KR101685109B1 - A use of Nogo-A related to differentiation, regeneration, or disease condition of muscle - Google Patents

Abstract

And to the use of Noge-Ai in relation to muscle differentiation, regeneration, or disease state. The nongo-ai can be used to identify the degree of muscle differentiation or regeneration, or disease state.

Description

A use of Nogo-A for muscle differentiation, regeneration, or disease state,

And to the use of Noge-Ai in relation to muscle differentiation, regeneration, or disease state.

Nogo-A is the longest of the three isoforms associated with labor. These isomers are made by the differences in promoter or splicing during gene transcription, and they have a common part that is well conserved at the 3'end. Except this, the 5 'terminal part has a difference between the isomers, and thus their action effect and expression pattern are greatly different.

The effect of Nogon-A in the present invention on the central nervous system is well known by previous studies. In the central nervous system, Nogoe-ei inhibits the formation of neuronal axons, inhibits the growth of neuronal axons, and inhibits the regenerative effects of neuronal tissue.

However, the effect of Nogeo-Ae outside the nervous system is not well known. Muscle atrophy sclerosis, commonly known as Lou Gehrig's disease, is a disease in which the muscles of the whole body weaken and atrophy due to the selective death of motor neurons, which tend to match the expression of Nogo- . Thus, the action of Nogeo-A in the muscles is still at the level of explanations related to the nervous system. There has been no ongoing research on the expression of the nongo-ae in the muscle itself in its entirety.

Accordingly, the present inventors provide contents on the expression pattern of Noga-ai in muscle, and provide new functions as muscle differentiation and regeneration and disease status markers.

One aspect provides a composition or kit for identifying the degree of differentiation of muscle precursor cells into muscle cells.

Another aspect provides a method of identifying the degree of differentiation of muscle precursor cells into muscle cells.

Another aspect provides a composition or kit for identifying the degree of regeneration of damaged muscles.

Another aspect provides a method of determining the degree of regeneration of injured muscles.

Another aspect provides a composition or kit for diagnosing muscle injuries.

Another aspect provides a method of providing information to diagnose an individual's muscle damage disease.

One aspect provides a composition for confirming the degree of differentiation of muscle precursor cells into muscle cells, including an agent for measuring the expression level of mRNA or protein of Nogo-A gene.

The Nogo-A can be named reticulon 4 isoform A, or RTN4-A, as a neurite outgrowth inhibitor A. The Noge-A gene may be one that encodes the polypeptide of SEQ ID NO: 1. The Noge-A gene may contain the polynucleotide of SEQ ID NO: 2 as a transcript.

Muscle precursor cells are cells that can develop or differentiate into muscle cells. Muscle progenitor cells include progenitor cells and muscle stem cells. The muscle precursor cells may be satellite cells or myoblasts. Muscle cells can be myocytes or myotubes.

The agent may be a primer pair that specifically binds to the gene, a probe, or an antibody that specifically binds to the protein. The term "primer" means a short oligonucleotide that has a free hydroxyl group at the 3 ' end and functions as a starting point for template replication by forming a base pair with a template having a complementary sequence. Each sequence of the primer pair specifically binding to this gene, the conditions of the polymerase chain reaction, and the length of each primer can be suitably designed or modified by a person skilled in the art using the nucleotide sequence of the Nogo-A gene .

The term "probe" means a nucleic acid fragment capable of detecting the presence of a target nucleotide having a sequence complementary to its sequence in a sample. The presence of the target nucleotide can be detected by denaturing the labeled probe with a single stranded nucleic acid and then inducing hybridization with the target nucleic acid. The conditions for hybridization with probes specifically binding to this gene and its target sequence can be suitably designed or modified by a person skilled in the art.

The term "antibody" refers to a specific protein molecule that recognizes a specific region of an antigen. An antibody that specifically binds to Nogo-id protein is an antibody having high specificity and affinity to the protein and little cross-reactivity to other proteins, and may be a monoclonal antibody, a polyclonal antibody or a recombinant antibody.

