JP2017009329A - Marker for assessing motor functions - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide biomarkers for evaluating motor functions of individuals, muscle mass, a degree of skeletal muscle cell differentiation, or degree of myotube hypertrophy.SOLUTION: Disclosed are a motor function assessment marker, muscle mass assessment marker, skeletal muscle cell differentiation assessment marker, and myotube hypertrophy assessment marker, which contain myosin-binding protein H derived from a skeletal muscle sample taken from an individual, or a gene that codes for the protein.SELECTED DRAWING: None

Description

  The present invention relates to a marker for determining motor function, a marker for determining muscle mass, a marker for determining differentiation of skeletal muscle cells, or a marker for determining myotube hypertrophy.

Locomotive syndrome refers to musculoskeletal diseases caused by aging. The prevalence of locomotive syndrome increases with age, and as the locomotive syndrome progresses, the risk of needing long-term care increases greatly.
To date, research on genes and proteins related to age-related diseases of motor organs such as locomotive syndrome has progressed. However, the factors related to musculoskeletal diseases have not been identified so far.

On the other hand, for myosin binding protein H (hereinafter also simply referred to as “MYBPH”), cell migration is promoted when MYBPH is knocked down in a human lung adenocarcinoma cell line, and MYBPH is inhibited in a canine kidney tubular epithelial cell line. It is known that cell migration and invasion are suppressed when overexpressed (see Non-Patent Document 1). In addition, it is known that when MYBPH and myosin-binding protein C are double knocked down in a rat heart-derived cell line, cell contraction due to adrenaline stimulation is suppressed (see Non-Patent Document 2). Furthermore, the use of MYBPH as a protein biomarker for detecting a skeletal muscle disorder caused by a side effect (muscle toxicity) caused by administration of a pharmaceutical or the like is also disclosed (see Patent Document 1).
However, it is not known that MYBPH can be used as a biomarker for determining and evaluating motor functions necessary for daily life and age-related diseases of musculoskelets.

International Publication No. 2007/0666129 Pamphlet

Yasuyuki Hosono, et al., The EMBO Journal, 2012, vol. 31, p. 481-493 Jomien Mouton, et al., Exp. Cell Res., 2015, vol. 331 (2), p. 338-351

An object of the present invention is to provide a biomarker for evaluating an individual's motor function, muscle mass, degree of skeletal muscle cell differentiation, or degree of myotube hypertrophy.
Another object of the present invention is to provide a method for evaluating motor function, muscle mass, degree of skeletal muscle cell differentiation, or degree of myotube hypertrophy using the biomarker.

The present inventors have intensively studied in view of the above problems.
It is known that many proteins are expressed in the skeletal muscle that is one of the motor organs and binds to the skeleton that moves the body. Among these proteins, the expression level of skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same (hereinafter also referred to as “MYBPH gene”) is determined by the individual's motor function, muscle mass, and skeletal muscle cell differentiation. It was found that there is a correlation between the degree of myotubes and myotube hypertrophy. The present inventors have found that the MYBPH or MYBPH gene is useful as a biomarker for evaluating an individual's motor function, muscle mass, degree of skeletal muscle cell differentiation, or myotube hypertrophy. Furthermore, the expression level of the MYBPH or MYBPH gene collected from the individual can be measured, and the motor function, muscle mass, skeletal muscle cell differentiation level, and myotube hypertrophy level can be evaluated based on the measured expression level. I found.
The present invention has been completed based on these findings.

The present invention relates to a marker for evaluation of motor function, which includes MYBPH derived from skeletal muscle collected from an individual or a gene encoding the same.
The present invention also relates to a muscle mass evaluation marker containing MYBPH derived from skeletal muscle collected from an individual or a gene encoding the same.
The present invention also relates to a skeletal muscle cell differentiation evaluation marker comprising skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same.
The present invention also relates to a marker for evaluating myotube hypertrophy comprising MYBPH derived from skeletal muscle collected from an individual or a gene encoding the same.

The present invention also relates to a method for evaluating motor function, which measures the expression level of skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same, and evaluates the motor function of the individual based on the measured expression level.
The present invention also relates to a method for evaluating muscle mass, in which the expression level of skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same is measured, and the muscle mass is evaluated based on the measured expression level.
The present invention also provides a method for measuring skeletal muscle cell-derived MYBPH collected from an individual or a gene encoding the same, and evaluating the degree of skeletal muscle cell differentiation based on the measured expression level. The present invention relates to a method for evaluating differentiation.
Furthermore, the present invention measures the expression level of MYBPH derived from skeletal muscle collected from an individual or a gene encoding the same, and evaluates the degree of myotube hypertrophy based on the measured expression level. About.

The biomarker of the present invention can be used to evaluate an individual's motor function, muscle mass, degree of skeletal muscle cell differentiation, or degree of myotube hypertrophy.
The evaluation method of the present invention can accurately and easily evaluate motor function, muscle mass, degree of skeletal muscle cell differentiation, or degree of myotube hypertrophy. Furthermore, based on the evaluation method of the present invention, it is suitably applied to screening of substances that improve motor function, substances that increase muscle mass, substances that promote differentiation of skeletal muscle cells, or substances that promote myotube hypertrophy. be able to.

It is a graph which shows the muscle mass of the soleus muscle of each mouse | mouth of a control group and an exercise group 2 months after the exercise | movement start measured in Test Example 1. FIG. It is a graph which shows the muscle strength of the soleus muscle of each mouse | mouth of the control group and exercise group 2 months after the exercise | movement start measured in Test Example 1. FIG. It is a graph which shows the expression level of the MYBPH gene in the soleus muscle of the mouse | mouth of a control group and the exercise | movement group of 1 month and 2 months after the exercise | movement start measured in Experiment 1. It is a graph which shows the amount of muscles of the plantar muscle of each mouse | mouth of the non-operation group of the joint muscle excision of a plantar muscle measured in Experiment 2, and each treatment group. It is a graph which shows the expression level of the MYBPH gene in the plantar muscle of each mouse | mouth of the non-operation group of the joint muscle excision of a plantar muscle measured in Experiment 2, and each treatment group. It is a graph which shows the amount of muscles of the soleus of each mouse | mouth measured by the test example 3 after tail suspension perpendicular | vertical and after re-grounding. It is a graph which shows the expression level of the MYBPH gene in the soleus muscle of each mouse | mouth after the tail-pick vertical and re-grounding measured in Experiment 3. It is a graph which shows the expression level of the MYBPH gene in the growth section of a mouse | mouth skeletal muscle origin cell strain measured in Experiment 4, and a differentiation process. It is a figure which shows the expression level of MYBPH in the growth section and differentiation process of the mouse | mouth skeletal muscle origin cell strain measured in Experiment 4. FIG. 10 is a graph showing changes in the expression level of the MYBPH gene due to the influence of RNA interference measured in Test Example 5. FIG. 6 is a graph showing changes in the expression level of MYBPH due to the influence of RNA interference measured in Test Example 5. 12 is a graph showing changes in the expression level of myosin heavy chain due to the influence of RNA interference measured in Test Example 5. It is the microscope picture which image | photographed the mode of expression of the myosin heavy chain in the mouse | mouth skeletal muscle origin cell line measured in Test Example 5, (a) is the microscope picture photographed 48 hours after differentiation induction, (b) It is the microscope picture image | photographed 72 hours after differentiation induction, (c) is the microscope picture image | photographed 120 hours after differentiation induction. The upper row is a photomicrograph when RNA interference is not performed, and the lower row is a photomicrograph when RNA interference is performed. 6 is a graph showing changes in mean myotube diameter measured by Test Example 5 due to the influence of RNA interference. FIG. 15A shows a micrograph of zebrafish in the control group, and FIG. 15B shows a micrograph of zebrafish microinjected with TALEN mRNA. It is an electrophoresis photograph which shows the mode of expression of the MYBPHa gene and the MYBPHb gene in the zebrafish which knocked out the MYBPHa gene and the MYBPHb gene performed in Test Example 6.

