JP6539511B2 - Marker for judgment of motor function - Google Patents

Description

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

Locomotive syndrome refers to a musculoskeletal disorder caused by aging and the like. The prevalence of locomotive syndrome increases with age, and as locomotive syndrome progresses, the risk of needing nursing care is greatly increased.
So far, studies on genes and proteins related to age-related diseases of exercise organs such as locomotive syndrome have been advanced. However, to date no factor has been identified that is related to motor disease.

On the other hand, with regard to myosin binding protein H (hereinafter also referred to simply as "MYBPH"), knocking down MYBPH in human lung adenocarcinoma cell lines promotes cell migration, and MYBPH in canine renal tubular epithelium-derived cell lines It is known that overexpression suppresses cell migration and invasion (see Non-patent Document 1). In addition, it is known that double knockdown of MYBPH and myosin binding protein C in a rat heart-derived cell line suppresses cell contraction due to adrenergic stimulation (see non-patent document 2). Furthermore, the use of MYBPH as a protein biomarker for detecting skeletal muscle damage caused by side effects (myotoxicity) by administration of pharmaceuticals and 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 age-related diseases of motor function and exercise equipment necessary for daily life.

WO 2007/066129 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 the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy in an individual.
Another object of the present invention is to provide a method for evaluating the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy using the above-mentioned biomarkers.

The present inventors diligently studied in view of the above problems.
It is known that many proteins are expressed in skeletal muscle which is one of the movement organs and is connected to a 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") indicates the motor function of the individual, muscle mass, differentiation of skeletal muscle cells It was found that there is a correlation between the degree of hypertrophy and myotube hypertrophy. And, it was found that this MYBPH or MYBPH gene is useful as a biomarker for evaluating the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy in an individual. Furthermore, the expression level of the MYBPH or MYBPH gene collected from the individual can be measured, and the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, and the degree of myotube hypertrophy can be evaluated based on the measured expression level. Found out.
The present invention has been completed based on these findings.

The present invention relates to a marker for evaluating motor function, which comprises skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same.
The present invention also relates to a marker for evaluating muscle mass, which comprises skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same.
The present invention also relates to a marker for evaluating differentiation of skeletal muscle cells, which comprises 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, which comprises skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same.

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

The biomarkers of the invention can be used to assess the individual's motor function, muscle mass, degree of differentiation of skeletal muscle cells, or degree of myotube hypertrophy.
The evaluation method of the present invention can accurately and simply evaluate the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy. Further, based on the evaluation method of the present invention, the present invention 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 the mouse | mouth of each of a control group and an exercise group two months after an exercise | movement measurement measured by Experiment 1. FIG. It is a graph which shows the muscle strength of the soleus muscle of the mouse | mouth of each of a control group and an exercise group 2 months after an exercise start measured by Experiment 1. FIG. It is a graph which shows the expression level of the MYBPH gene in the flatfish muscle of the mouse | mouth of the control group and the exercise group 1 month and 2 months after an exercise start measured by Experiment 1. FIG. It is a graph which shows the muscle mass of the plantar muscle of the mouse | mouth of each non-operation group of the joint muscle excision of a plantar muscle and each operation group which were measured by Experiment 2. FIG. It is a graph which shows the expression level of the MYBPH gene in the plantar muscles of the non-operation group of the joint muscle excision of the plantar muscle and the operation group of each measured in Test Example 2. It is a graph which shows the muscle mass of the soleus muscle of each mouse after tail suspension vertical and reground measured in Experiment 3. It is a graph which shows the expression level of the MYBPH gene in the soleus muscle of each mouse after tail suspension vertical and reground measured in Test Example 3. It is a graph which shows the expression level of the MYBPH gene in the growth section and differentiation process of the mouse | mouth skeletal muscle origin cell line measured by Experiment 4. FIG. FIG. 7 is a view showing the expression level of MYBPH in the growth zone and differentiation process of the mouse skeletal muscle-derived cell line measured in Test Example 4. It is a graph which shows the change of the expression level of MYBPH gene by the influence of RNA interference measured by Experiment 5. It is a graph which shows the change of the expression level of MYBPH by the influence of RNA interference measured by Experiment 5. It is a graph which shows the change of the expression level of myosin heavy chain by the influence of RNA interference measured by Experiment 5. It is the microscope picture which image | photographed the mode of the expression of myosin heavy chain in the mouse | mouth skeletal muscle origin cell line measured by Experiment 5, (a) is a microscope picture image | photographed 48 hours after differentiation induction, (b) It is a microscope picture image | photographed 72 hours after differentiation induction, (c) is a microscope picture image | photographed 120 hours after differentiation induction. The upper part is a photomicrograph when no RNA interference was performed, and the lower part is a photomicrograph when RNA interference was performed. It is a graph which shows the change of the average myotube diameter by the influence of RNA interference measured in Experiment 5. FIG. 15 (a) shows a photomicrograph of the zebrafish in the control group, and FIG. 15 (b) shows a photomicrograph of the zebrafish microinjected with TALEN mRNA. It is an electrophoresis photograph which shows the appearance of the expression of the MYBPHa gene and the MYBPHb gene in the zebrafish which knocked out the MYBPHa gene and the MYBPHb gene performed by Experiment 6. FIG.

In the present specification, "evaluation" or "determination" regarding data or measured values etc. refers to analysis of data or measured values obtained from a certain sample (group) according to a predetermined purpose, and based on the analyzed information. It is meant to conclude whether the sample (s) have specific characteristics, trends, trends.
Further, in the present invention, the term "motor function" refers to the exercise ability of a motion organ that constitutes, supports and enables physical exercise.