Another aspect provides a kit for determining the degree of differentiation of muscle precursor cells into muscle cells. The kit may include a composition for confirming the degree of differentiation of muscle precursor cells into muscle cells. The composition is as described above. The kit may be a reverse transcription polymerase chain reaction kit, a DNA chip kit, or an ELISA kit. The kit may further comprise one or more other compositions, solutions, or devices depending on the assay method. The RT-PCR kit may include a primer pair specific to Nogo-A gene, an enzyme such as a reaction buffer, deoxynucleotide, Taq polymerase and reverse transcriptase, and sterilized water. The DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to Nogo-A gene or a fragment thereof is attached, and a reagent, a preparation, and an enzyme for producing a labeled probe. The ELISA kit may include an antibody that specifically binds to Nogo-id protein.

Another aspect provides a method of identifying the degree of differentiation of muscle precursor cells into muscle cells, including measuring the level of expression of Noga-A in cells that are differentiating from muscle precursor cells to muscle cells. The muscle precursor cells and muscle cells are as described above.

Measurement of the expression level can be performed using an antibody that specifically binds to a primer pair, a probe, or Nogo-id protein that specifically binds Nogo-Ag gene. The above primers, probes, and antibodies are as described above. Measurement of the expression level can be performed using reverse transcriptase polymerase, real-time RT-PCR, Northern blotting, Western blotting, ELISA, immunoprecipitation analysis, or immunohistochemistry.

As a result of the measurement, when the expression level of Nogo- A in the differentiating cells is higher than the expression level of Nogo- A in the control group, it can be determined that the cell is a cell differentiated more than the control. In addition, when the expression level of noga-ai in the differentiating cells is lower than the expression level of noga-ai in the control group, it can be determined that the cells are less differentiated than the control.

Another aspect provides a composition for identifying the degree of regeneration of damaged muscle, comprising an agent that measures the level of expression of the mRNA or protein of the nongonase gene. The Nogon-A gene and the above-described preparation are as described above.

Another aspect provides a kit for determining the degree of regeneration of damaged muscles. The kit may include a composition for confirming the degree of regeneration of the injured muscle. The composition is as described above. The kit may be a reverse transcription polymerase chain reaction kit, a DNA chip kit, or an ELISA kit. Each kit is as described above.

Another aspect provides a method of identifying the degree of regeneration of damaged muscle, comprising measuring the level of expression of Nogo- A in a cell or tissue isolated from the injured muscle.

The measurement of the expression level can be performed using an antibody that specifically binds to a primer pair, a probe, or Nogo-id protein that specifically binds Nogo-Ag gene. The above primers, probes, and antibodies are as described above.

As a result of the measurement, if the expression level of Noga-A in cells or tissues separated from the injured muscle is lower than the expression level of Noga-A in the control group, the injured muscle can be determined to be more regenerated than the control group. Where the control group may be a cell or tissue isolated from the muscle immediately after injury.

Another aspect provides a composition for diagnosing muscle injurious disease, which comprises an agent that measures the level of expression of the mRNA or protein of the nongonase gene.

The term " muscle injury disorder "refers to any disease in which muscle tissue or muscle cells are damaged and muscle tissue is lost. The muscle damage disorder may be muscular dystrophy, muscular atrophy, myositis, polymyositis, peripheral vascular disease, or fibrosis. The muscular dystrophies include, for example, Becker's muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facial palsy, muscular dystrophy, dystrophy muscular dystrophy, ankle dysentery dystrophy, dystrophy muscular dystrophy, sarco gliosis, spinal muscular atrophy, or Brown-Vialetto-Van Laere syndrome (BVVL). The Nogon-A gene and the above-described preparation are as described above.

Another aspect provides a kit for diagnosing muscle injury disorders. The kit may include the composition for diagnosing an injured muscle disorder. The composition is as described above. The kit may be a reverse transcription polymerase chain reaction kit, a DNA chip kit, an ELISA kit, or a protein chip kit. Each kit is as described above.

Another aspect is a method for measuring the level of expression of a mRNA or protein of a nogeo-gene in a sample separated from an individual; And comparing the measured expression level with an expression level of the mRNA or protein of the nongo-a gene in the control sample, to provide information for diagnosing muscle damage disease of an individual.

The sample may be isolated from the muscle tissue of the individual. The subject may be a mammal. The mammal may be a human, mouse, rat, guinea pig, rabbit, monkey, pig, horse, cattle, sheep, nurse, dog, or cat.

The measurement of the above-mentioned muscle injury disease, the Nogo-A gene, and the expression level is as described above. The control sample may be an individual without muscle injury disease. When the level of Nogon-a expression in a sample isolated from an individual is increased relative to the level of Nogeo-a expression in a control sample, the individual can be diagnosed as having muscle damage disease.