In this specification, “evaluation” or “determination” relating to data or measurement values, etc. refers to analyzing data or measurement values obtained from a sample (group) according to a predetermined purpose, and based on the analyzed information. It means to conclude whether a sample (s) has a specific property, trend or trend.
Further, in the present invention, “motor function” refers to the motor ability of a motor organ that constitutes and supports the body and enables physical motion.

  Motor function evaluation marker, muscle mass evaluation marker, skeletal muscle cell differentiation evaluation marker, and myotube hypertrophy evaluation marker of the present invention (hereinafter collectively referred to as "the marker of the present invention") ) Includes MYBPH or MYBPH genes derived from skeletal muscle collected from individuals.

As shown in the examples below, the expression levels of MYBPH and MYBPH genes derived from skeletal muscle collected from an individual included in the marker of the present invention are the motor function, muscle mass, and degree of skeletal muscle cell differentiation of the individual. , And myotube hypertrophy. The present inventors have found that the expression levels of MYBPH and MYBPH genes are significantly increased with improvement of motor function, increase in muscle mass, progression of skeletal muscle cell differentiation, and myotube hypertrophy.
Therefore, the marker of the present invention can be used as an index of motor function, muscle mass, degree of skeletal muscle cell differentiation, and degree of myotube hypertrophy. Furthermore, the marker of the present invention can be suitably used in a method for simply and accurately evaluating motor function, muscle mass, degree of skeletal muscle cell differentiation, and degree of myotube hypertrophy.

  Patent Document 1 describes a biomarker for detecting a skeletal muscle disorder caused by a side effect (muscle toxicity) caused by administration of a pharmaceutical or the like, which is composed of MYBPH. On the other hand, the marker of the present invention is a biomarker for determining and evaluating motor functions necessary for daily life and age-related diseases of musculature. Thus, the object of determination or evaluation in the present invention is completely different from the invention described in Patent Document 1.

Information on the amino acid sequence of MYBPH can be obtained from various databases. For example, the amino acid sequence of MYBPH derived from mouse is NCBI RefSeq as ID: NP_058029, and the amino acid sequence of MYBPH derived from zebrafish (MYBPHa and MYBPHb) is NCBI RefSeq as ID: NP_956852.1 and NM_001100137.1 (http: // www.ncbi.nlm.nih.gov/gene/393530, http://www.ncbi.nlm.nih.gov/gene/321053), the amino acid sequence of human-derived MYBPH is NCBI RefSeq as ID: NP_004988, Each is registered.
The MYBPH used in the present invention is not limited to these. In these amino acid sequences, one or several, usually 1 to 100, preferably 1 to 75, more preferably 1 to 50, and still more preferably 1-25 amino acids, particularly preferably 1-10 amino acids, most preferably 1-5 amino acids are deleted, substituted, inserted and / or added amino acid sequences and include functionally equivalent proteins It is. Further, these amino acid sequences have a homology of 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. Proteins consisting of amino acid sequences and functionally equivalent are also included. Here, in this specification, the homology of amino acid sequences is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it is calculated by performing an analysis with Unit size to compare (ktup) set to 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

  The marker of the present invention comprises a polypeptide having a part of the amino acid sequence of MYBPH described above, and is a polypeptide having a length that is specifically recognized by an antibody or peptide aptamer for obtaining information on the expression level of MYBPH. Preferably there is. Here, the “polypeptide having a part of the amino acid sequence of MYBPH” means a part of the amino acid sequence of MYBPH described above, preferably 5 or more, more preferably 7 or more, still more preferably 10 or more, particularly preferably. Means a polypeptide having 12 or more, preferably 40 or less, more preferably 30 or less, still more preferably 20 or less, particularly preferably 15 or less.

Information on the base sequence of the MYBPH gene can be obtained from various databases. For example, the nucleotide sequence of the mouse-derived MYBPH gene is NCBI RefSeq as ID: NM_016749, and the zebrafish-derived MYBPH gene (MYBPHa gene and MYBPHb gene) is NCBI RefSeq as ID: NM_200558.1 and NP_001093607.1 ( http://www.ncbi.nlm.nih.gov/gene/393530, http://www.ncbi.nlm.nih.gov/gene/321053), the nucleotide sequence of human-derived MYBPH is assigned to NCBI RefSeq : NM_004997, respectively.
The MYBPH gene used in the present invention is not limited to these. In these base sequences, one or several, usually 1 to 389, preferably 1 to 292, more preferably 1 to 195, and even more preferably 1 to 98, particularly preferably 1 to 39, most preferably 1 to 20 nucleotides in which the nucleotide sequence is deleted, substituted, inserted or added, and also includes the above-mentioned nucleic acid encoding MYBPH. It is. In addition, these base sequences have a homology of 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. A nucleic acid consisting of a base sequence and encoding the aforementioned MYBPH is also included. Here, in the present specification, the homology of the nucleotide sequence is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it is calculated by performing an analysis with Unit size to compare (ktup) set to 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).
In the present invention, the MYBPH gene may be either DNA or RNA, and may be any of MYBPH gene itself (DNA), mRNA, cDNA, and cRNA.

  The marker of the present invention comprises a polyoligonucleotide having a part of the base sequence of the MYBPH gene described above, under stringent conditions with a nucleic acid probe, nucleic acid aptamer, or nucleic acid primer for obtaining information on the expression level of the MYBPH gene. It is preferable that the polynucleotide has a length capable of hybridizing. Here, as “stringent conditions”, for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W., et al. Russell., Cold Spring Harbor Laboratory Press], for example, 6 × SSC (composition of 1 × SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% Examples include conditions in which a solution containing SDS, 5 × Denhart and 100 mg / mL herring sperm DNA is incubated with a probe at 65 ° C. for 8 to 16 hours and hybridized.

  The method for evaluating motor function, the method for evaluating muscle mass, the method for evaluating differentiation of skeletal muscle cells, and the method for evaluating myotube hypertrophy (hereinafter collectively referred to as “the evaluation method of the present invention”) Using the inventive marker, the expression level of MYBPH or MYBPH gene derived from skeletal muscle collected from an individual is measured. Then, based on the measured expression level of the MYBPH or MYBPH gene, the individual's motor function, muscle mass, degree of skeletal muscle cell differentiation, or degree of myotube hypertrophy is evaluated.

  In the evaluation method of the present invention, first, a biological sample collected from an animal, preferably a human or non-human animal, more preferably a human or a laboratory animal (mouse, rat, guinea pig, hamster, rabbit, zebrafish, etc.), or an artificial The MYBPH or MYBPH gene is detected from a cell line that has been cultured automatically. The biological sample for detecting the MYBPH or MYBPH gene is not particularly limited, but the MYBPH or MYBPH gene is collected from skeletal muscle, preferably soleus muscle, hifuku muscle or plantar muscle.

  The timing for collecting the MYBPH or MYBPH gene from an individual can be appropriately selected. For example, it is preferable to collect the MYBPH or MYBPH gene 1 to 6 hours after an external stimulus that increases muscle mass such as exercise is applied to the individual.

  The expression level of the MYBPH or MYBPH gene thus collected is measured. As a method for measuring the expression level, the expression of the MYBPH or MYBPH gene can be appropriately selected from the conventional methods for qualitatively or quantitatively measuring, that is, the presence / absence and expression level of the MYBPH or MYBPH gene.

  The method for measuring the expression level of skeletal muscle-derived MYBPH collected from an individual using immunoprecipitation, mass spectrometry, RIA using an antibody or peptide aptamer that specifically binds to MYBPH prepared according to a conventional method. (Radioimmunoassay), Western blotting, ELISA (enzyme-linked immunosorbent) method, ECLIA (electrochemiluminescence immunoassay) method, and a method of measuring according to a conventional method such as a microarray.