  The marker for evaluating motor function, the marker for evaluating muscle mass, the marker for evaluating differentiation of skeletal muscle cells, and the marker for evaluating myotube hypertrophy according to the present invention (hereinafter collectively referred to simply as "the marker of the present invention" 2.) contains skeletal muscle-derived MYBPH or MYBPH gene collected from an individual.

As shown in Examples described later, the expression levels of skeletal muscle-derived MYBPH and MYBPH genes contained in the marker of the present invention are the motor function of the individual, muscle mass, and the degree of differentiation of skeletal muscle cells. And myotube hypertrophy have correlation. The present inventors found that the expression levels of MYBPH and MYBPH genes are significantly increased with the improvement of motor function, the increase of muscle mass, the progress of differentiation of skeletal muscle cells, and myotube hypertrophy.
Therefore, the marker of the present invention can be used as an indicator of motor function, muscle mass, degree of differentiation of skeletal muscle cells, and degree of myotube hypertrophy. Furthermore, the marker of the present invention can be suitably used in a method for simply and accurately evaluating the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, and the degree of myotube hypertrophy.

  Patent Document 1 describes a biomarker composed of MYBPH for detecting a skeletal muscle disorder caused by a side effect (muscle toxicity) by administration of a drug or the like. On the other hand, the marker of the present invention is a biomarker for determining and evaluating age-related diseases of motor function and exercise equipment necessary for daily life. 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 is available from various databases. For example, the amino acid sequence of mouse-derived MYBPH is NCBI RefSeq as ID: NP_058029, the amino acid sequence of zebrafish-derived MYBPH (MYBPHa and MYBPHb) as NCBI RefSeq as ID: NP_956852.1 and NM_001100137.1 (http: // www.ncbi.nlm.nih.gov/gene/393530 (see 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, and in these amino acid sequences, one or several, usually 1 to 100, preferably 1 to 75, more preferably 1 to 50, more preferably 1 to 25, particularly preferably 1 to 10, most preferably 1 to 5 amino acids consist of amino acid sequences with deletion, substitution, insertion and / or addition, and functionally equivalent proteins are also included Be In addition, it has homology of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, most preferably 99% or more to these amino acid sequences. Also included are proteins that consist of amino acid sequences and that are functionally equivalent. Here, the homology of amino acid sequences herein is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it is calculated by performing analysis with Unit size to compare (ktup) of 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

  The marker of the present invention consists of a polypeptide having a part of the amino acid sequence of MYBPH as described above, and is a polypeptide of a length specifically recognized by an antibody or peptide aptamer for obtaining information on the expression level of MYBPH. Is preferred. Here, “a polypeptide having a portion of the amino acid sequence of MYBPH” is a portion 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 nucleotide sequence of the MYBPH gene is available from various databases. For example, the nucleotide sequence of mouse-derived MYBPH gene is NCBI RefSeq as ID: NM_016749, the nucleotide sequence of zebrafish-derived MYBPH gene (MYBPHa gene and MYBPHb gene) is NCBI RefSeq as ID: NM_200558.1 and NP_001093607.1 ( Refer to http://www.ncbi.nlm.nih.gov/gene/339530, http://www.ncbi.nlm.nih.gov/gene/321053), and the nucleotide sequence of human-derived MYBPH as the ID of NCBI RefSeq. : Registered as NM_004997, respectively.
The MYBPH gene used in the present invention is not limited to these, and in these base sequences, one or several, usually 1 to 389, preferably 1 to 292, more preferably 1 to 195, more preferably Is composed of a nucleotide sequence having 1 to 98, particularly preferably 1 to 39, and most preferably 1 to 20, deletions, substitutions, insertions or additions of bases, and also includes the above-mentioned nucleic acid encoding MYBPH Be In addition, they have homology of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, most preferably 99% or more to these base sequences. Also included are nucleic acids consisting of base sequences and encoding the aforementioned MYBPH. Here, the homology of the nucleotide sequence is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it is calculated by performing analysis with Unit size to compare (ktup) of 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 is composed of a polyoligonucleotide having a part of the base sequence of the MYBPH gene described above, and is under stringent conditions with nucleic acid probes and nucleic acid aptamers for obtaining information on the expression level of the MYBPH gene It is preferred that the polynucleotide is a hybridizable length. 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% Conditions in which a solution containing SDS, 5 × Denhardt and 100 mg / mL herring sperm DNA is incubated with a probe at 65 ° C. for 8 to 16 hours and hybridized are mentioned.

  The method of evaluating motor function, the method of evaluating muscle mass, the method of evaluating differentiation of skeletal muscle cells, and the method of evaluating myotube hypertrophy according to the present invention (hereinafter collectively referred to as "the evaluation method of the present invention") The expression level of skeletal muscle-derived MYBPH or MYBPH gene collected from an individual is measured using the marker of the invention. Then, based on the measured expression level of the MYBPH or MYBPH gene, the motor function of the individual, the muscle mass, the degree of differentiation of skeletal muscle cells, or the 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 an experimental animal (mouse, rat, guinea pig, hamster, rabbit, zebrafish, etc.) The MYBPH or MYBPH gene is detected from cell lines cultured in culture. There is no particular limitation on the biological sample for detecting the MYBPH or MYBPH gene, but the MYBPH or MYBPH gene is collected from skeletal muscle, preferably soleus muscle, hip muscle or plantar muscle.

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

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

  As a method of measuring the expression level of skeletal muscle-derived MYBPH collected from an individual, immunoprecipitation, mass spectrometry, RIA using an antibody or peptide aptamer specifically binding to MYBPH prepared according to a conventional method (Radioimmunoassay), Western blotting, ELISA (enzyme-linked immunosorbent assay), ECLIA (electrochemiluminescence immunoassay), a method of measuring according to a conventional method such as microarray, etc. may be mentioned.

