO: 3, when the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 1 are aligned, the nucleotide sequence of SEQ ID NO: 1 is located at positions 131 to 262. The base sequence corresponding to the base sequence; the base sequence at positions 286 to 388 of the base sequence of SEQ ID NO: 3; or the base sequence of SEQ ID NO: 1 and the base of SEQ ID NO: 3 among the base sequences of SEQ ID NO: 1. When aligned with the sequence, the base sequence of SEQ ID NO: 5 or SEQ ID NO: 7 of the Glut3 gene (particularly, the base sequence), the base sequence corresponding to the base sequences of positions 286 to 388 of the base sequence of SEQ ID NO: 3. Nucleotide sequence of positions 280 to 424 of the base sequence of No. 5; When the base sequence of SEQ ID NO: 7 and the base sequence of SEQ ID NO: 5 are aligned among the base sequences of SEQ ID NO: 7, SEQ ID NO: 5 The base sequence corresponding to the base sequence of positions 280 to 424 of the base sequence of the above; the base sequence of positions 254 to 337 of the base sequence of SEQ ID NO: 7; or the base sequence of SEQ ID NO: 5 , When the base sequence of SEQ ID NO: 5 and the base sequence of SEQ ID NO: 7 are aligned, the base sequence corresponding to the base sequences of positions 254 to 337 of the base sequence of SEQ ID NO: 7), the sequence of the MCT4 gene The base sequence of No. 9 or SEQ ID NO: 11 (particularly, the base sequence of positions 70 to 156 of the base sequence of SEQ ID NO: 9; the base sequence of SEQ ID NO: 11 and the base sequence of SEQ ID NO: 9 among the base sequences of SEQ ID NO: 11 When aligned with the base sequence of SEQ ID NO: 9, the base sequence corresponding to the base sequence of positions 70 to 156 of the base sequence of SEQ ID NO: 9; Base sequence; Alternatively, when the base sequence of SEQ ID NO: 9 and the base sequence of SEQ ID NO: 11 are aligned among the base sequences of SEQ ID NO: 9, positions 176 to 274 of the base sequence of SEQ ID NO: 11 The base sequence corresponding to the base sequence), 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 of the base sequence of SEQ ID NO: 13; the base sequence of SEQ ID NO: 15 Of these, when the base sequence of SEQ ID NO: 15 and the base sequence of SEQ ID NO: 13 are aligned, the base sequence of positions 894 to 1042 of the base sequence of SEQ ID NO: 3 The base sequence corresponding to; the base sequence at positions 893 to 1053 of the base sequence of SEQ ID NO: 15; or the base sequence of SEQ ID NO: 13 and the base sequence of SEQ ID NO: 15 among the base sequences of SEQ ID NO: 13. When aligned, the base sequence of SEQ ID NO: 15 (base sequence corresponding to the base sequence of positions 893 to 1053) or the base sequence of SEQ ID NO: 17 or SEQ ID NO: 19 of the PDK1 gene (particularly, SEQ ID NO: Of the 17 base sequences, the base sequences of positions 475 to 594; among the base sequences of SEQ ID NO: 19, when the base sequence of SEQ ID NO: 19 and the base sequence of SEQ ID NO: 17 are aligned, the base sequence of SEQ ID NO: 17 The base sequence corresponding to the base sequence of positions 475 to 594 of the base sequence; the base sequence of positions 1011 to 1127 of the base sequence of SEQ ID NO: 19; or the base sequence of the base sequence of SEQ ID NO: 17 When the base sequence of SEQ ID NO: 17 and the base sequence of SEQ ID NO: 19 are aligned, 10 or more bases contained in the base sequence of positions 1011 to 1127 of the base sequence of SEQ ID NO: 19) It can contain a nucleic acid fragment containing a continuous partial base sequence of 15 bases or more, more preferably 17 bases or more, more preferably 20 bases or more, 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 bases or less, 40 bases or less, or 30 bases or less. The probe may be one in which the nucleic acid fragment is modified with another substance such as a labeling substance.

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

The reaction substrate for measuring the enzyme activity of the one or more proteins can be appropriately selected according to the activity of the protein to be measured.

The kit according to the above embodiment may further include a buffer solution for dilution or reaction containing components necessary for measurement, a washing solution, a coloring reagent, a reaction vessel, and the like.

The kit according to the embodiment preferably further contains a reagent and / or a column for separating mononuclear cells from the sample. Examples of the reagent and / or column include Ficoll-Paque PLUS (manufactured by GE Healthcare), Lymphoprep (manufactured by Abbott Laboratories Technologies), and Human Peripheral Blood Monologic Laboratory (manufactured by Human Biological Blood Mononulus Technology). Registered trademark) CPT mononuclear cell separation blood collection tube (manufactured by Becton Dickinson), purriMate (manufactured by purriSelect), SepMate (manufactured by STEMCELL Technologies) and the like can be mentioned. The mononuclear cells and granulocytes in the sample can be separated, and a mononuclear cell-containing sample for measurement can be efficiently obtained.

<5. Kit for determining the degree of aging in a subject>
Another embodiment of the present invention
A kit for determining the degree of aging in a subject.
The present invention relates to a kit containing a reagent for measuring gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.

The determination of the degree of the aging phenomenon is preferably a determination of age, a determination of the degree of aging phenomenon of brain tissue, a degree of aging phenomenon of peripheral blood, or a determination of the degree of aging phenomenon of bone marrow cells.

The bone marrow cells are preferably hematopoietic stem cells, mesenchymal stem cells, and immature cells.

As the reagent, a reagent that can be used in the above-mentioned method for measuring gene expression or enzyme activity of one or more proteins is preferable.