As a method of measuring the expression level of skeletal muscle-derived MYBPH gene collected from an individual, using a nucleic acid probe, nucleic acid aptamer, or nucleic acid primer that is produced according to a conventional method and binds to the MYBPH gene under stringent conditions. , Such as polymerase chain reaction (PCR) method, RT-PCR method, real-time PCR method, nucleic acid amplification method such as LAMP (Loop-mediated isothermal amplification) method, hybridization method such as Southern hybridization, Northern hybridization, microarray method, etc. The method of measuring according to a conventional method is mentioned. The nucleic acid probe, nucleic acid aptamer, and nucleic acid primer may be labeled with a labeling substance such as a radioisotope, a fluorescent substance, or an enzyme.
Among these, in the present invention, it is preferable to measure the expression level of the MYBPH gene by PCR using a nucleic acid primer. As the nucleic acid primer, any one of nucleic acid primer sets 1 to 3 shown in Table 1 below can be preferably used.

The expression level of MYBPH and MYBPH genes increases with improvement of motor function, increase in muscle mass, progression of skeletal muscle cell differentiation, and myotube hypertrophy. In contrast, the expression level of the MYBPH and MYBPH genes decreases with a decrease in motor function, a decrease in muscle mass, a progression of skeletal muscle cell differentiation, and a myotube contraction.
Therefore, the expression level of MYBPH or MYBPH gene derived from skeletal muscle collected from an individual is measured, and MYBPH collected from a standard individual with respect to motor function, muscle mass, degree of skeletal muscle cell differentiation, or myotubes or Comparison is made with the expression level of the MYBPH gene. When the measured expression level increases, it is determined that the motor function is improved, the muscle mass is increased, the differentiation of skeletal muscle cells is advanced, or the myotube is enlarged. On the other hand, when the expression level of the MYBPH or MYBPH gene decreases, it is determined that the motor function has decreased, the muscle mass has decreased, skeletal muscle cell differentiation has not progressed, or the myotubes have contracted. If no change is confirmed in the expression level of MYBPH or MYBPH gene, it is determined that motor function, muscle mass, degree of skeletal muscle cell differentiation, and myotubes are in a standard state. Alternatively, if the individual does not increase the expression level of MYBPH or MYBPH gene even if an external stimulus that increases muscle mass, such as exercise, is performed, the individual suffers from locomotive syndrome or suffers from locomotive syndrome Judge that the risk is high.

When measuring the expression level of MYBPH in the present invention and evaluating the motor function, muscle mass, degree of skeletal muscle cell differentiation, or degree of myotube hypertrophy based on the measured expression level, protein molecules of skeletal muscle It is preferable to compare the expression level of a substance usually used as a marker with the expression level of MYBPH and correct the measured expression level of MYBPH. By correcting the expression level in this manner, it is possible to accurately evaluate each individual's motor function, muscle mass, degree of skeletal muscle cell differentiation, and myotube hypertrophy.
Examples of commonly used protein molecular markers for skeletal muscle include β-actin.

When measuring the expression level of the MYBPH gene in the present invention and evaluating the individual's motor function, muscle mass, degree of skeletal muscle cell differentiation, or degree of myotube hypertrophy based on the measured expression level, a housekeeping gene ( It is preferable that the expression level of the MYBPH gene is corrected by comparing the expression level of the internal standard gene) with the expression level of the MYBPH gene. Specifically, the expression level of the MYBPH gene is measured, corrected with the separately measured expression level of the housekeeping gene, and based on the corrected value, the individual's motor function, muscle mass, the degree of skeletal muscle cell differentiation, Alternatively, it is preferable to evaluate the degree of myotube hypertrophy. By comparing the expression levels in this way, it is possible to accurately evaluate each individual's motor function, muscle mass, degree of skeletal muscle cell differentiation, and myotube hypertrophy.
Commonly used housekeeping genes include 36B4 gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, β-actin gene and the like.

The present invention relates to an individual's motor function, muscle mass, degree of skeletal muscle cell differentiation, or an antibody, peptide aptamer, nucleic acid probe, nucleic acid aptamer or nucleic acid primer for measuring the expression level of MYBPH or MYBPH gene. A kit for evaluating the degree of myotube hypertrophy is also provided.
This kit may contain the aforementioned antibody, peptide aptamer, nucleic acid probe, nucleic acid aptamer, or nucleic acid primer for measuring the expression level of the protein molecular marker or housekeeping gene.

The present invention is based on the evaluation method described above, the preventive or ameliorating agent for locomotive syndrome, the promoter for improving the motor function, the agent for preventing or improving the motor function, the promoter for increasing the muscle mass, the agent for preventing or improving the muscle mass decrease Also provided are screening methods for screening for an agent for promoting differentiation of skeletal muscle cells, an agent for promoting myotube hypertrophy, and an agent for preventing or improving myotube contraction. In this screening method, a human or non-human animal, or a skeletal muscle-derived cell line is administered or ingested with a substance that is a candidate for the agent, and before or after the administration or ingestion of the substance, Substances that improve motor function, substances that increase muscle mass, substances that promote differentiation of skeletal muscle cells, or muscles that evaluate motor function, muscle mass, degree of skeletal muscle cell differentiation, or myotube hypertrophy Substances that enlarge the tube can be selected as these agents.
In the present specification, “prevention” means prevention or delay of the onset of a disease or symptom in an individual or reduction of the risk of onset of a disease or symptom in an individual. In the present specification, “improvement” refers to improvement of disease, symptom or condition, prevention or delay of deterioration of disease, symptom or condition, or reversal, prevention or delay of progression of disease, symptom or condition.

The biomarker of the present invention can also be suitably used in a method for detecting skeletal muscle cells in which differentiation has progressed and a method for detecting skeletal muscle cells in which myotubes are enlarged. In this detection method, the expression level of MYBPH or MYBPH gene expressed in skeletal muscle cells can be measured, and cells with high expression levels can be detected as skeletal muscle cells with advanced differentiation or skeletal muscle cells with enlarged myotubes. .
The biomarker of the present invention can also be suitably used in a method for evaluating the presence or absence of a locomotive syndrome or the risk of being affected. In this evaluation method, the expression level of MYBPH or MYBPH gene derived from skeletal muscle cells of an individual who has performed external stimulation to increase muscle mass such as exercise is measured, and when the expression level of MYBPH or MYBPH gene does not increase, It can be assessed that the patient is suffering from or has a high risk of suffering from locomotive syndrome.
Furthermore, the biomarker of the present invention can be suitably used for a method for evaluating the therapeutic or preventive effect of locomotive syndrome. When the treatment or prevention of locomotive syndrome is effective for a subject suffering from locomotive syndrome, the expression level of MYBPH or MYBPH gene is increased accordingly. Therefore, the expression level of the MYBPH or MYBPH gene is measured before and after the treatment or prevention of locomotive syndrome, and when the expression level increases, it can be evaluated that the treatment or prevention is effective.

  The present invention further discloses the following markers, uses, methods, evaluation methods, screening methods, and kits relating to the above-described embodiments.

<1> A marker for determining motor function, a marker for determining muscle mass, a marker for determining differentiation of skeletal muscle cells, or a marker for determining myotube hypertrophy, including MYBPH or MYBPH gene derived from skeletal muscle collected from an individual .

<2> The marker according to <1>, comprising a polypeptide having a part of the amino acid sequence of the MYBPH or a polyoligonucleotide having a part of the base sequence of the MYBPH gene.
<3> The marker according to <1> or <2> above, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene derived from a soleus muscle, a cypress muscle or a plantar muscle.
<4> The MYBPH is a protein registered in NCBI RefSeq as NP_058029, NP_956852.1, NM_001100137.1, or NP_004988, and one or several, usually 1 to 100, preferably 1 to 100 in the amino acid sequence of these proteins 1 to 75, more preferably 1 to 50, further preferably 1 to 25, particularly preferably 1 to 10, most preferably 1 to 5 amino acids deleted, substituted, inserted and / or added 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably, a protein comprising the amino acid sequence and functionally equivalent to the protein, or the amino acid sequence of these proteins <1> to <1>, wherein the protein is a functionally equivalent protein having an amino acid sequence having a homology of 98% or more, most preferably 99% or more Marker according to any one of 3>.
<5> The MYBPH gene has one or several, usually 1 to 389, preferably 1 to 389 genes in the nucleotide sequence of these genes registered in NCBI RefSeq as NM_016749, NM_200558.1, NP_001093607.1, or NM_004997. 1 to 292, more preferably 1 to 195, further preferably 1 to 98, particularly preferably 1 to 39, most preferably 1 to 20 bases are deleted, substituted, inserted or added. 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% of the nucleic acid consisting of the nucleotide sequence and encoding MYBPH or the nucleotide sequence of these genes As described above, the marker according to any one of <1> to <4>, which is a nucleic acid consisting of a base sequence having homology of 99% or more and encoding MYBPH.
<6> The marker according to any one of <1> to <5>, wherein the MYBPH is detected by an antibody or peptide aptamer specifically recognized as MYBPH, preferably an antibody.
<7> The marker according to any one of <1> to <6>, wherein the MYBPH gene is detected by a nucleic acid probe, a nucleic acid aptamer or a nucleic acid primer, preferably a nucleic acid primer.
<8> The marker according to <7>, wherein the MYBPH gene is detected by any one of the nucleic acid primer sets 1 to 3 shown in Table 1.
<9> The <1> to <7>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene collected 1 to 6 hours after an external stimulus such as exercise is applied to an individual to increase muscle mass. The marker of any one of Claims.