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

The expression levels of the MYBPH and MYBPH genes rise with the improvement of motor function, the increase of muscle mass, the progress of differentiation of skeletal muscle cells, and myotube hypertrophy. On the other hand, the expression levels of the MYBPH and MYBPH genes decrease with the decrease in motor function, the decrease in muscle mass, the non-progression of differentiation of skeletal muscle cells, and the contraction of myotubes.
Therefore, the expression level of the MYBPH or MYBPH gene derived from skeletal muscle collected from an individual is measured, and the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, or MYBPH collected from a standard individual with respect to myotubes Compare with the expression level of MYBPH gene. Then, when the measured expression level is increased, it is determined that the motor function has been improved, the muscle mass has been increased, the differentiation of skeletal muscle cells has progressed, or the myotube has been enlarged. On the other hand, when the expression level of the MYBPH or MYBPH gene is reduced, it is judged that the motor function is reduced, the muscle mass is reduced, the differentiation of skeletal muscle cells is not progressing, or the myotubes are contracted. Also, if no change in the expression level of MYBPH or MYBPH gene can be confirmed, the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, and myotubes are judged to be in a standard state. Alternatively, if the individual does not increase the expression level of the MYBPH or MYBPH gene even if an external stimulus such as exercise to increase muscle mass is given to the individual, the individual suffers from locomotive syndrome or suffers from locomotive syndrome Decide that the risk is high.

In the present invention, the expression level of MYBPH is measured, and a protein molecule of skeletal muscle is used to evaluate the motor function, muscle mass, the degree of differentiation of skeletal muscle cells or the degree of myotube hypertrophy based on the measured expression level. 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 the individual's motor function, muscle mass, the degree of differentiation of skeletal muscle cells, and the degree of myotube hypertrophy.
Examples of the protein molecule marker for skeletal muscle that is usually used include β-actin and the like.

In the present invention, the expression level of the MYBPH gene is measured, and the housekeeping gene (the muscle function, muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy is assessed based on the measured expression level. It is preferable to compare the expression level of the internal control gene) with the expression level of the MYBPH gene and correct the measured expression level of the MYBPH gene. Specifically, the expression level of the MYBPH gene is measured, corrected with the expression level of the housekeeping gene measured separately, and based on the corrected value, the exercise function of the individual, the muscle mass, the degree of differentiation of skeletal muscle cells, Alternatively, it is preferable to evaluate the degree of myotube hypertrophy. By comparing the expression levels in this manner, it is possible to accurately evaluate the individual's motor function, muscle mass, the degree of differentiation of skeletal muscle cells, and the degree of myotube hypertrophy.
Examples of 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 antibody for measuring the expression level of MYBPH or MYBPH gene, a peptide aptamer, a nucleic acid probe, a nucleic acid aptamer or a nucleic acid primer, the exercise function of an individual, the muscle mass, the degree of differentiation of skeletal muscle cells or Also provided is a kit for evaluation of the degree of myotube hypertrophy.
This kit may contain the aforementioned antibody for measuring the expression level of a protein molecule marker or a housekeeping gene, a peptide aptamer, a nucleic acid probe, a nucleic acid aptamer or a nucleic acid primer.

The present invention provides a preventive or ameliorating agent for locomotive syndrome, a promoter for improving the motor function, a promoter for preventing or improving the motor function decline, a promoter for increasing the muscle mass, a promoter for preventing or improving the muscle mass loss based on the above evaluation method. Also provided are screening methods for screening for skeletal muscle cell differentiation promoting agents, myotube hypertrophy promoting agents, and agents 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 a candidate substance of the agent, and before or after administration or intake of the substance, the individual evaluates the individual according to the evaluation method of the present invention. Substances that improve motor function, substances that increase muscle mass, substances that promote differentiation of skeletal muscle cells, or muscle that evaluate motor function, muscle mass, degree of differentiation of skeletal muscle cells, or degree of myotube hypertrophy Substances that enlarge the tube can be selected as these agents.
As used herein, "prevention" refers to preventing or delaying the onset of a disease or condition in an individual, or reducing the risk of developing an individual's disease or condition. Moreover, in the present specification, “improvement” refers to amelioration 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 for a method of detecting skeletal muscle cells in which differentiation has progressed and a method of 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 in which differentiation has progressed or skeletal muscle cells in which myotubes are enlarged .
The biomarkers of the present invention can also be suitably used as a method for evaluating the presence or absence of the risk of having the locomotive syndrome. In this evaluation method, the expression level of MYBPH or MYBPH gene derived from skeletal muscle cells of an individual subjected to external stimulation such as exercise to increase muscle mass is measured, and the expression level of MYBPH or MYBPH gene does not increase. It can be evaluated that the patient is suffering from or at high risk of suffering from locomotive syndrome.
Furthermore, the biomarkers of the present invention can also be suitably used in methods for evaluating the therapeutic or prophylactic effect of locomotive syndrome. When a subject suffering from locomotive syndrome is effective in the treatment or prevention of locomotive syndrome, the expression level of the MYBPH or MYBPH gene is also elevated accordingly. Therefore, the expression level of MYBPH or MYBPH gene can be measured before and after treatment or preventive measures for locomotive syndrome, and treatment or preventive measures can be evaluated to be effective if the expression levels are elevated.

  With regard to the embodiments described above, the present invention further discloses the following markers, uses, methods, evaluation methods, screening methods, and kits.