Specifically, a primer pair for amplifying a nucleic acid (genomic DNA, mRNA or a cDNA prepared based on mRNA) containing a gene of the one or more proteins to be measured, and the one or more proteins to be measured. A probe that hybridizes with a nucleic acid containing the gene (mRNA or a cDNA prepared based on mRNA), an antibody that specifically binds to the one or more proteins, and a reaction substrate for measuring the enzymatic activity of the one or more proteins. Etc. can be exemplified. The genomic DNA may also include an untranslated region, an intron region, a signal sequence region, and the like. Preferably, the nucleic acid is mRNA or cDNA. The mRNA or cDNA may contain 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 capable of amplifying at least a part of the base sequences of CDS and UTR. An example of the probe is a probe that can hybridize to at least a part of the base sequences of CDS and UTR.

The primer pair can be selected from the same range as the preferred example of the primer pair described in the above "4. Kit for determination of dementia or determination of brain function".

The probe can be selected from the same range as the preferred example of the blow part described in "4. Kit for determining dementia or determining brain function" above.

The present invention will be specifically described with reference to the following examples, but the present invention is not limited to the examples.

All animal experiments described below are approved by the Animal Experiment Review Committee of the Foundation for Biomedical Research and Innovation, Kobe, and comply with the basic guidelines for conducting animal experiments at research institutions, etc. established by the Ministry of Education, Culture, Sports, Science and Technology. It was carried out. Experiments and results will be reported according to the ARRIVE guidelines prepared by the UK 3Rs Center (NC3Rs).

1. 1. Comparative Example 1
<Method>
In Comparative Example 1, an index for determining dementia and brain function in a subject was examined using a body fluid sample derived from a healthy mouse.

1-1. Preparation of experimental animals Male CB-17 strain mice (Oriental Yeast Co., Ltd.) 5 weeks old or younger were subjected to the following experiments.

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.

1-2-1. Open field test The open field test uses the average number of mouse movements (the average value of the total number of horizontal movements and vertical movements) in the light and dark states as an index, and the responsiveness of the mouse to changes in light and dark. It is a behavioral test to evaluate. The open field test was carried out by the following method.

The mouse was placed in a transparent acrylic box (bottom surface: length 30 cm x width 30 cm). The mouse in the acrylic box was freely searched for 30 minutes with the white light turned on (bright state). The lights were then turned off and kept dark for 30 minutes. The number of horizontal movements of the mouse was evaluated by measuring the number of times the mouse blocked infrared beams provided at intervals of 10 cm along the vertical and horizontal axes at a height of 2 cm from the floor. The number of vertical movements (number of rises) of the mouse was evaluated by measuring the number of times the mouse blocked infrared beams provided at intervals of 3 cm along the vertical axis at a height of 5 cm from the floor. The test was carried out 9 times or more, and the average number of movements was calculated.

1-2-2. Wire hang test The wire hang test is a behavioral test that evaluates the muscle strength of a mouse by using the average value (average fall time) of the time until the mouse suspended from the wire net falls as an index. The wire hang test was carried out by the following method.

The mouse was placed on a wire mesh with an interval of 12 mm. Next, the mouse was turned over together with the wire mesh and hung at a height of 20 cm. The time until the mouse fell from the wire mesh (falling time) was recorded. The test was carried out 8 times or more, and the average value (average fall time) of those fall times was calculated.

1-2-3. Passive avoidance test In the passive avoidance test, learning and memory of mice that have been subjected to aversive stimuli in advance using the average value of the response latency (mean response latency) at the time of this test as an index. It is a behavioral test that evaluates the function of the mouse. The passive avoidance test was conducted by the following method.

The passive avoidance test consisted of a light room and a dark room separated by a plate with a hole through which the mouse could pass, and was conducted using a box with an electrical stimulator installed on the floor of the dark room. As a preliminary step, a mouse was placed in a bright room with white lighting, and the inside of the box was freely explored. Mice gradually entered the darkroom because they preferred darkrooms, but when they entered the darkroom, they were given a light electrical stimulus. The time the mouse stayed in the bright room (ie, the time it took to enter the dark room) was measured and recorded. Twenty-four hours later, as a main test, the mice were placed in the bright room again, and the time for the mice to stay in the bright room (that is, the time until entering the dark room) was measured again. The maximum time for staying in the bright room (time to enter the dark room) was 180 seconds. The reaction latency was calculated as the absolute value of the difference between the time spent in the bright room in this test and the time spent in the bright room in the preliminary stage. The test was carried out 5 times or more, and the average value of their reaction latency was calculated.

1-2-4. Open space swimming test The open space swimming test uses the average swimming distance (average swimming distance) of mice placed in a large pool of water as an index to measure the coping behavior and depressive tendency of mice. It is a behavioral test to evaluate. The open space swimming test was conducted by the following method.

A mouse was placed in a pool with a diameter of 120 cm and allowed to swim freely. The state of swimming was photographed with a video camera, and the swimming distance for 60 seconds was continuously analyzed for 10 minutes using image analysis software.

1-2-5. Rotarod test The Rotarod test is a behavioral test that evaluates the motor function and sense of balance of a mouse using the average value (average fall time) of the time it takes for the mouse to fall from the rotating rotor rod as an index. .. The rotor rod test was carried out by the following method.

The mouse was placed in the lane of the rotor rod that rotates 20 times per minute, and the time (fall time) until the mouse fell from the rotating rotor rod was measured. The test was carried out three times, and the average value (average fall time) of those fall times was calculated.