The nongo-ai can be used to identify the degree of muscle differentiation or regeneration, or disease state. In addition, the present invention can contribute to widening the ecopathological understanding of muscles.

FIGS. 1 and 2 are the results of observing the expression of Nogon-A through immunofluorescent staining in the myoblasts of the early differentiation stage and the differentiation stage, respectively.
FIG. 3 is a graph showing the expression patterns of nogeo-a and other muscle differentiation factors during the differentiation process.
FIG. 4A shows the results of analysis of expression of Noga-A in the regenerating muscle using RT-PCR.
FIG. 4B shows the results of analysis of expression of Noga-A in the muscle of the disease state using RT-PCR.
FIG. 5 shows the results of analysis of the expression level of Noga-A in muscles in the regenerating muscle and disease state using q-PCR.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example  One: Muscular  Analysis of nogeo-a expression during differentiation

One. Muscular  Induction of differentiation

Mouse-derived myoblast C2C12 was used to test muscle cell differentiation (ATCC®, USA). (FBS, ATLAS, USA), and 1% penicillin-streptomycin (PS, Life technologies, USA) in the presence of high glucose (Dulbecco's Modified Eagle Medium Proliferation was maintained in conditioned medium. After that, low-density glucose-DMEM (L-DMEM, GE healthcare, USA), 2% horse serum (HS, Life technologies, USA) and 1% PS The differentiation was induced by replacing with the differentiation condition medium containing. And cultured in an incubator at 37 ° C and 5% CO ₂ . The expression of Noga-Ae during the differentiation process of myoblasts was analyzed as follows.

2. Immunofluorescent staining  Analysis of Nongo-eo Expression Using

Immunofluorescent staining was performed on the myoblasts before and 14 days after the replacement with the differentiation condition medium, that is, in the early stage and the late stage of differentiation. Pax7, MyoD, Myogenin, and MYH2 proteins were co-stained to compare the differentiation stage.

The cultured cells were fixed with 4% paraformaldehyde (Sigma, USA) for 15 minutes at room temperature, and then washed three times with 0.01M phosphate buffered saline (PBS, Gendepot, USA) for 5 minutes. The fixed cells were then incubated with methanol at -20 [deg.] C for 10 minutes. After washing three times for 5 minutes with PBS, the cells were blocked with 3% bovine serum albumin (Sigma, USA) in 0.01 M PBS for 1 hour at room temperature. Myogen (1:20; DSHB, USA), MyoD (1: 500; Abcam, USA), Pax7 (1:50; Developmental Studies Hybridoma Bank, And MYH2 (1: 200; Santacruz, USA) were each diluted in PBS (PBS / T) supplemented with 0.1% Triton X-100 (Sigma, USA). The prepared cells were stained with each antibody diluent at 4 ° C for about 18 hours. After washing three times for 5 minutes with PBS, the cells were washed with PBS (1: 1) supplemented with fluorescein isocyanate (FITC, 1: 500; Life Technologies, USA) and tetramethylrhodamine (TRITC, Lt; / RTI > at room temperature. After washing three times for 5 minutes with PBS, the cells were finally stained with 4 ', 6-diamidino-2-phenylindole (DAPI, 0.1 μg / ml; Sigma, USA). Photographs were taken using a confocal laser scanning microscope (Carl Zeiss, Germany). Images were analyzed with Zen 2009 (Carl Zeiss, Germany) software.

FIGS. 1 and 2 are the results of observing the expression of Nogon-A through immunofluorescent staining in the myoblasts of the early differentiation stage and the differentiation stage, respectively. As shown in FIGS. 1 and 2, expression of Nogon-AE was significantly consistent with the expression of MYH2, a marker protein of myotube, a differentiated muscle cell. In contrast, expression of Noga-Ae partially coincided with the differentiated muscle cell marker proteins MyoD and Myogenin, and did not coincide with PAX7, a muscle cell-associated protein.

3. RT - PCR  And q- PCR Analysis of Expression-expression Using

RNA was extracted from differentiation-induced myoblasts at intervals of 1 day and RT-PCR or q-PCR was performed to observe the expression of Nogo-

The PCR reaction conditions were as follows. 1 μl each, 1 μl cDNA, and 17 μl of ultrapure water were mixed with AccuPower® PCR premix (Bioneer, Korea) for each of the conditions-free primer sets (SEQ ID NOS: 3 and 4) and Gapdh primer sets (SEQ ID NOS: 5 and 6) . Using a Biorad T100 instrument, the reaction was carried out at 95 ° C for 4 minutes, followed by 35 cycles of 95 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 30 seconds, and reaction at 72 ° C for 7 minutes.