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

The marker as described in said <1> which consists of a polypeptide which has a part of amino acid sequence of <2> said MYBPH, or the polynucleotide which has a part of base sequence of said MYBPH gene.
<3> The marker according to <1> or <2>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene derived from flatfish muscle, hippocampus muscle or plantar muscle.
<4> Proteins whose MYBPH is 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 the amino acid sequences of these proteins 1 to 75, more preferably 1 to 50, more preferably 1 to 25, particularly preferably 1 to 10, most preferably 1 to 5 amino acids are deleted, substituted, inserted and / or added Or a functionally equivalent protein, or an amino acid sequence of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably an amino acid sequence of these proteins A functionally equivalent protein consisting of an amino acid sequence having a homology of 98% or more, most preferably 99% or more, which is the above <1> to Marker according to any one of 3>.
<5> Genes whose MYBPH genes are registered in NCBI RefSeq as NM_016749, NM_200558.1, NP_001093607.1, or NM_004997, and the nucleotide sequences of these genes: one or several, usually 1 to 389, preferably Is 1 to 292, more preferably 1 to 195, still more preferably 1 to 98, particularly preferably 1 to 39, most preferably 1 to 20 bases are deleted, substituted, inserted or added. And a nucleic acid encoding MYBPH, or a nucleotide sequence of these genes, 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% The marker according to any one of the above <1> to <4>, which is a nucleic acid consisting of a nucleotide sequence having homology of 99% or more, most preferably 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 that is 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 the above <7>, wherein the MYBPH gene is detected by any one of the nucleic acid primer sets 1 to 3 shown in Table 1 above.
<9> The above <1> to <7>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene collected 1 to 6 hours after the individual is loaded with an external stimulus that increases muscle mass such as exercise. The marker according to any one of the preceding claims.

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

<13> The polypeptide according to any one of <10> to <12>, wherein a polypeptide having a portion of the amino acid sequence of MYBPH or a polyoligonucleotide having a portion 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 an MYBPH or MYBPH gene derived from flatfish muscle, hippocampus muscle or plantar muscle.
<15> Proteins whose MYBPH is 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 the amino acid sequences of these proteins 1 to 75, more preferably 1 to 50, more preferably 1 to 25, particularly preferably 1 to 10, most preferably 1 to 5 amino acids are deleted, substituted, inserted and / or added Or a functionally equivalent protein, or an amino acid sequence of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably an amino acid sequence of these proteins A functionally equivalent protein consisting of an amino acid sequence having a homology of 98% or more, most preferably 99% or more, Use or method according to any one of - <14>.
<16> Genes whose MYBPH genes are registered in NCBI RefSeq as NM_016749, NM_200558.1, NP_001093607.1, or NM_004997, and the nucleotide sequences of these genes: one or several, usually 1 to 389, preferably Is 1 to 292, more preferably 1 to 195, still more preferably 1 to 98, particularly preferably 1 to 39, most preferably 1 to 20 bases are deleted, substituted, inserted or added. And a nucleic acid encoding MYBPH, or a nucleotide sequence of these genes, 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% The use according to any one of the above <10> to <15>, which is a nucleic acid consisting of a nucleotide sequence having the homology as described above, most preferably 99% or more and encoding MYBPH Method.
<17> The use according to any one of the above <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 the 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 above.
<20> The above <10> to <19>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene collected 1 to 6 hours after the individual is loaded with an external stimulus that increases muscle mass such as exercise. Use or method according to any one of the preceding paragraphs.

<21> measuring the expression level of MYBPH or MYBPH gene derived from skeletal muscle collected from an individual;
Evaluate the motor function, muscle mass, degree of differentiation of skeletal muscle cells, or degree of myotube hypertrophy based on the expression level measured, motor function, muscle mass, degree of differentiation of skeletal muscle cells, or myotube How to assess the degree of hypertrophy.

&Lt; 22 &gt; Motor function of the individual based on the expression level measured by measuring the expression level of the polypeptide having a part of the amino acid sequence of MYBPH or the polynucleotide having a part of the base sequence of the MYBPH gene 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 amount of the MYBPH or a gene encoding the same.
<24> The method according to any one of <21> to <23>, wherein the MYBPH or MYBPH gene is an MYBPH or MYBPH gene derived from a flatfish muscle, a calf muscle or a plantar muscle.
<25> Proteins whose MYBPH is 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 the amino acid sequences of these proteins 1 to 75, more preferably 1 to 50, more preferably 1 to 25, particularly preferably 1 to 10, most preferably 1 to 5 amino acids are deleted, substituted, inserted and / or added Or a functionally equivalent protein, or an amino acid sequence of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably an amino acid sequence of these proteins A functionally equivalent protein consisting of an amino acid sequence having a homology of 98% or more, most preferably 99% or more, The method according to any one of - <24>.
<26> Genes whose MYBPH genes are registered in NCBI RefSeq as NM_016749, NM_200558.1, NP_001093607.1, or NM_004997, and one or several, usually 1 to 389, in the base sequences of these genes, preferably Is 1 to 292, more preferably 1 to 195, still more preferably 1 to 98, particularly preferably 1 to 39, most preferably 1 to 20 bases are deleted, substituted, inserted or added. And a nucleic acid encoding MYBPH, or a nucleotide sequence of these genes, 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% The method according to any one of <21> to <25> above, which is a nucleic acid encoding a MYBPH-encoding nucleic acid consisting of a nucleotide sequence having homology of 99% or more, most preferably 99% or more.
The antibody or peptide aptamer specifically recognized as <27> MYBPH, preferably an antibody, detects MYBPH and measures the expression level of MYBPH according to any one of the above <21> to <26>. Method.
<28> The method according to any one of <21> to <27>, 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, 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 above, and the expression level of the MYBPH gene is measured.
<30> When the expression level of the measured MYBPH or MYBPH gene is increased, it is judged that the motor function is improved, the muscle mass is increased, the differentiation of skeletal muscle cells is advanced, or the myotube is expanded. The method according to any one of <21> to <29>.
<31> It is judged that the motor function was reduced, the muscle mass was decreased, the differentiation of skeletal muscle cells was not progressed, or the myotube was contracted when the expression level of the measured MYBPH or MYBPH gene was decreased The method according to any one of <21> to <30>.
<32> If no change is observed in the measured expression level of MYBPH or MYBPH gene, motor function, muscle mass, degree of differentiation of skeletal muscle cells, or myotubes are judged to be in a standard state, 21. The method according to any one of <31>.
<33> The expression level of skeletal muscle-derived MYBPH or MYBPH gene collected from an individual subjected to external stimulation to increase muscle mass is measured, and if the measured expression level does not increase, the individual has locomotive syndrome The method according to any one of <21> to <32>, which is judged to be afflicted with or at high risk of suffering from locomotive syndrome.
Any one of the above <21> to <33> which compares the expression level of a protein molecule marker of <34> skeletal muscle, preferably β-actin, with the expression level of MYBPH and corrects the measured expression level of MYBPH Method according to paragraph 1.
<35> The expression level of the MYBPH gene is compared with the expression level of the MYBPH gene by comparing the expression level of the housekeeping gene, preferably the 36B4 gene, preferably the GAPDH gene, or the β-actin gene, with the above <21>. The method according to any one of <34>.
<36> The above <21> to <35>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene collected 1 to 6 hours after the individual is loaded with an external stimulus that increases muscle mass such as exercise. The method according to any one of the preceding claims.