<10> As a marker for determining motor function, a marker for determining muscle mass, a marker for determining differentiation of skeletal muscle cells, or a marker for determining myotube hypertrophy of MYBPH or MYBPH gene derived from skeletal muscle collected from an individual Use of.
<11> A skeletal muscle-derived MYBPH or MYBPH gene collected from an individual as a marker for determining motor function, a marker for determining muscle mass, a marker for determining differentiation of skeletal muscle cells, or a marker for determining myotube hypertrophy How to use.
<12> A method for determining motor function, a method for determining muscle mass, a method for determining differentiation of skeletal muscle cells, or a method for determining myotube hypertrophy using MYBPH or MYBPH gene derived from skeletal muscle collected from an individual.

<13> The polypeptide according to any one of <10> to <12>, wherein a polypeptide having a part of the amino acid sequence of MYBPH or a polyoligonucleotide having a part of the base sequence of the gene is used. Use or method.
<14> The use or method according to any one of the above <10> to <13>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene derived from a soleus muscle, a cypress muscle or a plantar muscle.
<15> The MYBPH is a protein registered in NCBI RefSeq as NP_058029, NP_956852.1, NM_001100137.1, or NP_004988, one or several, usually 1 to 100, preferably 1 to 100 in the amino acid sequence of these proteins 1 to 75, more preferably 1 to 50, further preferably 1 to 25, particularly preferably 1 to 10, most preferably 1 to 5 amino acids deleted, substituted, inserted and / or added 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably, a protein comprising the amino acid sequence and functionally equivalent to the protein, or the amino acid sequence of these proteins <10, wherein the protein is composed of 98% or more, most preferably 99% or more homologous amino acid sequences and is functionally equivalent. Use or method according to any one of - <14>.
<16> The MYBPH gene has one or several, usually 1 to 389, preferably 1 to 389 genes in the nucleotide sequence of these genes registered in NCBI RefSeq as NM_016749, NM_200558.1, NP_001093607.1, or NM_004997 1 to 292, more preferably 1 to 195, further preferably 1 to 98, particularly preferably 1 to 39, most preferably 1 to 20 bases are deleted, substituted, inserted or added. 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% of the nucleic acid consisting of the nucleotide sequence and encoding MYBPH or the nucleotide sequence of these genes The use according to any one of <10> to <15> above, which is a nucleic acid consisting of a nucleotide sequence having homology of 99% or more, and most preferably encoding MYBPH. Method.
<17> The use according to any one of <10> to <16>, wherein the MYBPH is detected by an antibody or peptide aptamer specifically recognized as MYBPH, preferably an antibody. Method.
<18> The use or method according to any one of <10> to <17>, wherein the MYBPH gene is detected by a nucleic acid probe, a nucleic acid aptamer or a nucleic acid primer, preferably a nucleic acid primer.
<19> The use or method according to <18>, wherein the MYBPH gene is detected by any one of the nucleic acid primer sets 1 to 3 shown in Table 1.
<20> The <10> to <19>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene collected 1 to 6 hours after an external stimulus such as exercise is applied to an individual to increase muscle mass. Use or method according to any one of the preceding claims.

<21> measure the expression level of MYBPH or MYBPH gene derived from skeletal muscle collected from the individual,
Evaluate the individual's motor function, muscle mass, degree of skeletal muscle cell differentiation, or myotube hypertrophy based on the measured expression level, motor function, muscle mass, degree of skeletal muscle cell differentiation, or myotube Evaluation method of the degree of hypertrophy.

<22> The expression level of a polypeptide having a part of the amino acid sequence of the MYBPH or a polyoligonucleotide having a part of the base sequence of the MYBPH gene is measured, and the motor function of the individual based on the measured expression level, The method according to <21>, wherein the muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy is evaluated.
<23> The method according to <21> or <22>, wherein the expression level is the presence or absence of expression of the MYBPH or MYBPH gene, or the expression level of the MYBPH or a gene encoding the same.
<24> The method according to any one of the above <21> to <23>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene derived from a soleus muscle, a hifuku muscle, or a plantar muscle.
<25> Proteins registered in NCBI RefSeq as NP_058029, NP_956852.1, NM_001100137.1, or NP_004988 in the amino acid sequence of these proteins, one or several, usually 1 to 100, preferably 1 to 75, more preferably 1 to 50, further preferably 1 to 25, particularly preferably 1 to 10, most preferably 1 to 5 amino acids deleted, substituted, inserted and / or added 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably, a protein comprising the amino acid sequence and functionally equivalent to the protein, or the amino acid sequence of these proteins <21, which is a functionally equivalent protein consisting of amino acid sequences having homology of 98% or more, most preferably 99% or more The method according to any one of - <24>.
<26> The MYBPH gene is a gene registered in NCBI RefSeq as NM_016749, NM_200558.1, NP_001093607.1, or NM_004997, one or several, usually 1 to 389 in the base sequence of these genes, preferably 1 to 292, more preferably 1 to 195, further preferably 1 to 98, particularly preferably 1 to 39, most preferably 1 to 20 bases are deleted, substituted, inserted or added. 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% of the nucleic acid consisting of the nucleotide sequence and encoding MYBPH or the nucleotide sequence of these genes The method according to any one of <21> to <25> above, which is a nucleic acid consisting of a nucleotide sequence having homology of 99% or more, and most preferably encoding MYBPH.
<27> The antibody according to any one of <21> to <26>, wherein MYBPH is detected by an antibody or peptide aptamer specifically recognized as MYBPH, preferably an antibody, and the expression level of MYBPH is measured. Method.
<28> The method according to any one of <21> to <27>, wherein the MYBPH gene is detected with a nucleic acid probe, a nucleic acid aptamer, or a nucleic acid primer, preferably a nucleic acid primer, and the expression level of the MYBPH gene is measured. .
<29> The method according to <28>, wherein the MYBPH gene is detected by any one of the nucleic acid primer sets 1 to 3 shown in Table 1 and the expression level of the MYBPH gene is measured.
<30> When the measured expression level of MYBPH or MYBPH gene is increased, the motor function is improved, the muscle mass is increased, the differentiation of skeletal muscle cells is advanced, or the myotube is enlarged, The method according to any one of <21> to <29>.
<31> When the measured expression level of MYBPH or MYBPH gene is decreased, it is determined that motor function is decreased, muscle mass is decreased, skeletal muscle cell differentiation is not progressing, or myotube is contracted The method according to any one of <21> to <30>.
<32> If no change can be confirmed in the measured expression level of MYBPH or MYBPH gene, the motor function, muscle mass, degree of skeletal muscle cell differentiation, or myotube is determined to be in a standard state, 21. The method according to any one of <31> to <31>.
<33> When the expression level of a skeletal muscle-derived MYBPH or MYBPH gene collected from an individual subjected to external stimulation that increases muscle mass is measured and the measured expression level does not increase, the individual has locomotive syndrome The method according to any one of <21> to <32>, wherein it is determined that the patient is at risk of suffering from or having a locomotive syndrome.
<34> Any one of <21> to <33>, wherein the expression level of skeletal muscle protein molecular marker, preferably β-actin, is compared with the expression level of MYBPH and the measured expression level of MYBPH is corrected 2. The method according to item 1.
<35> The expression level of the housekeeping gene, preferably 36B4 gene, GAPDH gene, or β-actin gene is compared with the expression level of the MYBPH gene to correct the measured expression level of the MYBPH gene, <21> The method of any one of-<34>.
<36> The <21> to <35>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene collected 1 to 6 hours after an external stimulus such as exercise is applied to an individual to increase muscle mass. The method according to any one of the above.