The qPCR reaction conditions were as follows. 12.5 μl of 2X SYBR green master mix (TAKARA, USA), 2.5 μl each of primer set, 3 μl cDNA, and 7 μl DEPC water were mixed at 25 μl reaction volume. (SEQ ID NOS: 7 and 8), Myogenin (Qiagen, USA), MyoD (Qiagen, USA), and Gapdh (Qiagen, USA). The reaction was carried out at 95 ° C for 5 minutes using Qiagen Rotor-Gene Q, followed by 40 cycles of reaction at 95 ° C for 30 seconds and 60 ° C for 10 seconds.

FIG. 3 is a graph showing the expression patterns of nogeo-a and other muscle differentiation factors during the differentiation process. As shown in FIG. 3, as the differentiation progresses, the Noge-A gene also has a tendency to continuously increase its expression with Myogenin and MyoD.

Example  2: In the process of muscle regeneration, Ai  Expression analysis

1. induction of muscle regeneration

To induce muscle regeneration, Antonio SJ Lee, BioArchithecture, 3: 2, 25-37; According to the method described in 2013, muscle injury was induced by injecting myotoxin, notexin, into muscles. After shaved skin of both legs of 8-week-old male C57BL / 6 mice (n = 9) (Central laboratory animal, Korea), nortexin (Sigma-aldrich, USA) was injected into the anterior tibialis muscle adjacent to the shin bone mg / l in saline and injected 20 ㎕ per leg (0.1 ㎍ / 1 leg).

2. RT - PCR  And q- PCR Analysis of Expression-expression Using

Three, three, and two mice were autopsied at 6, 9, and 12 days after nontexin injection to separate the muscles. RNA was extracted from isolated muscle samples and RT-PCR or q-PCR was performed to observe the expression of Nogo-

FIG. 4A shows the results of analysis of expression of Noga-A in the regenerating muscle using RT-PCR. The negative control, the complete muscle and the positive control, brain tissue were muscle samples and brain tissue isolated from 8 week old male C57BL / 6 mice, respectively, which were not injured.

FIG. 5 shows the results of analysis of the expression level of Noga-A in the regenerating muscle using q-PCR. As shown in FIG. 5, as the regeneration of injured muscle progressed, the expression of nogeo-ae decreased, and the tendency was shown to be close to that of uninjured muscle.

Example  3: Nervousness in the muscles of the diseased state - Ai  Expression analysis

Expression of intracellular noga-ae was measured by RT-PCR in the same manner as in Example 2, using an animal model of 3 months, 6 months, or 15 months of age in a male MDX mouse (n = 3) (distribution at Jacques P.Tremblay Lab) And q-PCR. Male C57BL / 6 mice at 1, 3, 6, or 15 months of age and their brain tissues were used as controls.

FIG. 4B shows the results of analysis of expression of Noga-A in the muscle of the disease state using RT-PCR. As shown in FIG. 4B, the expression level of Noga-A in the muscle of the diseased state was higher than that of the control muscle without the disease.

FIG. 5 shows the results of analysis of the expression level of Noga-A in the muscle of the disease state using q-PCR. As shown in Fig. 5, the expression of noga-ai was found to be higher in the muscle with disease than in the muscle of the control group without disease.