<37> Antibody for measuring the expression level of MYBPH or MYBPH gene, peptide aptamer, nucleic acid probe, nucleic acid aptamer or nucleic acid primer, motor function of the individual, muscle mass, degree of differentiation of skeletal muscle cells or muscle Evaluation kit for the degree of ductal hypertrophy.
<38> Antibody, peptide aptamer, nucleic acid probe for measuring the expression level of protein molecule marker of skeletal muscle, preferably β-actin, or 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> An agent for preventing or improving locomotive syndrome, an agent for improving motor function, an agent for preventing or improving motor function decrease, an agent for increasing muscle mass, an agent for preventing or improving muscle mass loss, a differentiation promoter for skeletal muscle cells A screening method for screening an agent for promoting myotube hypertrophy and an agent for preventing or improving myotube contraction,
Administration or ingestion of a candidate for the agent to a human or non-human animal or a cell line derived from skeletal muscle,
Before and after administration or intake of substance Based on the evaluation method according to any one of the above <21> to <35>, the individual's motor function, muscle mass, degree of differentiation of skeletal muscle cells, or myotube hypertrophy Evaluate 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 a myotube is selected as the agent.
<40> The expression level of MYBPH or MYBPH gene expressed in skeletal muscle cells is measured using the marker described in any one of <1> to <9>.
Detecting cells with high expression level of MYBPH or MYBPH gene as skeletal muscle cells or myotubes with enlarged differentiation;
A method of detecting a skeletal muscle cell in which differentiation has progressed or a skeletal muscle cell in which a myotube is enlarged.
<41> The expression level of MYBPH or MYBPH gene derived from a skeletal muscle cell is measured using the marker according to any one of <1> to <9>.
Measuring the expression level of MYBPH or MYBPH gene derived from skeletal muscle cells,
If external stimulation to increase muscle mass does not increase the expression level of MYBPH or MYBPH gene, the individual is evaluated as suffering from or at high risk of suffering from locomotive syndrome.
Method of evaluating presence or absence of or risk of suffering from locomotive syndrome.
<42> The expression level of the MYBPH or MYBPH gene expressed in skeletal muscle cells is measured using the marker described in any one of <1> to <9>.
If the expression level of MYBPH or MYBPH gene is increased, the treatment or preventive measure is evaluated as effective.
A method for evaluating the therapeutic or preventive effect of locomotive syndrome.

EXAMPLES The present invention will be described in more detail based on examples given below, but the invention is not meant to be limited by these.
Here, the base sequences of the primers used for amplification of various genes used in the following test examples and comparative examples are shown in Table 2. The following primers were designed to amplify a target gene fragment at a size of about 100 to 200 bp.

Test Example 1 Measurement of muscle mass of soleus muscle and expression level of MYBPH gene in solei muscle after exercise test of mouse (1) Measurement of muscle mass and muscle strength of solei muscle room temperature 23 ± 2 ° C., humidity 55 ± 10% Under the breeding environment where the lighting time is set to 7 to 19 o'clock, one week of environmental acclimation is performed on 7-week-old male C57BL / 6J mice (purchased from Oriental Bioservices Co., Ltd.), and then individualized breeding I got used to it. Thereafter, 32 mice were selected based on body weight. These 32 mice were divided into 16 non-motor condition rearing groups (target group: individual rearing) and 16 locomotion condition rearing groups (exercise group: individual rearing in a cage with a rotating cage). These two groups of mice are bred under a free diet on a test meal (trade name: CE-2, manufactured by CLEA Japan, Inc.), and eight animals in each group are dissected one month and two months after the start of exercise. did. The autopsy was performed under anesthesia with sebofurene (trade name, manufactured by Maruishi Pharmaceutical Co., Ltd.) under free feeding conditions, and then whole blood was collected from the lower abdominal vena cava to collect soleus muscle. The muscle mass and muscle strength of the collected soleus muscle were measured, immediately frozen with liquid nitrogen and stored at -80 ° C. The results of muscle mass of the flatfish muscle two months after the start of exercise are shown in FIG. 1, and the results of muscle strength of the flatfish muscle two months after the start of exercise are shown in FIG.
To measure the muscle strength of the soleus muscle, fix the soleus muscle of the left leg with a suture (# 5-0 silk) to a transducer (trade name: FORT 100, manufactured by World Precision Instruments), and use a 37 ° C Krebs solution (95%) After soaking in O ₂ , 5% CO ₂ aeration, pH 7.2) and electrical stimulation from two platinum electrodes to give twitch stimulation, the signal obtained from the transducer was evaluated as muscle force.