<37> Motor function, muscle mass, degree of skeletal muscle cell differentiation, or muscle comprising an antibody, peptide aptamer, nucleic acid probe, nucleic acid aptamer or nucleic acid primer for measuring the expression level of MYBPH or MYBPH gene Evaluation kit for the extent of tube enlargement.
<38> Antibody, peptide aptamer, nucleic acid probe for measuring the expression level of a skeletal muscle protein molecular marker, preferably β-actin, or a housekeeping gene, preferably 36B4 gene, GAPDH gene, or β-actin gene The kit according to <37>, further comprising a nucleic acid aptamer or a nucleic acid primer.

<39> Locomotive syndrome prevention or improvement agent, motor function improvement promoter, motor function decrease prevention or improvement agent, muscle mass increase promoter, muscle mass decrease prevention or improvement agent, skeletal muscle cell differentiation promoter A screening method for screening for an agent for promoting myotube hypertrophy, an agent for preventing or improving myotube contraction,
A human or non-human animal, or a skeletal muscle-derived cell line is administered or ingested with a substance that is a candidate for the agent
Based on the evaluation method according to any one of <21> to <35> before and after administration or ingestion of a substance, an individual's motor function, muscle mass, degree of skeletal muscle cell differentiation, or myotube hypertrophy Assess the degree,
A screening method, wherein a substance that improves motor function, a substance that increases muscle mass, a substance that promotes differentiation of skeletal muscle cells, or a substance that enlarges myotubes is selected as the agent.
<40> Using the marker according to any one of <1> to <9>, the expression level of MYBPH or MYBPH gene expressed in skeletal muscle cells is measured,
Detecting cells with high expression level of MYBPH or MYBPH gene as skeletal muscle cells with advanced differentiation or skeletal muscle cells with enlarged myotubes,
A method for detecting skeletal muscle cells in which differentiation has progressed or skeletal muscle cells in which myotubes are enlarged.
<41> Using the marker according to any one of <1> to <9>, measuring the expression level of skeletal muscle cell-derived MYBPH or MYBPH gene,
Measure the expression level of MYBPH or MYBPH gene derived from skeletal muscle cells,
If an individual does not increase the expression level of the MYBPH or MYBPH gene even if an external stimulus that increases muscle mass is performed, the individual is evaluated as having or having a high risk of suffering from locomotive syndrome,
A method for evaluating the presence or risk of suffering from locomotive syndrome.
<42> Using the marker according to any one of <1> to <9>, the expression level of MYBPH or MYBPH gene expressed in skeletal muscle cells is measured,
When the expression level of MYBPH or MYBPH gene is increased, the treatment or prevention measures are evaluated to be effective.
A method for evaluating the therapeutic or preventive effect of locomotive syndrome.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.
Here, Table 2 shows the base sequences of the primers used for amplification of various genes used in the following test examples and comparative examples. The following primers were designed to amplify the target gene fragment with a size of about 100 to 200 bp.

Test Example 1 Measurement of soleus muscle mass and expression level of MYBPH gene in soleus muscle after exercise test of mice (1) Measurement of soleus muscle mass and muscle strength Room temperature 23 ± 2 ℃, humidity 55 ± 10% In a breeding environment where the lighting time was set to 7-19 o'clock, after acclimatizing for 7 weeks to male C57BL / 6J mice (purchased from Oriental Bioservice) for one week, individual breeding Accustomed. Thereafter, 32 mice were selected based on body weight. These 32 mice were divided into 16 non-exercise condition breeding groups (subject group: individual breeding) and 16 voluntary exercise condition breeding groups (exercise group: individual breeding in a cage with a rotating cage). Test mice (trade name: CE-2, manufactured by CLEA Japan, Inc.) Breeding these 2 groups of mice under free feeding, dissecting 8 animals in each group in the first and second months of exercise did. For anatomy, sevoflurane (trade name, manufactured by Maruishi Pharmaceutical Co., Ltd.) was anesthetized under free feeding conditions, and then whole blood was collected from the inferior vena cava and the soleus muscle was collected. The muscle mass and strength of the collected soleus were measured, and immediately frozen with liquid nitrogen and stored at -80 ° C. FIG. 1 shows the result of the muscle mass of the soleus two months after the start of exercise, and FIG. 2 shows the result of the muscle strength of the soleus two months after the start of exercise.
The soleus muscle strength was measured by fixing the soleus muscle of the left limb to the transducer (trade name: FORT100, manufactured by World Precision Instruments) with a suture thread (# 5-0 silk), and 37 ° C Krebs solution (95% After soaking in O ₂ , 5% CO ₂ aeration, pH 7.2) and applying twitch contraction by applying electrical stimulation from two platinum electrodes, the signal obtained from the transducer was evaluated as muscle strength.

(2) Measurement of expression level of MYBPH gene Total RNA was extracted from the preserved soleus muscle using RNeasy Fibrous Mini Kit (trade name, manufactured by Qiagen). The extracted total RNA was prepared at the same concentration, heat-treated at 65 ° C. for 10 minutes, quenched, and used for the reverse transcription reaction shown below.
Reverse transcription reaction solution {1 × PCR buffer 2 (trade name, manufactured by Applied Biosystems), 5 mM MgCl ₂ , 1 mM dNTP mix, 2.5 μM oligo d (T) 18 (trade name, New England) Biolabs), 1U / μL Rnase inhibitor (trade name, manufactured by Takara Bio Inc.) and mixed with 20 μL, (42 ° C, 1 hour) → (52 ° C, 30 minutes) → (99 ° C, 5 minutes) → Reverse transcription reaction was performed at 4 ° C. The obtained cDNA sample was stored at -20 ° C.
Further, as a standard for real-time PCR shown below, reverse transcription reaction was performed on 500 ng of RNA in the same reaction system.

  Using the obtained cDNA as a template, mix with PCR reaction solution {Fast SYBR (trade name, Applied Biosystems), 1 μM Forward Primer (SEQ ID NO: 1), 1 μM Reverse Primer (SEQ ID NO: 2)}, 7500 Fast Real-time PCR was performed on MYBPH mRNA using Real-Time PCR System (trade name, manufactured by Applied Biosystems). Real-time PCR was performed using a cDNA prepared by diluting the cDNA obtained by reverse transcription for the standard in 7 steps as a standard cDNA, and the expression level of MYBPH mRNA was analyzed based on the prepared calibration curve. The obtained analysis results were corrected by the expression level of the internal standard gene (36B4 gene) measured using the primers having the nucleotide sequences shown in SEQ ID NOs: 3 and 4, and expressed as relative mRNA expression levels. The obtained results are shown in FIG.

As shown in FIG. 1, the soleus weight increased significantly in the exercise group in the second month from the start of exercise. However, as shown in FIG. 2, no significant difference was found in the soleus muscle strength even after two months from the start of exercise.
In addition, as shown in FIG. 3, the expression level of MYBPH mRNA was significantly increased in the exercise group in the first month from the start of exercise.
These results indicate that when the expression level of the MYBPH gene increases during muscle formation (about 1 month from the start of exercise), the weight of the soleus muscle increases 2 months after the start of exercise. In other words, these results show that transient expression of MYBPH is required at any stage of the process of increasing soleus muscle mass, and individuals who cannot increase MYBPH expression during myogenesis This suggests that muscle mass and motor function cannot be maintained or enhanced.