<110> Kyungpook National University industry academic cooperation foundation <120> A use of Nogo-A related to differentiation, regeneration, or          disease condition of muscle <130> PN107894 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 1162 <212> PRT <213> Mus musculus <400> 1 Met Glu Asp Ile Asp Gln Ser Ser Leu Val Ser Ser Ser Ala Asp Ser   1 5 10 15 Pro Pro Arg Pro Pro Pro Ala Phe Lys Tyr Gln Phe Val Thr Glu Pro              20 25 30 Glu Asp Glu Glu Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Asp Glu          35 40 45 Asp Leu Glu Glu Leu Glu Val Leu Glu Arg Lys Pro Ala Ala Gly Leu      50 55 60 Ser Ala Ala Pro Val Pro Pro Ala Ala Pro Leu Leu Asp Phe Ser  65 70 75 80 Ser Asp Ser Val Pro Pro Ala Pro Arg Gly Pro Leu Pro Ala Ala Pro                  85 90 95 Pro Thr Ala Pro Glu Arg Gln Pro Ser Trp Glu Arg Ser Pro Ala Ala             100 105 110 Ser Ala Pro Ser Leu Pro Pro Ala Ala Ala Val Leu Pro Ser Lys Leu         115 120 125 Pro Glu Asp Asp Glu Pro Pro Ala Arg Pro Pro Ala Pro Ala Gly Ala     130 135 140 Ser Pro Leu Ala Glu Pro Ala Ala Pro Pro Ser Thr Pro Ala Ala Pro 145 150 155 160 Lys Arg Arg Gly Ser Gly Ser Val Asp Glu Thr Leu Phe Ala Leu Pro                 165 170 175 Ala Ala Ser Glu Pro Val Ile Pro Ser Ser Ala Glu Lys Ile Met Asp             180 185 190 Leu Lys Glu Gln Pro Gly Asn Thr Val Ser Ser Gly Gln Glu Asp Phe         195 200 205 Pro Ser Val Leu Phe Glu Thr Ala Ala Ser Leu Pro Ser Leu Ser Pro     210 215 220 Leu Ser Thr Val Ser Phe Lys Glu His Gly Tyr Leu Gly Asn Leu Ser 225 230 235 240 Ala Val Ala Ser Thr Glu Gly Thr Ile Glu Glu Thr Leu Asn Glu Ala                 245 250 255 Ser Arg Glu Leu Pro Glu Arg Ala Thr Asn Pro Phe Val Asn Arg Glu             260 265 270 Ser Ala Glu Phe Ser Val Leu Glu Tyr Ser Glu Met Gly Ser Ser Phe         275 280 285 Asn Gly Ser Pro Lys Gly Glu Ser Ala Met Leu Val Glu Asn Thr Lys     290 295 300 Glu Glu Val Ile Val Arg Ser Ser Lys Asp Lys Glu Asp Leu Val Cys Ser 305 310 315 320 Ala Ala Leu His Asn Pro Gln Glu Ser Pro Ala Thr Leu Thr Lys Val                 325 330 335 Val Lys Glu Asp Gly Val Met Ser Pro Glu Lys Thr Met Asp Ile Phe             340 345 350 Asn Glu Met Lys Met Ser Val Val Ala Pro Val Arg Glu Glu Tyr Ala         355 360 365 Asp Phe Lys Pro Phe Glu Gln Ala Trp Glu Val Lys Asp Thr Tyr Glu     370 375 380 Gly Ser Arg Asp Val Leu Ala Ala Arg Ala Asn Met Glu Ser Lys Val 385 390 395 400 Asp Lys Lys Cys Phe Glu Asp Ser Leu Glu Gln Lys Gly His Gly Lys                 405 410 415 Asp Ser Glu Ser Arg Asn Glu Asn Ala Ser Phe Pro Arg Thr Pro Glu             420 425 430 Leu Val Lys Asp Gly Ser Arg Ala Tyr Ile Thr Cys Asp Ser Phe Ser         435 440 445 Ser Ala Thr Glu Ser Thr Ala Ala Asn Ile Phe Pro Val Leu Glu Asp     450 455 460 His Thr Ser Glu Asn Lys Thr Asp Glu Lys Lys Ile Glu Glu Arg Lys 465 470 475 480 Ala Gln Ile Ile Thr Glu Lys Thr Ser Pro Lys Thr Ser Asn Pro Phe                 