1-3. Quantitative RT-PCR
Approximately 1 mL of peripheral blood was collected from the mice subjected to the above behavioral test, and 0.4 mL of which was used as a body fluid sample to be analyzed in the following experiments. 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, 1 μg using a cDNA synthesis kit (PrimeScript (registered trademark) II 1st reverse cDNA Synthesis Kit (Takara Bio Inc./model number: 6210B (A × 4))) according to the manufacturer's protocol. CDNA was synthesized (reverse transcription) from the total RNA of. Using the obtained cDNA as a template, a real-time PCR reaction was carried out using a nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / model number: A25777)). As a real-time PCR system, Agilent AliaMx Real-Time PCR System (Agilent) was used. The real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C. for 30 seconds for 1 cycle. , And 95 ° C. for 30 seconds was defined as one cycle. The 18S ribosomal RNA gene, which is a housekeeping gene, was used as the endogenous control, and the relative expression level of each target gene with respect to the endogenous control was analyzed by the Pfaffl method. The Pfaffl method is a kind of relative quantification method of the comparative Ct method in which the PCR amplification efficiency of the housekeeping gene and the target gene is taken into consideration. Nucleic Acids Res. 2001; 29 (9): e45. The method described in can be used. The test was carried out 5 times or more, and the average value (average relative expression level) of their relative expression levels was calculated. Table 1 shows the base sequences of primers for each gene.

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

2. Example 1
<Method>
In Example 1, an index for determining dementia and brain function in a subject was examined using a body fluid sample derived from a mouse that developed dementia.

2-1. Preparation of experimental animals Male CB-17 strain mice (Oriental Yeast Co., Ltd.) 80 weeks old or older were subjected to the following experiments.

2-2. Behavior test Five behavior tests (open field test, wire hang test, passive avoidance test, open space swimming test and rotarod test) were carried out in the same manner as in "1-2. Behavior test" of Comparative Example 1.

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

<Result>
The results of the behavioral tests of Example 1 and Comparative Example 1 are shown below.
In the open field test, the average number of movements of the mouse of Comparative Example 1 in the bright state 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 of the mouse in Example 1 in the bright state 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 of light and darkness was lower than that of the mouse of Comparative Example 1.

In the wire hang test, the average fall time of the mouse of Comparative Example 1 was 84.9 ± 41.1 seconds, whereas the average fall time of the mouse of Example 1 was 25.6 ± 22.9 seconds. rice field. Therefore, it was suggested that the muscle strength of the mouse of Example 1 was lower than that of the mouse of Comparative Example 1.

In the passive avoidance test, the average value of the time spent in the bright room in the pre-stage of the mouse of Comparative Example 1 was 31.5 ± 22.9 seconds, whereas the average value of the time spent in the pre-stage of the mouse of Example 1 was 31.5 ± 22.9 seconds. The average time spent in the room was 20.0 ± 14.1 seconds. In addition, the average value of the time spent in the bright room of the mouse of Comparative Example 1 in this test was 180.0 ± 0.0 seconds, whereas the mouse of Example 1 stayed in the bright room in the preliminary stage. The average time was 47.8 ± 67.4 seconds. Therefore, the average response latency of the mouse of Comparative Example 1 was calculated to be 148.5 ± 22.9 seconds, and the average response latency of the mouse of Example 1 was calculated to be 32.8 ± 63.5 seconds. From the above, the mouse of Comparative Example 1 was able to learn and memorize the invasion into the dark room and the fear of aversive stimulus in association with each other, whereas the mouse of Example 1 had a deteriorated learning and memory function. It has been suggested.

In the open space swimming test, the average swimming distance of the mouse of Comparative Example 1 was 794 ± 192 cm, whereas the average swimming distance of the mouse of Example 1 was 1193 ± 154 cm. Compared to the mouse of Comparative Example 1, the mouse of Example 1 floats on the water in an immobile posture (that is, without struggling, making only the minimum movement necessary to get the head out of the water surface. There was a tendency to shorten the time to indicate the condition), and there was a tendency to swim around more than necessary. From the above, it was suggested that the coping behavior of the mouse of Example 1 was attenuated as compared with the mouse of Comparative Example 1 and showed a depressive tendency.

In the rotor rod test, the average fall time of the mouse of Comparative Example 1 was 41.6 ± 25.2 seconds, whereas the average fall time of the mouse of Example 1 was 6.8 ± 4.5 seconds. rice field. Therefore, it was suggested that the motor function and the sense of balance of the mouse of Example 1 were lower than those of the mouse of Comparative Example 1.

From the results of the above behavioral tests, it was determined that the mouse of Comparative Example 1 did not develop dementia and the brain function did not deteriorate, whereas the mouse of Example 1 developed dementia. And / or brain function was determined to be impaired.
The results of quantitative RT-PCR of Comparative Example 1 and Example 1 are shown below.

1 and 2 show the average value (mean) of the relative expression level of each target gene with respect to the expression level of the 18S ribosomal RNA gene calculated by the Pfaffl method in the body fluid samples of the mice of Comparative Example 1 and Example 1. Relative expression level) is shown.

As shown in FIGS. 1 and 2, in the mouse body fluid sample of Comparative Example 1, the average relative expression of the Glut1 gene was 0.56, the average relative expression of the Glut3 gene was 0.67, and the average relative expression of the MCT4 gene. The amount 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 mouse body fluid sample 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, and the PHD3 gene. The average relative expression level of PDK1 gene was 2.33, and the average relative expression level of PDK1 gene was 3.03. Compared with the mouse of Comparative Example 1, the gene expression of the Glut1 gene, the Glut3 gene, the MCT4 gene, the PHD3 gene and the PDK1 gene in the body fluid sample of the mouse of Example 1 was significantly increased. Therefore, as genes whose expression levels change remarkably with the onset of dementia and / or deterioration of brain function, genes of proteins involved in glucose transport (Glut1 gene and Glut3 gene) and genes of proteins involved in lactic acid transport. (MCT4 gene), a gene for a protein involved in the glycolytic system (PHD3 gene), and a gene for a protein involved in the TCA circuit (PDK1 gene) were identified.

Therefore, the possibility of using the above gene and / or the protein encoded by the above gene as an index in determining the onset of dementia and / or the decrease in brain function of the subject was shown. In addition, the possibility of using a gene for a protein involved in energy metabolism and / or a protein involved in energy metabolism as an index for determining the onset of dementia and / or a decrease in brain function of a subject was shown. ..