Test example 2 Measurement of the expression level of MYBPH when the cooperative muscle is excised (1) Measurement of the muscle mass of the plantar muscle
C57BL / 6J mice (purchased from Oriental Bioservice, 16 weeks old) were divided into 3 non-treated groups and 3 treated groups. Mice and soleus muscles of the mice in the treatment group were excised (SA, Synergist Ablation operation) to increase the load on the plantar muscles.
Mice of the non-treated group and the treated group were bred, and plantar muscles were collected from the mice of each group 3 days after the excision of the scissor and soleus muscles of the treated group of mice. The muscle mass of the collected plantar muscles was measured, and immediately frozen with liquid nitrogen and stored at -80 ° C. The result of the measured muscle mass of the plantar muscle is shown in FIG.

(2) Measurement of expression level of MYBPH gene The expression level of MYBPH mRNA in the preserved plantar muscle was measured in the same manner as in Test Example 1. The obtained results are shown in FIG.

As shown in FIGS. 4 and 5, the weight of plantar muscles increased significantly in the treatment group, and the expression level of MYBPH mRNA increased significantly.
When the soleus and hifuku muscles, which are the cooperative muscles of the plantar muscles, are excised, a large load is applied to the plantar muscles. Therefore, the results shown in FIGS. 4 and 5 indicate that the expression level of the MYBPH gene increases when a large load is applied to the plantar muscles and the muscle mass is increased in a short period of time.

Test Example 3 Measurement of MYBPH expression level when re-grounding after tail suspension
Balb / c mice (purchased from Charles River, 10 weeks old) were deactivated by hindlimb suspension for 1 week, and then the tail suspension was released (reground). Soleus muscle was collected 3 days after re-grounding.
The muscle mass of the mouse after tail suspension and the soleus after re-grounding was measured, and immediately frozen with liquid nitrogen and stored at -80 ° C. The result of the measured amount of soleus muscle is shown in FIG.

(2) Measurement of expression level of MYBPH gene The expression level of MYBPH mRNA in the preserved soleus muscle was measured in the same manner as in Test Example 1. The obtained results are shown in FIG.

As shown in FIGS. 6 and 7, the weight of soleus muscle significantly increased after re-grounding, and the expression level of MYBPH mRNA significantly increased.
These results indicate that when the muscle mass is increased in a short period of time by re-grounding after tail suspension, the expression level of the MYBPH gene increases with increasing muscle mass.

Test Example 4 Measurement of MYBPH expression during skeletal muscle cell proliferation and differentiation (1) Measurement of mRNA expression Mouse skeletal muscle cell line C2C12 was measured at 0.5 × 10 ⁵ cells / well or 1.5 × 10 ⁵ cells / well. In a 24-well plate (n = 3) and DMEM medium containing 10% fetal bovine serum (FBS, AusGeneX) {growth medium, 10% FBS-DMEM; 10% FBS, 1% Pen Strep (Gibco) Made)}. 24 hours after seeding of the cells, cell samples in the growth area were collected (cell density: 80% confluent). Furthermore, DMEM medium (differentiation induction medium, 2% HS) containing 2% horse serum (HS, manufactured by Kohjin Bio) 48 hours after seeding (cell density: 100% confluent) for cell samples in the differentiation induction zone. -DMEM: 2% HS, 1% Pen Strep (Gibco)} was used to induce differentiation. Cell samples were collected 0 hours, 8 hours, 24 hours, 48 hours, 72 hours, and 120 hours after differentiation induction.

Total RNA was extracted from the collected cell samples using RNeasy Mini Kit (trade name, manufactured by QIAGEN), reverse transcription reaction was performed in the same manner as in Test Example 1, and the expression level of MYBPH mRNA was analyzed. The obtained analysis results were corrected with the expression level of the internal standard gene (GAPDH) measured using the primers consisting of the nucleotide sequences shown in SEQ ID NOs: 5 and 6, and expressed as relative mRNA expression levels. The results obtained in this way are shown in FIG.
As shown in FIG. 8, the expression level of MYBPH mRNA increased with skeletal muscle cell differentiation.

(2) Measurement of protein expression level The mouse skeletal muscle cell line C2C12 was seeded on a 6-well plate at 0.5 × 10 ⁵ cells / well or 1.5 × 10 ⁵ cells / well (n = 3), and 10% bovine It was maintained using a DMEM medium {Growth medium, 10% FBS-DMEM; 10% FBS, 1% Pen Strep (Gibco)} containing fetal serum (FBS, AusGeneX). 24 hours after seeding of the cells, cell samples in the growth area were collected (cell density: 80% confluent). Furthermore, DMEM medium (differentiation induction medium, 2% HS) containing 2% horse serum (HS, manufactured by Kohjin Bio) 48 hours after seeding (cell density: 100% confluent) for cell samples in the differentiation induction zone. -DMEM: 2% HS, 1% Pen Strep (Gibco)} was used to induce differentiation. Cell samples were collected 0 hours, 8 hours, 24 hours, 48 hours, 72 hours, and 120 hours after differentiation induction.

  Each collected cell sample was mixed with 100 μL of CelLytic M (trade name, manufactured by Sigma) containing 1% Protease Inhibitor Cocktail (manufactured by Sigma) and stored at −80 ° C. The obtained mixed solution was centrifuged at 15,000 rpm and 4 ° C. for 15 minutes, and the supernatant was recovered to prepare a protein solution. The concentration of the protein solution was adjusted, 4 × SDS Sample Buffer was added (protein solution: (Sample Buffer) = 3: 1 (volume ratio)), and heat treatment was performed at 99 ° C. for 10 minutes.

SDS-PAGE was performed on 20 μg of the protein solution treated with SDS, and the protein was transferred to Immobilon-P (PVDF membrane, manufactured by Millipore) treated with methanol for 1 minute. The transferred PVDF membrane was immersed in PVDF Blocking Reagent (trade name, manufactured by TOYOBO) and subjected to blocking treatment at room temperature for 1 hour. Then, anti-MYBPH antibody (1: 1000, manufactured by Aviva Systems Biology), anti-myosin heavy chain (hereinafter also referred to as “MyHC”) antibody (1: 600, manufactured by Developmental studies hybridoma bank; MF20), anti-β-actin The antibody (1: 1000, manufactured by Cell Signaling) is diluted with Can Get Signal Solution 1 (trade name, manufactured by TOYOBO) to a concentration suitable for each, and these solutions are used at 4 ° C overnight. Primary antibody treatment was performed.
After washing the membrane with 0.1% Tween20-TBS (T-TBS, manufactured by Bio-Rad), anti-rabbit IgG antibody (for detecting MYBPH and β-actin, manufactured by Cell signaling) or anti-mouse IgG antibody (for detecting MyHC) Cell Signal) was diluted 1000 times with Can Get Signal Solution 2 (trade name, manufactured by TOYOBO) to prepare a secondary antibody solution, which was then subjected to secondary antibody treatment at room temperature for 1 hour. After washing with T-TBS, treatment with LumiGLO Reagent and Peroxide (trade name, manufactured by Cell signaling) for 5 minutes causes chemiluminescence, and the luminescence intensity is measured with ChemiDoc XRS (trade name, manufactured by Bio Rad). And expression levels of MyHC and β-actin.

The results obtained in this way are shown in FIG. Here, the upper part of FIG. 9 is a graph showing the relative value of the expression level of MYBPH with respect to the expression level of β-actin. is there.
As shown in FIG. 9, the expression level of MYBPH increased with the differentiation of myocytes.

Test Example 5 Influence of RNA interference on MYBPH expression level and myotube hypertrophy in skeletal muscle cells (1) Preparation of siRNA solution
Two types of small interfering RNA (siRNA) solutions (MYBPH siRNA1 and MYBPH siRNA2) (Silencer Select Pre-designed and Validated siRNA, Ambion) and negative control siRNA solution (Silencer Select) specifically designed for MYBPH mRNA Negative Control # 2 siRNA (manufactured by Ambion) was prepared to a final concentration of 25 nM.