485 490 495 Leu Val Ala Ile His Asp Ser Glu Ala Asp Tyr Val Thr Thr Asp Asn             500 505 510 Leu Ser Lys Val Thr Glu Ala Val Val Ala Thr Met Pro Glu Gly Leu         515 520 525 Thr Pro Asp Leu Val Glu Glu Ala Cys Glu Ser Glu Leu Asn Glu Ala     530 535 540 Thr Gly Thr Lys Ile Ala Tyr Glu Thr Lys Val Asp Leu Val Gln Thr 545 550 555 560 Ser Glu Ala Ile Gln Glu Ser Ile Tyr Pro Thr Ala Gln Leu Cys Pro                 565 570 575 Ser Phe Glu Glu Ala Glu Ala Thr Pro Ser Pro Val Leu Pro Asp Ile             580 585 590 Val Met Glu Ala Pro Leu Asn Ser Leu Leu Pro Ser Thr Gly Ala Ser         595 600 605 Val Ala Gln Pro Ser Ala Ser Pro Leu Glu Val Ser Ser Pro Val Ser     610 615 620 Tyr Asp Gly Ile Lys Leu Glu Pro Glu Asn Pro Pro Pro Tyr Glu Glu 625 630 635 640 Ala Met Ser Val Ala Leu Lys Thr Ser Asp Ser Lys Glu Glu Ile Lys                 645 650 655 Glu Pro Glu Ser Phe Asn Ala Ala Ala Gln Glu Ala Glu Ala Pro Tyr             660 665 670 Ile Ser Ile Ala Cys Asp Leu Ile Lys Glu Thr Lys Leu Ser Thr Glu         675 680 685 Pro Ser Pro Glu Phe Ser Asn Tyr Ser Glu Ile Ala Lys Phe Glu Lys     690 695 700 Ser Val Pro Asp His Cys Glu Leu Val Asp Asp Ser Ser Pro Glu Ser 705 710 715 720 Glu Pro Val Asp Leu Phe Ser Asp Asp Ser Ile Pro Glu Val Pro Gln                 725 730 735 Thr Gln Glu Glu Ala Val Met Leu Met Lys Glu Ser Leu Thr Glu Val             740 745 750 Ser Glu Thr Val Thr Gln His Lys His Lys Glu Arg Leu Ser Ala Ser         755 760 765 Pro Gln Glu Val Gly Lys Pro Tyr Leu Glu Ser Phe Gln Pro Asn Leu     770 775 780 His Ile Thr Lys Asp Ala Ala Ser Asn Glu Ile Pro Thr Leu Thr Lys 785 790 795 800 Lys Glu Thr Ile Ser Leu Gln Met Glu Glu Phe Asn Thr Ala Ile Tyr                 805 810 815 Ser Asn Asp Leu Leu Ser Ser Lys Glu Asp Lys Met Lys Glu Ser             820 825 830 Glu Thr Phe Ser Asp Ser Ser Pro Ile Glu Ile Ile Asp Glu Phe Pro         835 840 845 Thr Phe Val Ser Ala Lys Asp Asp Ser Pro Lys Glu Tyr Thr Asp Leu     850 855 860 Glu Val Ser Asn Lys Ser Glu Ile Ala Asn Val Gln Ser Gly Ala Asn 865 870 875 880 Ser Leu Pro Cys Ser Glu Leu Pro Cys Asp Leu Ser Phe Lys Asn Thr                 885 890 895 Tyr Pro Lys Asp Glu Ala His Val Ser Asp Glu Phe Ser Lys Ser Arg             900 905 910 Ser Ser Val Ser Ser Val Ser Ala Leu         915 920 925 Glu Ser Gln Ile Glu Met Gly Asn Ile Val Lys Pro Lys Val Leu Thr     930 935 940 Lys Glu Ala Glu Glu Lys Leu Pro Ser Asp Thr Glu Lys Glu Asp Arg 945 950 955 960 Ser Leu Thr Ala Val Leu Ser Ala Glu Leu Asn Lys Thr Ser Val Val                 965 970 975 Asp Leu Leu Tyr Trp Arg Asp Ile Lys Lys Thr Gly Val Val Phe Gly             980 985 990 Ala Ser Leu Phe Leu Leu Leu Ser Leu Thr Val Phe Ser Ile Val Ser         995 1000 1005 Val Thr Ala Tyr Ile Ala Leu Ala Leu Leu Ser Val Thr Ile Ser Phe    1010 1015 1020 Arg Ile Tyr Lys Gly Val Ile Gln Ala Ile Gln Lys Ser Asp Glu Gly 1025 1030 1035 1040 His Pro Phe Arg Ala