In addition, the average relative expression level of each target gene in the body fluid sample of the mouse of Example 1 was higher than each reference value shown in the following (a) to (e), and each of them in the mouse of Comparative Example 1 It was found that the average relative expression level of the target gene of (a) to (e) below was lower than the respective reference values shown in (a) to (e) below.
(A) Reference value of gene expression of Glut1 gene: 1.30
(B) Reference value of gene expression of Glut3 gene: 1.30
(C) Reference value of gene expression of MCT4 gene: 1.40
(D) Reference value of gene expression of PHD3 gene: 1.20
(E) Reference value for gene expression of PDK1 gene: 1.30

In the body fluid sample of the mouse of Example 1, the ratio of the average relative expression level of the Glut1 gene to the above reference value was 2.6. In the body fluid sample of the mouse of Example 1, the ratio of the average relative expression level of the Glut3 gene to the above reference value was 1.3. In the body fluid sample of the mouse of Example 1, the ratio of the average relative expression level of the MCT4 gene to the above reference value was 2.3. In the body fluid sample of the mouse of Example 1, the ratio of the average relative expression level of the PHD3 gene to the above reference value was 1.9. In the body fluid sample of the mouse of Example 1, the ratio of the average relative expression level of the PDK1 gene to the above reference value was 2.3.

Therefore, by using the relative expression level of one or more of the above genes as an index higher than the above reference value, it is possible to obtain an index for determining dementia or brain function in a subject. That is, by measuring the relative expression level of one or more of the above genes in the body fluid sample derived from the subject and comparing the relative expression level with the reference value, the determination of dementia in the subject or the brain function can be determined. An index for judgment can be obtained.

In addition, the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction in the sample derived from the subject is measured, and the measured values of the gene expression or enzyme activity of the one or more proteins are used as reference values. By comparing, an index for determining dementia or determining brain function in a subject can be obtained.

3. 3. Example 2
<Method>
In Example 2, an index for determining the degree of the aging phenomenon in the subject was examined using a body fluid sample derived from a plurality of mice having a known degree of the aging phenomenon.

3-1. Preparation of experimental animals 1, 5, 10, 52, 104-week-old male CB-17 strain mice (Oriental Yeast Co., Ltd.) were subjected to the following experiments.

3-2. Quantitative RT-PCR
Approximately 1 mL of peripheral blood was collected from the above mice, of which 0.4 mL was used as a body fluid sample to be analyzed in the following experiments. 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, 1 μg using a cDNA synthesis kit (PrimeScript (registered trademark) II 1st reverse cDNA Synthesis Kit (Takara Bio Inc./model number: 6210B (A × 4))) according to the manufacturer's protocol. CDNA was synthesized (reverse transcription) from the total RNA of. Using the obtained cDNA as a template, a real-time PCR reaction was carried out using a nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / model number: A25777)). As a real-time PCR system, Agilent AliaMx Real-Time PCR System (Agilent) was used. The real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C. for 30 seconds for 1 cycle. , And 95 ° C. for 30 seconds was defined as one cycle. The 18S ribosomal RNA gene, which is a housekeeping gene, was used as the endogenous control, and the relative expression level of each target gene with respect to the endogenous control was analyzed by the Pfaffl method. The Pfaffl method is a kind of relative quantification method of the comparative Ct method in which the PCR amplification efficiency of the housekeeping gene and the target gene is taken into consideration. Nucleic Acids Res. 2001; 29 (9): e45. The method described in can be used. The test was carried out 5 times or more, and the average value (average relative expression level) of their relative expression levels was calculated. Table 3 shows the base sequences of primers for each gene.

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

<Result>
The results of quantitative RT-PCR of Example 2 are shown below.

2 and 4 show the average value (average relative expression level) of the relative expression level of each target gene with respect to the expression level of the 18S ribosomal RNA gene calculated by the Pfaffl method in the body fluid sample of each mouse of Example 2. Is shown.

As shown in FIGS. 2 and 4, in the body fluid sample of 1-week-old mice, the average relative expression of the Glut1 gene was 0.42, the average relative expression of the Glut3 gene was 0.27, and the average relative expression of the MCT4 gene. The amount was 0.20, the average relative expression level of the PHD3 gene was 0.35, and the average relative expression level of the PDK1 gene was 0.28. In the body fluid sample of 5-week-old mice, the average relative expression of the Glut1 gene was 0.68, the average relative expression of the Glut3 gene was 0.81, the average relative expression of the MCT4 gene was 0.54, and the average of the PHD3 gene. The relative expression level was 0.62, and the average relative expression level of the PDK1 gene was 0.83. In the body fluid sample of 10-week-old mice, the average relative expression of the Glut1 gene was 1.16, the average relative expression of the Glut3 gene was 1.18, the average relative expression of the MCT4 gene was 1.22, and the average of the PHD3 gene. The relative expression level was 1.12, and the average relative expression level of the PDK1 gene was 1.17. In the body fluid sample of 52-week-old mice, the average relative expression of the Glut1 gene was 1.47, the average relative expression of the Glut3 gene was 1.83, the average relative expression of the MCT4 gene was 2.47, and the average of the PHD3 gene. The relative expression level was 1.74, and the average relative expression level of the PDK1 gene was 1.71. In the body fluid sample of 104-week-old mice, the average relative expression of the Glut1 gene was 1.99, the average relative expression of the Glut3 gene was 1.96, the average relative expression of the MCT4 gene was 2.82, and the average of the PHD3 gene. The relative expression level was 2.35, and the average relative expression level of the PDK1 gene was 1.96. With the increase in age, the gene expression of the Glut1, Glut3, MCT4, PHD3 and PDK1 genes in the body fluid sample of the mouse was significantly increased. Therefore, as genes whose expression level changes remarkably as the degree of aging phenomenon increases, the gene of the protein involved in glucose transport (Glut1 gene and Glut3 gene) and the gene of the gene involved in lactic acid transport (MCT4 gene) are solved. A gene for a protein involved in the sugar system (PHD3 gene) and a gene for a protein involved in the TCA circuit (PDK1 gene) were identified.