(2) Measurement of Expression Level of MYBPH Gene From the stored flatfish muscle, total RNA was extracted using RNeasy Fibrous Mini Kit (trade name, manufactured by Qiagen). The concentration of extracted total RNA was made uniform, heat treatment was carried out at 65 ° C. for 10 minutes, and after rapid cooling, it was used for 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) using 125 ng of RNA as a template Biolabs), 1 U / μL Rnase inhibitor (trade name, manufactured by Takara Bio)} mixed with 20 μL, (42 ° C., 1 hour) → (52 ° C., 30 minutes) → (99 ° C., 5 minutes) → Reverse transcription was performed at 4 ° C. The resulting cDNA samples were stored at -20.degree.
In addition, reverse transcription was performed in the same reaction system on 500 ng of RNA as a standard for real-time PCR shown below.

  Using the obtained cDNA as a template, it is mixed with 20 μL of PCR reaction solution {Fast SYBR (trade name, manufactured by Applied Biosystems), 1 μM Forward Primer (SEQ ID NO: 1), 1 μM Reverse Primer (SEQ ID NO: 2)} to obtain 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 as a standard cDNA a cDNA prepared by 7-step dilution of reverse-transcribed cDNA for a standard, and analysis of the expression level of MYBPH mRNA was performed based on the prepared calibration curve. The analysis results obtained were corrected with the expression level of the internal standard gene (36B4 gene) measured using the primers consisting of the nucleotide sequences shown in SEQ ID NOS: 3 and 4, and expressed as relative mRNA expression levels. The obtained result is shown in FIG.

As shown in FIG. 1, the flatfish muscle weight significantly increased in the exercise group two months after the start of exercise. However, as shown in FIG. 2, no significant difference could be confirmed in the muscle power of the flatfish muscle even two months after the start of exercise.
In addition, as shown in FIG. 3, the expression level of MYBPH mRNA was significantly increased in the exercise group one month after the start of exercise.
These results indicate that when the expression level of the MYBPH gene increases during myogenesis (approximately one month from the start of exercise), the weight of soleus muscle increases at two months after the start of exercise. That is, these results indicate that the transient expression level of MYBPH needs to be increased at any stage of the process of increasing the mass of soleus muscle, and individuals who can not increase the expression of MYBPH during myogenesis It suggests that muscle mass and motor function can not be maintained or enhanced.

Test Example 2 Measurement of the expression level of MYBPH when excising muscle was removed (1) Measurement of muscle mass of plantar muscle
C57BL / 6J mice (purchased from Oriental Bioservices, 16 weeks old) were divided into 3 non-treatment groups and 3 treatment groups. The muscle of the treatment group mice were resected (SA, Synergist Ablation operation), and the load on the plantar muscles was increased.
The mice in the non-operation group and the operation group were bred, and the plantar muscles were collected from the mice in each group three days after the removal of the hippocampus muscle and soleus muscle of the operation group mice. The muscle mass of the plantar muscle collected was measured, immediately frozen with liquid nitrogen and stored at -80.degree. The results of the measured plantar muscle mass are shown in FIG.

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

As shown in FIGS. 4 and 5, the weight of the plantar muscles significantly increased in the treatment group, and the expression level of MYBPH mRNA significantly increased.
When the sole and muscle muscles which are the co-operating muscles of the plantar muscles are excised, the plantar muscles are heavily loaded. Therefore, the results shown in FIGS. 4 and 5 indicate that when a large load is applied to the plantar muscles to increase muscle mass in a short period, the expression level of the MYBPH gene increases.

Test Example 3 Measurement of the expression level of MYBPH when regrounding after tail suspension
Balb / c mice (purchased from Charles River, 10 weeks old) were rendered hindlimb inactive by 1 week of tail suspension, and then the tail suspension was released (reground). The soleus muscle was collected three days after re-contacting.
The muscle mass of the mouse after tail suspension vertical and reground was measured, and immediately frozen with liquid nitrogen and stored at -80 ° C. The results of the measured muscle mass of the flatfish muscle are shown in FIG.

(2) Measurement of the expression level of MYBPH gene The expression level of MYBPH mRNA in the stored flatfish muscle was measured in the same manner as in Test Example 1. The obtained result is shown in FIG.

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

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

Total RNA was extracted from the collected cell sample using RNeasy Mini Kit (trade name, manufactured by QIAGEN), reverse transcription was performed in the same manner as in Test Example 1, and expression amount of MYBPH mRNA was analyzed. The analysis results obtained were corrected with the expression level of the internal standard gene (GAPDH) measured using the primers consisting of the base 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 the differentiation of skeletal muscle cells.

(2) Measurement of protein expression level Seed the mouse skeletal muscle cell line C2C12 in a 6-well plate at 0.5 × 10 ⁵ cells / well or 1.5 × 10 ⁵ cells / well (n = 3), 10% bovine Maintenance was carried out using DMEM medium (growth medium, 10% FBS-DMEM; 10% FBS, 1% Pen Strep (Gibco)) containing fetal serum (FBS, AusGeneX). Twenty four hours after cell seeding, cell samples in the growth zone were collected (cell density: 80% confluence). Furthermore, DMEM medium containing 2% horse serum (HS, manufactured by Kohjin Bio) at 48 hours after seeding (cell density: 100% confluent) with respect to the cell sample of differentiation induction zone {differentiation induction medium, 2% HS Differentiation induction was performed using -DMEM; 2% HS, 1% Pen Strep (manufactured by Gibco)}. Cell samples were collected at 0, 8, 24, 48, 72 and 120 hours after induction of differentiation.

  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 resulting mixture was centrifuged at 15,000 rpm and 4 ° C. for 15 minutes, and the supernatant was collected to prepare a protein solution. The concentration of the protein solution was made uniform, 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.