Tyr Leu Glu Ser Glu Val Ala Ser Ser Glu Glu                1045 1050 1055 Leu Val Gln Lys Tyr Ser Asn Ser Ala Leu Gly His Val Asn Ser Thr            1060 1065 1070 Ile Lys Glu Leu Arg Arg Leu Phe Leu Val Asp Asp Leu Val Asp Ser        1075 1080 1085 Leu Lys Phe Ala Val Leu Met Trp Val Phe Thr Tyr Val Gly Ala Leu    1090 1095 1100 Phe Asn Gly Leu Thr Leu Leu Ile Leu Ala Leu Ile Ser Leu Phe Ser 1105 1110 1115 1120 Ile Pro Val Ile Tyr Glu Arg His Gln Ala Gln Ile Asp His Tyr Leu                1125 1130 1135 Gly Leu Ala Asn Lys Ser Val Lys Asp Ala Met Ala Lys Ile Gln Ala            1140 1145 1150 Lys Ile Pro Gly Leu Lys Arg Lys Ala Glu        1155 1160 <210> 2 <211> 6165 <212> RNA <213> Mus musculus <400> 2 cccctgccgg gaaggtgagt cacgccaaac tgggcggaga gtctgccctc ctctctcagt 60 tgctcatctg ggcggcggcg gctgctgcaa ctgaggacag ggcgggtggc gcatctcgag 120 cgcggaggca ggaggagaag tcttattgtt cctggagctg tcgcctttgc gggttcctcg 180 gcttggttcg gccagcccgg cctctgccag tcctgcccaa cccccacaac cgcccgcggc 240 tctgaggaga agtggcccgc ggcggcagta gctgcagcat catcgccgac catggaagac 300 atagaccagt cgtcgctggt ctcctcgtcc gcggatagcc cgccccggcc cccgcccgct 360 ttcaagtacc agttcgtgac ggagcccgag gacgaggagg acgaggaaga cgaggaggag 420 gaggaggacg acgaggacct ggaggaattg gaggtgctgg agaggaagcc cgcagccggg 480 ctgtccgcgg ctccggtgcc ccccgccgcc gcaccgctgc tggacttcag cagcgactcg 540 gtgccccccg cgccccgcgg gccgctgccg gccgcgcccc ccaccgcccc tgagaggcag 600 ccgtcctggg aacgcagccc cgcggcgtcc gcgccatccc tgccgcccgc tgccgcagtc 660 ctgccctcca agctcccgga ggacgacgag cctccagcgc ggcctccggc gccagccggc 720 gcgagccccc tagcggagcc cgccgcgccc ccttccacgc cggccgcgcc caagcgcagg 780 ggctcgggct cagtggatga gacccttttt gctcttcctg ctgcatctga gcctgtgata 840 ccctcctctg cagaaaaaat tatggatttg aaggagcagc caggtaacac tgtttcgtct 900 ggtcaagagg atttcccatc tgtcctgttt gaaactgctg cctctcttcc ttctctatct 960 cctctctcaa ctgtttcttt taaagaacac ggataccttg gtaacttatc agcagtggca 1020 tccacagaag gaactattga agaaacttta aatgaagctt ctagagaatt gccagagagg 1080 gcaacaaatc catttgtaaa tagagagtca gcagagtttt cagtattaga atactcagaa 1140 atgggatcat ctttcaatgg ctccccaaaa ggagagtcag ccatgttagt agaaaacact 1200 aaggaagaag taattgtgag gagtaaagac aaagaggatt tagtttgtag tgcagccctt 1260 cataatccac aagagtcacc tgcgaccctt actaaagtgg ttaaagaaga cggagttatg 1320 tctccagaaa agacaatgga catttttaat gaaatgaaaa tgtcagtggt agcacctgtg 1380 agggaagagt atgcagattt taagccattt gaacaagcat gggaagtgaa agatacttat 1440 gagggaagta gggatgtgct ggctgctaga gctaatatgg aaagtaaagt ggacaaaaaa 1500 tgctttgaag atagcctgga gcaaaaaggt catgggaagg atagtgaaag cagaaatgag 1560 aatgcttctt tccccaggac cccagaactt gtgaaggacg gctccagagc gtacatcacc 1620 tgtgattcct ttagctcagc aaccgagagt actgcagcaa acattttccc tgtgctagaa 1680 gatcacactt cagaaaacaa aacagatgaa aaaaaaatag aagaaaggaa ggcccaaatt 1740 ataacagaga agactagccc caaaacgtca aatcctttcc ttgtagcaat acatgattct 1800 gaggcagatt atgtcacaac agataattta tcaaaggtga ctgaggcagt agtggcaacc 1860 