Therefore, the possibility of using the above gene and / or the protein encoded by the above gene as an index for determining the degree of aging phenomenon in the subject was shown. In addition, the possibility of using the gene of the protein involved in energy metabolism and / or the protein involved in energy metabolism as an index for determining the degree of the aging phenomenon in the subject was shown.

FIG. 3 shows the relationship between the average relative expression level of each target gene in the body fluid sample of the mouse of Example 2 and the age of the mouse. As shown in FIG. 3, the relational expression between the average relative expression level of each target gene in the body fluid sample of the mouse of Example 2 and the age of the mouse is represented by the following relational expressions (A) to (E). Was done.
(A) Relational expression between the average relative expression level of the Glut1 gene and the age of the week: y = 0.3257 × ln (x) + 0.3316
(B) Relational expression between the average relative expression level of the Glut3 gene and the age of the week: y = 0.3788 × ln (x) + 0.2658
(C) Relational expression between the average relative expression level of the MCT4 gene and the age of the week: y = 0.6078 × ln (x) −0.0663
(D) Relationship between the average relative expression level of the PHD3 gene and the age of the week: y = 0.4283 × ln (x) + 0.1677
(E) Relationship between the average relative expression level of the PDK1 gene and the age of the week: y = 0.3626 × ln (x) +0.2848
Here, ln represents the natural logarithm, x represents the age of the mouse, and y represents the average relative expression level of each target gene in the body fluid sample of the mouse.

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

In addition, by measuring the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction in the sample derived from the subject, it is possible to obtain an index for determining the degree of the aging phenomenon in the subject. can.

4. Example 3
<Manufacturing of dementia / brain function judgment kit>
A kit for determining dementia or determining brain function (dementia / brain function determination kit) is manufactured by the following procedure.

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

<Use of dementia / brain function judgment kit>
The dementia / brain function determination kit can be used by the following procedure.

Peripheral blood is collected from the mouse that is the subject, and a part of it 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 determination kit. Using the obtained total RNA as a template, cDNA is synthesized using the cDNA synthesis reagent constituting the dementia / brain function determination kit. Using the obtained cDNA as a template, a real-time PCR reaction is performed using a primer pair solution for Glut1 gene amplification, a primer pair solution for 18S ribosomal RNA gene amplification, and a nucleic acid amplification reagent that constitute the dementia / brain function determination kit. The real-time PCR system uses Agilent AliaMx Real-Time PCR System (Agilent). The real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C. for 30 seconds for 1 cycle. , And 95 ° C. for 30 seconds as one cycle. The relative expression level of the Glut1 gene with respect to the 18S ribosomal RNA gene is analyzed by the Pfaffl method. By comparing the relative expression level of the Glut1 gene with respect to the 18S ribosomal RNA gene with 1.30, which is the reference value of the gene expression of the Glut1 gene, the presence or absence of dementia in mice can be determined.

5. Example 4
<Manufacturing of aging degree judgment kit>
A kit for determining the degree of the aging phenomenon (aging degree determination kit) is manufactured by the following procedure.

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

<Use of aging degree judgment kit>
The aging degree determination kit can be used by the following procedure.
Peripheral blood is obtained from multiple mice of known age and some of them are used as body fluid samples for standard preparation. Total RNA is extracted from the analysis sample using the RNA extraction reagent constituting the aging degree determination kit. Using the obtained total RNA as a template, cDNA is synthesized using the cDNA synthesis reagent constituting the aging degree determination kit. Using the obtained cDNA as a template, a real-time PCR reaction is carried out using a primer pair solution for Glut3 gene amplification, a primer pair solution for 18S ribosomal RNA gene amplification, and a nucleic acid amplification reagent that constitute the aging degree determination kit. The real-time PCR system uses the Agilent AriaMx Real-Time PCR System (Agilent). The real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C. for 30 seconds for 1 cycle. , And 95 ° C. for 30 seconds as one cycle. The relative expression level of the Glut3 gene with respect to the 18S ribosomal RNA gene is analyzed by the Pfaffl method. The relational expression between the obtained relative expression level and the age of the week is obtained.

Next, peripheral blood is collected from the mouse as the subject, and a part of it is used as a body fluid sample to be analyzed. Using the body fluid sample to be analyzed, the relative expression level of the Glut3 gene with respect to the 18S ribosomal RNA gene is measured by the same method. Using the relative expression level of the Glut3 gene in the body fluid sample to be analyzed as an index, the age of the subject is determined based on the above relational expression.

6. Comparative Example 2
<Method>
In Comparative Example 2, an index for determining dementia and brain function in a subject was examined using a body fluid sample derived from a human (volunteer donor).

6-1. Recruitment of subjects The following experiments were conducted 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 years old, respectively.

6-2. Dementia Screening Test A Mini-Mental State Examination (MMSE) was performed on volunteer donors to assess dementia and / or brain function. The MMSE is a cognitive function test consisting of a total of 11 questions in the form of questions. Time orientation, location orientation, immediate recall, attention and calculation ability, delayed reproduction (short-term memory), linguistic ability, and graphic. Cognitive functions such as ability (spatial cognition) can be evaluated. In the MMSE, the maximum score of the MMSE score was 30 points, and a case of 27 points or less was judged to be mild cognitive impairment (MCI), and a case of 23 points or less was judged to be dementia.

6-3. Blood cell analysis Peripheral blood was collected from volunteer donors and the proportion of mononuclear cells in the peripheral blood sample was measured using an automatic blood cell counter.

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

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

7. Example 5
<Method>
In Example 5, a body fluid sample derived from a human (volunteer donor) was used to examine an index for determining dementia and brain function in the subject.

7-1. Recruitment of subjects The following experiments were conducted with the cooperation of four volunteer donors (volunteer donors FI). The ages of volunteer donors FI were 82, 81, 58, and 51, respectively.