The 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 PVDF membrane after transfer was immersed in PVDF Blocking Reagent (trade name, manufactured by TOYOBO) and subjected to blocking treatment at room temperature for 1 hour. After that, anti-MYBPH antibody (1: 1000, manufactured by Aviva Systems Biology), anti-myosin heavy chain (hereinafter also referred to as "MyHC") antibody (1: 600, Developmental studies hybrida bank; MF20), anti-β-actin The antibody (1: 1000, made by Cell Signaling) is diluted with Can Get Signal Solution 1 (trade name, made by TOYOBO) to a concentration suitable for each, and these solutions are used overnight at 4 ° C. Primary antibody treatment was performed.
After washing the membrane with 0.1% Tween 20-TBS (T-TBS, manufactured by Bio-Rad), an anti-rabbit IgG antibody (for detecting MYBPH and β-actin, manufactured by Cell signaling) or an anti-mouse IgG antibody (for detecting MyHC) A second antibody solution was prepared by diluting Cell Signaling Co., Ltd.) with Can Get Signal Solution 2 (trade name, manufactured by TOYOBO) 1000 times to prepare a secondary antibody solution, and the secondary antibody treatment was performed 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 by ChemiDoc XRS (trade name, manufactured by Bio Rad), MYBPH , MyHC and β-actin expression levels.

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 to the expression level of β-actin, and the lower part is a chart showing the result of Western blotting performed after SDS-PAGE and the bands detected by chemiluminescence. is there.
As shown in FIG. 9, the expression level of MYBPH increased with myocyte differentiation.

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

(2) Measurement of mRNA Expression The mouse skeletal muscle 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).
Twenty-four hours after seeding of the cells, C2C12 was transfected with siRNA using the siRNA solution and Lipofectamine RNAiMAX Transfection Reagent (trade name, manufactured by Invitrogen) according to the attached protocol as described below.
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 a concentration of 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 sit for 5 minutes. Both solutions were then mixed and allowed to react for 10 minutes. 100 μL of the mixed solution after reaction was added to a 24 well plate.

Twenty-four hours after initiation of siRNA transfection (cell density: 100% confluence), the cells were induced to differentiate using 2% HS-DMEM, and cell samples were collected 48, 72, 120 hours later.
Total RNA was extracted from the collected cell sample using RNeasy Mini Kit (trade name, manufactured by QIAGEN), reverse transcription was performed in the same manner as in Test Example 1, and expression amount of MYBPH mRNA was analyzed. . The analysis results obtained were corrected with 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, RNA interference reduced the expression level of MYBPH mRNA.

(3) Measurement of protein expression amount 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). Twenty-four hours after seeding of the cells, C2C12 was transfected with siRNA in the same manner as described above using the siRNA solution and Lipofectamine RNAiMAX Transfection Reagent (trade name, Invitrogen). Then, 24 hours after initiation of siRNA transfection (cell density: 100% confluence), differentiation induction was performed on the cells using 2% HS-DMEM, and cell samples were collected 48, 72, 120 hours later.
For each cell sample collected, 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 (expression level of MYBPH) and FIG. 12 (expression level of MyHC).
As shown in FIGS. 11 and 12, RNA interference reduced the expression levels of MYBPH and MyHC, which is a differentiation marker of skeletal muscle cells.

(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). Twenty-four hours after seeding of the cells, C2C12 was transfected with siRNA using the above-mentioned siRNA solution and Lipofectamine RNAiMAX Transfection Reagent (trade name, manufactured by Invitrogen) in the same manner as described above. Then, 24 hours after initiation of siRNA transfection (cell density: 100% confluence), differentiation induction was performed on the cells using 2% HS-DMEM, and cell samples were collected 48, 72, 120 hours later.

Culture cover glass (Poly-L-Lysine coat, manufactured by Matsunami Glass 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). After fixing the cells with 4% paraformaldehyde phosphate buffer (manufactured by Wako Pure Chemical Industries, Ltd.), the cells were washed with PBS (manufactured by GIBCO). Thereafter, the cells were subjected to a solubilization treatment for 10 minutes at room temperature using a 0.2% Triton X-100 (manufactured by 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, Developmental studies hybrid bank; MF20) was diluted with 5% BSA-PBS to 6 μg / mL, and this solution was used at room temperature for 1 hour.
After washing with PBS, secondary antibody treatment was performed for 1 hour using a secondary antibody solution {Alexa Fluor 488 Donkey Anti-mouse IgG (manufactured by Invitrogen) diluted 500 folds with 5% BSA-PBS} .
After washing with PBS, the cells were sealed using Prolong Gold antifade reagent with DAPI (manufactured by Invitrogen), and morphological observation and photography of the cells were performed to measure the myotube diameter. 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. About 30 images of myotube diameter per image for 5 images taken per well Were measured, and the average value was calculated.

Photomicrographs of the cells 48 hours, 72 hours and 120 hours after induction of differentiation are shown in FIG. When treated with a negative control siRNA solution, myotubes became thick with induction of differentiation (for example, FIG. 13 (b)). On the other hand, when treated with the MYBPH siRNA solution, the myotube reached the culture limit and peeled off without being able to thicken the myotube (see, for example, FIG. 13 (c)). Thus, by suppressing the expression of MYBPH mRNA by RNA interference, hypertrophy of myotubes was suppressed.
Also, the mean myotube diameter 72 hours and 120 hours after induction of differentiation 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 A gene having homology to the mouse-derived MYBPH gene is identified by NCBI (National Center for Biotechnology Information, http: //www.ncbi.nlm The Zebrafish MYBPH gene was searched using the HomoloGene search of .nih.gov /). As a result, it was found that zebrafish has two homologous genes (MYBPHa gene and MYBPHb gene) present in different chromosomes. Furthermore, sequencing was performed in the vicinity of the start codons of the MYBPHa gene and the MYBPHb gene. As a result, the base sequences of these genes are different from the information and differences of NCBI (http://www.ncbi.nlm.nih.gov/) and Ensembl (http://asia.ensembl.org/index.html). I confirmed that there was not.
Therefore, as shown below, a double knockout zebrafish was created in which all these two genes were knocked out.