(2) Measurement of mRNA expression level The mouse skeletal muscle-derived cell line C2C12 was seeded on a 24-well plate at 0.5 × 10 ⁵ cells / well or 1.5 × 10 ⁵ cells / well (n = 3).
24 hours after cell seeding, C2C12 was transfected with siRNA as described below using the siRNA solution and Lipofectamine RNAiMAX Transfection Reagent (trade name, manufactured by Invitrogen) according to the attached protocol.
First, 500 μL of 10% FBS-DMEM without antibiotics was added to a 24-well plate. Then, the 20 μM siRNA stock solution was diluted with Opti-MEM I Reduced-Serum Medium (trade name, manufactured by Invitrogen) to 300 μM (12 times the final concentration). On the other hand, Lipofectamine RNAiMAX Transfection Reagent was diluted to 2% with Opti-MEM I Reduced-Serum Medium (trade name, manufactured by Invitrogen). Each of these solutions was allowed to stand for 5 minutes. Thereafter, both solutions were mixed and reacted for 10 minutes. 100 μL of the mixed solution after the reaction was added to a 24-well plate.

24 hours after the start of siRNA transfection (cell density: 100% confluent), cells were induced to differentiate using 2% HS-DMEM, and cell samples were collected 48, 72, and 120 hours later.
Total RNA was extracted from the collected cell samples using RNeasy Mini Kit (trade name, manufactured by QIAGEN), reverse transcription reaction was performed in the same manner as in Test Example 1, and the expression level of MYBPH mRNA was analyzed. . The obtained analysis results were corrected by the expression level of the internal standard gene (GAPDH) and expressed as a relative mRNA expression level.
The results obtained in this way are shown in FIG. As shown in FIG. 10, the expression level of MYBPH mRNA decreased due to RNA interference.

(3) Measurement of protein expression level The mouse skeletal muscle-derived cell line C2C12 was seeded on a 6-well plate at 0.5 × 10 ⁵ cells / well or 1.5 × 10 ⁵ cells / well (n = 3). 24 hours after cell seeding, C2C12 was transfected with siRNA in the same manner as described above using the siRNA solution and Lipofectamine RNAiMAX Transfection Reagent (trade name, manufactured by Invitrogen). Then, 24 hours after the start of siRNA transfection (cell density: 100% confluent), cells were induced to differentiate using 2% HS-DMEM, and cell samples were collected 48, 72, and 120 hours later.
For each collected cell sample, the expression levels of MYBPH, MyHC and β-actin were measured in the same manner as in Test Example 2. The results thus obtained are shown in FIG. 11 (MYBPH expression level) and FIG. 12 (MyHC expression level).
As shown in FIGS. 11 and 12, the expression level of MYBPH and MyHC, which is a differentiation marker for skeletal muscle cells, decreased due to RNA interference.

(4) Detection of myosin heavy chain The mouse skeletal muscle-derived cell line C2C12 was seeded on a 6-well plate at 0.5 × 10 ⁵ cells / well or 1.5 × 10 ⁵ cells / well (n = 3). 24 hours after cell seeding, C2C12 was transfected with siRNA in the same manner as described above using the siRNA solution and Lipofectamine RNAiMAX Transfection Reagent (trade name, manufactured by Invitrogen). Then, 24 hours after the start of siRNA transfection (cell density: 100% confluent), cells were induced to differentiate using 2% HS-DMEM, and cell samples were collected 48, 72, and 120 hours later.

Cultured cover glass (Poly-L-Lysine coat, manufactured by Matsunami Glass Kogyo Co., Ltd.) was placed on the bottom, and the recovered cells were seeded on a 6-well plate coated with Fibronectin (used at 100-fold dilution, manufactured by Sigma). The cells were fixed with 4% paraformaldehyde / phosphate buffer (manufactured by Wako Pure Chemical Industries, Ltd.), and then washed with PBS (manufactured by GIBCO). Thereafter, the cells were solubilized for 10 minutes at room temperature using a 0.2% TritonX-100 (Sigma) -PBS solution. After washing with PBS, the cells were blocked for 30 minutes at room temperature using a 5% BSA (Sigma) -PBS solution. Thereafter, anti-MyHC antibody (1: 600, manufactured by Developmental studies hybridoma bank; MF20) was diluted with 5% BSA-PBS so as to be 6 μg / mL, and this solution was treated at room temperature for 1 hour.
After washing with PBS, the secondary antibody solution {Alexa Fluor 488 Donkey Anti-mouse IgG (manufactured by Invitrogen) was diluted 500-fold with 5% BSA-PBS} was used to treat the secondary antibody for 1 hour. .
After washing with PBS, cells were encapsulated using Prolong Gold antifade reagent with DAPI (manufactured by Invitrogen), cell morphology observation and photography were performed, and myotube diameter was measured. The myotube diameter was measured using an all-in-one microscope (trade name: BZ-9000, manufactured by Keyence Corporation) and the attached image analysis software, and about 30 myotube diameters per image for 5 images taken per well. Were measured, and the average value was calculated.

FIG. 13 shows micrographs of cells 48 hours, 72 hours and 120 hours after differentiation induction. When treated with a negative control siRNA solution, myotubes became thicker with differentiation induction (for example, FIG. 13 (b)). On the other hand, when treated with the MYBPH siRNA solution, the myotube reached the culture limit without being completely thickened and peeled off (see, for example, FIG. 13C). Thus, myotube hypertrophy was suppressed by suppressing the expression of MYBPH mRNA by RNA interference.
Moreover, the average myotube diameter after 72 hours and 120 hours after differentiation induction is shown in FIG. As shown in FIG. 14, by suppressing the expression of MYBPH mRNA by RNA interference, the average myotube diameter was decreased and myotube hypertrophy was suppressed.

Test example 6 Knockout test of MYBPH gene in zebrafish (1) Search for MYBPH gene in zebrafish NCBI (National Center for Biotechnology Information, http: //www.ncbi.nlm) Using the homoloGene search of .nih.gov /), the MYBPH gene of zebrafish was searched. As a result, it was found that zebrafish has two homologous genes (MYBPHa gene and MYBPHb gene) present in different chromosomes. Furthermore, the sequence near the start codon of MYBPHa gene and MYBPHb gene was performed. As a result, the base sequences of these genes differ from the information of NCBI (http://www.ncbi.nlm.nih.gov/) and Ensembl (http://asia.ensembl.org/index.html). Confirmed that there is no.
Therefore, as shown below, a double knockout zebrafish in which all these two genes were knocked out was prepared.

(2) Synthesis of TALEN mRNA
Amplification of MYBPHa gene and MYBPHb gene knockout TAL effector nuclease (TALEN) -encoded plasmid (Wako Pure Chemical Industries, Ltd.), and the amplified TALEN plasmid was subjected to a restriction enzyme treatment solution {10 μL of each TALEN plasmid, 10 × M buffer (TOYOBO Co.) 5μL, dH ₂ O 34μL, overnight restricted with 37 ° C. with the restriction enzyme Hin dIII (TOYOBO Co. Ltd.) 1 [mu] L}.
The obtained digest was purified using QIAquick PCR Purification Kit (trade name, manufactured by Qiagen). Then, using the purified solution as a template, TALEN mRNA subjected to RNA synthesis, cap structure addition, and PolyA addition was prepared using mMESSAGE mMACHINE Kit (trade name, manufactured by Applied Biosystems). Here, DNase treatment was performed to decompose the contaminated plasmid.
The product thus obtained was purified using RNeasy MiniElute Cleanup Kit (trade name, manufactured by Qiagen) to obtain a purified TALEN mRNA solution. The purified TALEN mRNA was analyzed with an Agilent 2100 bioanalyzer (trade name, manufactured by Agilent Technologies), and it was confirmed that RNA was not degraded.

(3) Microinjection of TALEN mRNA
Injection solution containing both MYBPHa-specific TALEN mRNA and MYBPHb-specific TALEN mRNA {TALEN (L) MYBPHa mRNA 3 μL, TALEN (R) MYBPHa mRNA 3 μL, TALEN (L) MYBPHb mRNA 3 μL, TALEN (R) MYBPHb mRNA 3 μL, A phenol red solution 2 μL, dH ₂ O 6 μL} was prepared and injected into about 100 zebrafish of several nL at a time in one cell stage embryos (excluding egg yolk). Here, the same injection operation was performed on about 100 control groups in which an injection solution containing no TALEN mRNA was injected.
An embryo (fertilized egg) after injection may be mixed with an unfertilized egg. In addition, it may be damaged by the injection operation and a dead egg may be generated. Therefore, the dead eggs were removed twice a day.