7-2. Dementia Screening Test A mini-mental state test was performed on volunteer donors in the same manner as in "6-2. Dementia Screening Test" in Comparative Example 2.

7-3. Blood cell analysis The ratio of mononuclear cells in the peripheral blood sample of the volunteer donor was measured in the same manner as in "6-3. Blood cell analysis" of Comparative Example 2.

7-4. Quantitative RT-PCR
The relative expression level of each target gene in the peripheral blood sample of the volunteer donor was analyzed in the same manner as in "6-4. Quantitative RT-PCR" of Comparative Example 2.

<Result>
The results of the Mini-Mental State Examination (MMSE) of Comparative Example 2 and Example 5 are shown below.
The MMSE scores of the volunteer donors A to E in Comparative Example 2 were 29 points or 30 points. On the other hand, the MMSE score of the volunteer donors FI of Example 5 was 25 points or 26 points. From the above, it was determined that the volunteer donors A to E of Comparative Example 2 did not develop dementia and the brain function did not deteriorate, whereas the volunteer donors FI of Example 5 were mild. It was determined that cognitive impairment was suspected, and a decline in brain function was suspected.

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

In Table 6, "age" is the age of each volunteer donor, and "mononuclear cells" is the ratio of mononuclear cells in the peripheral blood sample of each volunteer donor (relative to the number of all leukocytes contained in the peripheral blood sample). "MMSE" indicates the MMSE score.

As shown in Table 6, in the peripheral blood sample of volunteer donor A, the relative expression level of the Glut1 gene was 0.33, the relative expression level of the Glut3 gene was 0.25, the relative expression level of the MCT4 gene was 0.09, and PHD3. The relative expression level of the gene was 0.50, and the relative expression level of the PDK1 gene was 0.72. In the peripheral blood sample of volunteer donor B, the relative expression level of the Glut1 gene was 0.45, the relative expression level of the Glut3 gene was 0.26, the relative expression level of the MCT4 gene was 0.19, and the relative expression level of the PHD3 gene was 0. At .24, the relative expression level of the PDK1 gene was 0.35. In the peripheral blood sample of volunteer donor C, the relative expression level of the Glut1 gene was 0.12, the relative expression level of the Glut3 gene was 0.17, the relative expression level of the MCT4 gene was 0.72, and the relative expression level of the PHD3 gene was 0. At .24, the relative expression level of the PDK1 gene was 0.73. In the peripheral blood sample of volunteer donor D, the relative expression level of the Glut1 gene is 0.55, the relative expression level of the Glut3 gene is 0.32, the relative expression level of the MCT4 gene is 1.00, and the relative expression level of the PHD3 gene is 0. At .44, the relative expression level of the PDK1 gene was 0.58. In the peripheral blood sample of volunteer donor E, the relative expression level of the Glut1 gene was 0.92, the relative expression level of the Glut3 gene was 0.41, the relative expression level of the MCT4 gene was 0.75, and the relative expression level of the PHD3 gene was 0. At .76, the relative expression level of the PDK1 gene was 0.88.

As shown in Table 6, in the peripheral blood sample of volunteer donor F, the relative expression level of the Glut1 gene was 1.14, the relative expression level of the Glut3 gene was 1.15, the relative expression level of the MCT4 gene was 1.35, and PHD3. The relative expression level of the gene was 1.11 and the relative expression level of the PDK1 gene was 1.26. In the peripheral blood sample of volunteer donor G, the relative expression level of the Glut1 gene was 2.96, the relative expression level of the Glut3 gene was 0.97, the relative expression level of the MCT4 gene was 1.44, and the relative expression level of the PHD3 gene was 2. At .65, the relative expression level of the PDK1 gene was 2.28. In the peripheral blood sample of volunteer donor H, the relative expression level of the Glut1 gene is 1.52, the relative expression level of the Glut3 gene is 1.56, the relative expression level of the MCT4 gene is 1.38, and the relative expression level of the PHD3 gene is 1. At .59, the relative expression level of the PDK1 gene was 1.27. In the peripheral blood sample of Volunteer Donor I, the relative expression level of the Glut1 gene is 1.76, the relative expression level of the Glut3 gene is 0.62, the relative expression level of the MCT4 gene is 2.08, and the relative expression level of the PHD3 gene is 2. At .18, the relative expression level of the PDK1 gene was 1.28.

As shown in Table 6, the Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene and PDK1 gene in the peripheral blood samples of the volunteer donors FI of Example 5 as compared with the volunteer donors A to E of Comparative Example 2 Gene expression was significantly increased. Therefore, as genes whose expression levels change remarkably with the onset of dementia and / or deterioration of brain function, genes of proteins involved in glucose transport (Glut1 gene and Glut3 gene) and genes of proteins involved in lactic acid transport. (MCT4 gene), a gene for a protein involved in the glycolytic system (PHD3 gene), and a gene for a protein involved in the TCA circuit (PDK1 gene) were identified.

In addition, the relative expression level of each target gene in the peripheral blood samples of the volunteer donors FI of Example 5 is higher than the reference values shown in the following (a) to (e), and Comparative Example 2 It was found that the relative expression level of each target gene in the volunteer donors A to E was lower than the reference value shown in (a) to (e) below.
(A) Reference value of gene expression of Glut1 gene: 1.00
(B) Reference value of gene expression of Glut3 gene: 0.50
(C) Reference value of gene expression of MCT4 gene: 1.10
(D) Reference value for gene expression of PHD3 gene: 0.90
(E) Reference value for gene expression of PDK1 gene: 1.00

In the peripheral blood sample of volunteer donor F, the ratios of the relative expression levels of the Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene and PDK1 gene to the above reference values were 1.1, 2.3, 1.2 and 1, respectively. It was .2 and 1.3.

In the peripheral blood sample of volunteer donor G, the ratios of the relative expression levels of the Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene and PDK1 gene to the above reference values were 3.0, 1.9, 1.3 and 2, respectively. It was 9.9 and 2.3.