(2) Synthesis of TALEN mRNA
A plasmid (manufactured by Wako Pure Chemical Industries, Ltd.) encoding a TAL effector nuclease (TALEN) for knockout of MYBPHa gene and MYBPHb gene is amplified, and the amplified TALEN plasmid is treated with a restriction enzyme-treated reaction solution {10 μL of each TALEN plasmid, 10 × M buffer The mixture was subjected to restriction enzyme treatment overnight at 37 ° C. using 5 μL (manufactured by TOYOBO), 34 μL dH ₂ O, and 1 μL of restriction enzyme Hin dIII (manufactured by TOYOBO).
The obtained digested product was purified using QIAquick PCR Purification Kit (trade name, manufactured by Qiagen). Then, using the purified solution as a template, TALEN mRNA to which RNA synthesis, cap structure addition, and PolyA addition were performed using mMESSAGE mMACHINE Kit (trade name, manufactured by Applied Biosystems) was prepared. Here, DNase treatment was performed to degrade the contaminating plasmid.
The product thus obtained was purified using RNeasy MiniElute Cleanup Kit (trade name, manufactured by Qiagen) to obtain a TALEN mRNA purified solution. The TALEN mRNA purified solution was analyzed with an Agilent 2100 bioanalyzer (trade name, manufactured by Agilent Technologies) to confirm that the RNA was not degraded.

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

Five days after fertilization, the morphology and behavior of the larva were observed under a microscope, and a moving image was taken. The results are shown in FIG. Here, FIG. 15 (a) shows a photomicrograph of the zebrafish in the control group, and FIG. 15 (b) shows a photomicrograph of the zebrafish microinjected with TALEN mRNA.
The morphology and behavior of the zebrafish in the control group shown in FIG. 15 (a) were both normal. On the other hand, as shown in FIG. 15 (b), it was confirmed that individuals with low motor ability appeared when microinjection of TALEN mRNA into zebrafish.

(4) T7 endonuclease 1 assay
Among the larvae grown for 5 days after microinjection of TALEN mRNA, 1 individual was recovered from the control group and 3 from the individual of the double knockout group with reduced motor function.
Genomic DNA was extracted from the collected sample using Dneasy blood & tissue mini kit (trade name, manufactured by Qiagen). Using extracted genomic DNA as a template, Tks Gflex DNA Polymerase (trade name, manufactured by Takara Bio Inc.), and primers for detecting MYBPHa gene (SEQ ID Nos. 7 and 8) and primers for detecting MYBPHb gene (SEQ ID Nos. 9 and 10) PCR reaction solution {2 x 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.) PCR was performed using 1 μl to amplify the vicinity of the first exon of the MYBPHa gene and the MYBPHb gene. The 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 {10 × 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 from ° C to 85 ° C at -2 ° C / sec cooling → (cooling from 85 ° C to 25 ° C at -0.3 ° C / sec) → 25 ° C 10 sec to 22 ° C. Thereafter, 1 μL each 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 subjected to the T7 endonuclease 1 treatment 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 results are 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 genomic DNA extracted from control individuals. That is, cleavage at a mismatch of genomic DNA by T7 endonuclease 1 did not occur, and multiple bands from 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 double-stranded DNA in all individuals with reduced motor function. Therefore, in addition to the band of the PCR amplification product, two small bands of low molecular weight were detected because T7 endonuclease is cleaved at the mismatched portion of the DNA where the mismatch exists.

  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 motor function and the expression level of the MYBPH gene.

Test Example 7 Measurement of the Expression Level of MYBPH Gene in Hyukku Muscle after Exercise Test in Mice
A 10-week-old male C57BL / 6J mouse (purchased from Japan Charles River) is pre-reared for 1 week to acclimate to the environment, and then the training for getting used to treadmill exercise is as follows: went.

Day 1: 10 m / min (15 minutes) → 15 m / min (15 minutes)
Day 2: 10 m / min (10 minutes) → 15 m / min (20 minutes)
Third day: 15 m / min (15 minutes) → 20 m / min (15 minutes)
Fourth day: 15 m / min (5 minutes) → 20 m / min (15 minutes) → 25 m / min (10 minutes)
Day 5: 15 m / min (5 minutes) → 20 m / min (10 minutes) → 25 m / min (15 minutes)

Then, the mice were divided into 3 groups so that their weights would be equal (N = 4 / group). In the second group, the running speed of 25 m / min was performed for 30 minutes. The remaining group was the resting group.
The exercise group was dissected one hour and six hours after the exercise, and the muscle of each group was collected.

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

  As shown in Table 3, when mice were subjected to treadmill exercise, the expression level of MYBPH gene was increased.

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

Claims (4)

A marker for evaluating a change in muscle mass due to a change in myotube diameter, comprising skeletal muscle-derived myosin binding protein H collected from an individual or a gene encoding the same.   A marker for evaluating myotube hypertrophy, which comprises skeletal muscle-derived myosin binding protein H collected from an individual or a gene encoding the same. Measuring the expression level of skeletal muscle-derived myosin binding protein H or a gene encoding the same collected from an individual;
Evaluate changes in muscle mass due to changes in myotube diameter based on measured expression levels,
How to assess muscle mass. Measuring the expression level of skeletal muscle-derived myosin binding protein H or a gene encoding the same collected from an individual;
Assess the degree of myotube hypertrophy based on the measured expression levels,
Evaluation method of myotube hypertrophy.