Five days after fertilization, the morphology and behavior of the larvae were observed under a microscope, and a video was taken. The result is shown in FIG. Here, FIG. 15 (a) shows a micrograph of zebrafish in the control group, and FIG. 15 (b) shows a micrograph of zebrafish microinjected with TALEN mRNA.
The zebrafish in the control group shown in FIG. 15 (a) was normal in both form and behavior. On the other hand, as shown in FIG. 15 (b), when TALEN mRNA was microinjected into zebrafish, it was confirmed that individuals with low exercise ability appeared.

(4) T7 endonuclease 1 assay
Of the larvae that grew for 5 days after microinjection of TALEN mRNA, 1 individual from the control group and 3 individuals with reduced motor function were recovered from the double knockout group.
Genomic DNA was extracted from the collected samples using a Dneasy blood & tissue mini kit (trade name, manufactured by Qiagen). Using extracted genomic DNA as a template, including Tks Gflex DNA Polymerase (trade name, manufactured by Takara Bio Inc.), MYBPHa gene detection primer (SEQ ID NO: 7 and 8) and MYBPHb gene detection primer (SEQ ID NO: 9 and 10) PCR reaction solution {2 × Gflex PCR buffer (trade name, manufactured by Takara Bio Inc.) 1 μL, 100 μM Forward primer 0.1 μL, 100 μM Reverse primer 0.1 μL, dH ₂ O 23 μL, Tks Gflex DNA Polymerase (trade name, manufactured by Takara Bio Inc.) 1 μL} was used to amplify the vicinity of the first exon of the MYBPHa gene and the MYBPHb gene. PCR conditions were (94 ° C. for 1 minute) → (98 ° C. for 10 seconds → MYBPHa: 54 ° C./MYBPHb: 58 ° C. for 15 seconds → 68 ° C. for 1 minute 20 seconds) × 38 cycles → 68 ° C. for 3 minutes → 4 ° C.

The obtained PCR product was purified using QIAquick PCR Purification Kit (manufactured by Qiagen). Prepare T7 endonuclease 1 reaction solution {200 × NEB2 Buffer (manufactured by NEW ENGLAND BioLabs), 1.9 μL, (dH ₂ O + sample solution): 17.1 μL} containing 200 ng of the purified PCR product, 95 ° C. for 10 minutes → (95 Melting reaction and re-annealing reaction were carried out at -2 ° C / second from 85 ° C to 85 ° C-> (cooled at -0.3 ° C / second from 85 ° C to 25 ° C)-> 10 seconds-25 ° C. Thereafter, 1 μL of T7 endonuclease 1 (manufactured by NEW ENGLAND BioLabs) was added to each sample (19 μL), and T7 endonuclease 1 treatment was performed at 37 ° C. for 15 minutes.
The sample treated with T7 endonuclease 1 was subjected to electrophoresis using a 1% agarose gel, and a DNA band was photographed using BioDoc-It 220 Imaging System (trade name, manufactured by BM Instruments Co., Ltd.). The result is shown in FIG. FIG. 16 (a) shows the results for the MYBPHa gene, and FIG. 16 (b) shows the results for the MYBPHb gene.

As shown in FIG. 16, there is no mismatched double-stranded DNA in the genomic DNA extracted from the control individual. That is, cleavage by genomic DNA mismatch by T7 endonuclease 1 did not occur, and multiple bands derived from the MYBPHa gene and MYBPHb gene were not detected.
On the other hand, when the MYBPHa gene and the MYBPHb gene are knocked out by Talen mRNA, there is a mismatch in the double-stranded DNA in all individuals with reduced motor function. For this reason, since the mismatch portion of the DNA in which the mismatch exists is cleaved by T7 endonuclease, two low-molecular-weight bands were detected in addition to the PCR amplification product band.

  As described above, the expression of the MYBPH gene derived from skeletal muscle is suppressed in individuals with reduced motor function. Therefore, it was confirmed that there is a correlation between the motor function and the expression level of the MYBPH gene.

Test Example 7 Measurement of the expression level of MYBPH gene in cypress muscle after exercise test of mice
10 weeks old male C57BL / 6J mice (purchased from Charles River Japan) were trained for 1 week to acclimate to the environment, and then trained to acclimate to the treadmill running exercise for 5 days as follows went.

Day 1: 10m / min (15min) → 15m / min (15min)
Day 2: 10m / min (10min) → 15m / min (20min)
Day 3: 15m / min (15min) → 20m / min (15min)
Day 4: 15m / min (5min) → 20m / min (15min) → 25m / min (10min)
Day 5: 15m / min (5min) → 20m / min (10min) → 25m / min (15min)

Thereafter, the mice were divided into 3 groups so that the body weights were equal (N = 4 / group). Of these, two groups were run for 30 minutes with a running speed of 25m / min. The remaining one group was a rest group.
The exercise groups were dissected 1 hour and 6 hours after exercise, and the cypress muscles of each group were collected.

Total RNA was extracted from cypress muscle using an RNA extraction kit (trade name: RNeasy Fibrous Kit, manufactured by QIAGEN). For the total RNA quality check, BioAnalyzer (manufactured by Agilent technology) was used, and it was confirmed that the RIN (RNA integrity number) value of all the samples was 7.0 or more and the quality was sufficient for microarrays.
Using the total RNA after the quality check, microarray was performed as follows. As the array slide, Agilent SurePrint G3 manufactured by Agilent was used. The labeled cRNA was prepared according to the Agilent protocol. The array slides after hybridization and washing were immediately filled with nitrogen and shielded from light, and scanned by requesting Hokkaido System Science Co., Ltd.
After scanning, the QC report confirmed that all array slides had no technical problems and analyzed the data. Normalization was performed using the 75% method (Scaling) recommended by Agilent, using the analysis software GeneSpring. Thereafter, the signal value of MYBPH in the 1 hour group after exercise and the 6 hour group after exercise was compared with that in the rest group. The results are shown in Table 3.

  As shown in Table 3, when the mouse was loaded with treadmill exercise, the expression level of the MYBPH gene increased.

  As shown in Test Examples 1 to 7, there is a correlation between the expression level of the MYBPH or MYBPH gene and the motor function. Therefore, the marker of the present invention containing MYBPH or MYBPH gene can be used as an indicator of motor function, muscle mass, degree of skeletal muscle cell differentiation, and degree of myotube hypertrophy.

Claims (8)

  A marker for evaluating motor function, comprising myosin binding protein H derived from skeletal muscle collected from an individual or a gene encoding the same.   A marker for evaluating muscle mass, comprising myosin binding protein H derived from skeletal muscle collected from an individual or a gene encoding the same.   A marker for evaluating the differentiation of skeletal muscle cells, comprising skeletal muscle-derived myosin binding protein H collected from an individual or a gene encoding the same.   A marker for evaluating myotube hypertrophy comprising myosin binding protein H derived from skeletal muscle collected from an individual or a gene encoding the same. Measuring the expression level of myosin binding protein H derived from skeletal muscle collected from an individual or a gene encoding the same,
Evaluate the individual's motor function based on the measured expression level,
Evaluation method of motor function. Measuring the expression level of myosin binding protein H derived from skeletal muscle collected from an individual or a gene encoding the same,
Evaluate muscle mass based on measured expression levels,
Evaluation method of muscle mass. Measuring the expression level of myosin binding protein H derived from skeletal muscle collected from an individual or a gene encoding the same,
Evaluate the degree of differentiation of skeletal muscle cells based on the measured expression level,
A method for evaluating differentiation of skeletal muscle cells. Measuring the expression level of myosin binding protein H derived from skeletal muscle collected from an individual or a gene encoding the same,
Evaluate the degree of myotube hypertrophy based on the measured expression level,
Evaluation method of myotube hypertrophy.