In the peripheral blood sample of volunteer donor H, the ratios of the relative expression levels of the Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene and PDK1 gene to the above reference values were 1.5, 3.1, 1.3 and 1, respectively. It was .8 and 1.3.

In the peripheral blood sample of Volunteer Donor I, the ratios of the relative expression levels of the Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene and PDK1 gene to the above reference values were 1.8, 1.2, 1.9 and 2, respectively. It was .4 and 1.3.

8. Example 6
<Method>
In Example 6, based on the indexes for determining dementia and brain function obtained in Comparative Example 2 and Example 5, the dementia and brain function of the human are determined from the body fluid sample derived from a human (volunteer donor). Judged. In Example 6, the Glut1 gene and the PDK1 gene were used as indexes for determining dementia and brain function.

8-1. Recruitment of subjects The following experiments were conducted with the cooperation of nine volunteer donors (volunteer donors JR). The ages of volunteer donors JR were 52, 81, 88, 81, 53, 50, 84, 53 and 22 years old, respectively.

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

8-3. Judgment of dementia and brain function The relative expression levels of the Glut1 gene and PDK1 gene obtained by the above "8-2. Quantitative RT-PCR" and the reference values of the Glut1 gene and PDK1 gene (both are 1.00). The risk of developing dementia and the presence or absence of decreased brain function in each volunteer donor were determined by comparing.

8-4. Dementia Screening Test A mini-mental state test was performed on volunteer donors in the same manner as in "6-2. Dementia Screening Test" in Comparative Example 2.

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

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 the reference values of the Glut1 gene and the PDK1 gene obtained in Comparative Example 2 and Example 5 (whichever). Was also higher than 1.00). 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 of the Glut1 gene and the PDK1 gene (both were 1.00). rice field. Based on the above, it was determined that volunteer donors K and M have a risk of developing dementia and are suspected of having decreased brain function.

The results of the Mini-Mental State Examination (MMSE) of Example 6 are shown below.
Volunteer donor K had an MMSE score of 22 points, and volunteer donor M had an MMSE score of 25 points. The MMSE scores of other volunteer donors (J, L, N to R) were 28 to 30 points. Based on the above, Volunteer Donor K was determined to be suspected of having dementia, and was suspected of having decreased brain function. Volunteer donor M was also suspected of having mild cognitive impairment and was suspected of having decreased brain function. In addition, it was determined that other volunteer donors (J, L, N to R) did not develop dementia and their brain function did not deteriorate.

Therefore, by using the relative expression level of one or more of the above genes as an index higher than the above reference value, it was possible to obtain an index for determining dementia or brain function in the subject.

9. Example 7
<Manufacturing of dementia / brain function judgment kit>
A kit for determining dementia or determining brain function (dementia / brain function determination kit) is manufactured by the following procedure.

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 PDK1 gene amplification having a final concentration of 10 μM. do. 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 PDK1 gene amplification is used as a reagent for measuring gene expression of PDK1, which is a protein involved in the TCA cycle. Further, the PDK1 gene amplification primer pair solution, the 18S ribosomal RNA gene amplification primer pair solution, a mononuclear cell separation reagent (Filel-Paque PLUS (GE Healthcare Co., Ltd./model number: 17144002)), and RNA extraction. Reagents (Kaneka Simple RNA Extraction Kit (for RT-PCR) (Kaneka Co., Ltd./Model: KN-T110004)) and cDNA Synthesis Reagent (PrimeScript (Registered Trademark) II 1st Strand cDNA Synthesis Kit (Takara Bio Co., Ltd./Model::) 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 determination kit.

Collect peripheral blood from the human subject. Mononuclear cells are separated from peripheral blood using the mononuclear cell separation reagent constituting the dementia / brain function determination kit, and a part 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 determination kit. Using the obtained total RNA as a template, cDNA is synthesized using the cDNA synthesis reagent constituting the dementia / brain function determination kit. Using the obtained cDNA as a template, a real-time PCR reaction is performed using a primer pair solution for PDK1 gene amplification, a primer pair solution for 18S ribosomal RNA gene amplification, and a nucleic acid amplification reagent that constitute the dementia / brain function determination kit. The real-time PCR system uses Agilent AliaMx Real-Time PCR System (Agilent). The real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C. for 30 seconds for 1 cycle. , And 95 ° C. for 30 seconds as one cycle. The relative expression level of the PDK1 gene with respect to the 18S ribosomal RNA gene is analyzed by the Pfaffl method. By comparing the relative expression level of the PDK1 gene with respect to the expression level of the 18S ribosomal RNA gene with the reference value of the gene expression of the PDK1 gene, it is possible to determine the presence or absence of dementia in humans.

All publications, patents and patent applications cited herein are incorporated herein by reference as is.

Claims

A kit for determining dementia or brain function.
A kit containing a reagent for measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
The kit according to claim 1, wherein the determination of the dementia is a determination of the presence or absence of dementia, a determination of the risk of developing dementia, or a determination of the severity of dementia.
The above-mentioned determination of brain function includes determination of presence / absence of deterioration of brain function, determination of risk of deterioration of brain function, determination of degree of deterioration of brain function, determination of presence / absence of disorder of autonomic nervous system, disorder of autonomic nervous system. Judgment of risk, judgment of degree of autonomic nervous system disorder, judgment of presence or absence of memory disorder, judgment of risk of memory disorder, judgment of degree of memory disorder, judgment of presence or absence of movement disorder, risk of movement disorder Judgment, judgment of the degree of motor disorder, judgment of the presence or absence of cooperative movement disorder, judgment of the risk of cooperative movement disorder, judgment of the degree of cooperative movement disorder, judgment of the presence or absence of involuntary movement disorder, involuntary Judgment of the risk of motor disorder, judgment of the degree of involuntary movement disorder, judgment of the presence or absence of sensory disorder, judgment of the risk of sensory disorder, judgment of the degree of sensory disorder, judgment of the presence or absence of visual disorder, visual Judgment of the risk of olfactory disorder, judgment of the degree of visual disorder, judgment of the presence or absence of olfactory disorder, judgment of the risk of olfactory disorder, judgment of the degree of olfactory disorder, judgment of the presence or absence of hearing disorder, hearing Judgment of the risk of disorder, judgment of the degree of hearing disorder, judgment of the presence or absence of balance disorder, judgment of the risk of balance sense disorder, judgment of the degree of balance sense disorder, judgment of the presence or absence of sleep disorder , Judgment of risk of sleep disorder, judgment of degree of sleep disorder, judgment of presence or absence of speech disorder, judgment of risk of language disorder, judgment of degree of speech disorder, judgment of presence or absence of emotional dysregulation, judgment of emotional dysregulation Judgment of risk, judgment of emotional dysregulation, judgment of presence or absence of headache, judgment of risk of headache, judgment of degree of headache, judgment of presence or absence of convulsions, judgment of risk of convulsions, judgment of degree of convulsions , Judgment of the presence or absence of tremors, judgment of the danger of tremors, judgment of the degree of tremors, judgment of the presence or absence of illusions, judgment of the danger of illusions, judgment of the degree of illusions, judgment of the presence or absence of delusions, judgment of the danger of delusions Judgment, judgment of the degree of delusion, judgment of the presence or absence of illusion, judgment of the risk of illusion, judgment of the degree of illusion, judgment of the presence or absence of abnormal behavior, judgment of the risk of abnormal behavior, judgment of the degree of abnormal behavior, depression The kit according to claim 1, which is a determination of the presence or absence of illness, a determination of the risk of developing depression, or a determination of the severity of depression.
The kit according to any one of claims 1 to 3, wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3 and PDK1.
The reagent specifically binds to a primer pair for amplifying a nucleic acid containing the gene of the one or more proteins, a probe that hybridizes with the nucleic acid containing the gene of the one or more proteins, or the protein containing the one or more proteins. The kit according to any one of claims 1 to 4, which is an antibody to be used.
The kit according to any one of claims 1 to 5, wherein the kit further includes a reagent and / or a column for separating mononuclear cells from the sample.
It is a method of acquiring an index for determining dementia or brain function in a subject.
A method comprising measuring the gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
The method according to claim 7, wherein the determination of dementia is determination of the presence or absence of morbidity of dementia, determination of the risk of developing dementia, or determination of the severity of dementia.
The sample is a body fluid sample or a bone marrow sample,
It further comprises comparing the measured values of gene expression or enzyme activity of one or more proteins with reference values.
A measurement value of gene expression or enzyme activity of one or more proteins, which is higher than the reference value, indicates that the subject suffers from dementia and that the subject has a risk of developing dementia. Or, the method according to claim 8, which indicates the severity of dementia of the subject.
The above-mentioned determination of brain function includes determination of presence / absence of deterioration of brain function, determination of risk of deterioration of brain function, determination of degree of deterioration of brain function, determination of presence / absence of disorder of autonomic nervous system, disorder of autonomic nervous system. Judgment of risk, judgment of degree of autonomic nervous system disorder, judgment of presence or absence of memory disorder, judgment of risk of memory disorder, judgment of degree of memory disorder, judgment of presence or absence of movement disorder, risk of movement disorder Judgment, judgment of the degree of motor disorder, judgment of the presence or absence of cooperative movement disorder, judgment of the risk of cooperative movement disorder, judgment of the degree of cooperative movement disorder, judgment of the presence or absence of involuntary movement disorder, involuntary Judgment of the risk of motor disorder, judgment of the degree of involuntary movement disorder, judgment of the presence or absence of sensory disorder, judgment of the risk of sensory disorder, judgment of the degree of sensory disorder, judgment of the presence or absence of visual disorder, visual Judgment of the risk of olfactory disorder, judgment of the degree of visual disorder, judgment of the presence or absence of olfactory disorder, judgment of the risk of olfactory disorder, judgment of the degree of olfactory disorder, judgment of the presence or absence of hearing disorder, hearing Judgment of the risk of disorder, judgment of the degree of hearing disorder, judgment of the presence or absence of balance disorder, judgment of the risk of balance sense disorder, judgment of the degree of balance sense disorder, judgment of the presence or absence of sleep disorder , Judgment of risk of sleep disorder, judgment of degree of sleep disorder, judgment of presence or absence of speech disorder, judgment of risk of language disorder, judgment of degree of speech disorder, judgment of presence or absence of emotional dysregulation, judgment of emotional dysregulation Judgment of risk, judgment of emotional dysregulation, judgment of presence or absence of headache, judgment of risk of headache, judgment of degree of headache, judgment of presence or absence of convulsions, judgment of risk of convulsions, judgment of degree of convulsions , Judgment of the presence or absence of tremors, judgment of the danger of tremors, judgment of the degree of tremors, judgment of the presence or absence of illusions, judgment of the danger of illusions, judgment of the degree of illusions, judgment of the presence or absence of delusions, judgment of the danger of delusions Judgment, judgment of the degree of delusion, judgment of the presence or absence of illusion, judgment of the risk of illusion, judgment of the degree of illusion, judgment of the presence or absence of abnormal behavior, judgment of the risk of abnormal behavior, judgment of the degree of abnormal behavior, depression The method according to claim 7, wherein the presence or absence of illness, the risk of developing depression, or the severity of depression is determined.
The method according to any one of claims 7 to 10, wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3 and PDK1.
Claims 7 to 11 include measuring the gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from the subject, including (a) and (b) shown below. The method described in any one of the paragraphs:
(A) Obtaining a mononuclear cell-containing sample by separating mononuclear cells from the sample.
(B) To measure the gene expression or enzyme activity of one or more proteins in the mononuclear cell-containing sample.