atgcctgaag gtctaacgcc agatttagtt caggaagcat gtgaaagtga actgaacgaa 1920 gccacaggta caaagattgc ttatgaaaca aaagtggact tggtccagac atcagaagct 1980 atacaagagt caatttaccc cacagcacag ctttgcccat catttgagga agctgaagca 2040 actccgtcac cagttttgcc tgatattgtt atggaagcgc cattaaattc tctccttcca 2100 agcactggtg cttctgtagc gcagcccagt gcatccccac tagaagtacc gtctccagtt 2160 agttatgacg gtataaagct tgagcctgaa aatcccccac catatgaaga agccatgagt 2220 gtagcactaa aaacatcgga ctcaaaggaa gaaattaaag agcctgaaag ttttaatgca 2280 gctgctcagg aagcagaagc tccttatata tccattgcat gtgatttaat taaagaaaca 2340 aagctctcca ctgagccaag tccagagttc tctaattatt cagaaatagc aaaatttgag 2400 aagtcggtgc ctgatcactg tgagctcgtg gatgattcct cacccgaatc tgaaccagtt 2460 gacttattta gtgatgattc aattcctgaa gtcccacaaa cacaagagga ggctgtgatg 2520 ctaatgaagg agagtctcac tgaagtgtct gagacagtaa cacaacacaa acataaggag 2580 agacttagtg cttcacctca ggaggtagga aagccatatt tagagtcttt tcagcccaat 2640 ttacatatta caaaagatgc tgcatctaat gaaattccaa cattgaccaa aaaggagaca 2700 atttctttgc aaatggaaga gtttaatact gcaatttatt ccaatgatga cttactttct 2760 tctaaggaag acaaaatgaa agaaagtgaa acattttccg attcatctcc cattgagata 2820 atagatgagt ttcccacatt tgtcagtgct aaagatgatt ctcctaagga gtacactgac 2880 ctagaagtat ccaacaaaag tgaaattgct aatgtccaga gcggggccaa ttcgttgcct 2940 tgctcagaat tgccctgtga cctttctttc aagaatacat atcctaaaga tgaagcacat 3000 gtctcagatg aattctccaa aagtaggtcc agtgtatcta aggtgccctt attgcttcca 3060 aatgtttctg ctttggaatc tcaaatagaa atgggcaaca tagttaaacc caaagtactt 3120 acgaaagaag cagaggaaaa acttccttct gatacagaga aagaggacag atccctgaca 3180 gctgtattgt cagcagagct gaataaaact tcagttgttg acctcctgta ctggagagac 3240 attaagaaga ctggagtggt gtttggtgcc agcttattcc tgctgctgtc tctgacagtg 3300 ttcagcattg tcagtgtaac ggcctacatt gccttggccc tgctctctgt gactatcagc 3360 tttaggatat ataagggtgt gatccaagct atccagaaat cagatgaagg ccacccattc 3420 agggcatatt tggaatctga agttgccata tcagaggaat tggttcagaa atatagtaat 3480 tctgctcttg gtcatgtgaa cagcacaata aaagaattga ggcgtctctt cttagttgat 3540 gatttagttg attccctgaa gtttgcagtg ttgatgtggg tatttactta cgttggtgcc 3600 ttgttcaatg gtttgacact actgatttta gctctgatct cactcttcag tattcctgtt 3660 atatatgaac ggcatcaggc gcagatagat cattatctag gacttgcaaa caagagcgtt 3720 aaggatgcca tggccaaaat ccaagcaaaa atccctggat tgaagcgcaa agcagaatga 3780 aaaggcccca aacagtagac attcatcttt aaaggggaca ctcccttggt tacggggtgg 3840 gcgggtcagg ggtgagccct gggtggccgt gcagtttcag ttatttttag cagtgcactg 3900 tttgaggaaa aattacctgt cttgacttcc tgtgtttatc atcttaagta ttgtaagctg 3960 ctgtgtatgg atctcattgt agtcatactt gttttcccca gatgaggcac ttggtgaata 4020 aaggatgctg ggaaaactgt gtgttatatt ctgttgcagg tagtctggct gtatttggaa 4080 agttgcaaag aaggtagatt tgggggcagg aaaaacaacc cttttcacag tgtactgtgt 4140 ttggttggtg taaaactgat gcagattttt ctgaaatgag atgtttagat gagcatacta 4200 ctaaagcaga gtggaaaaat ctgtctttat ggtatgttct aggtgtattg tgatttactg 4260 ttagattgcc aatataagta aatatagaca taatctatat atagtgtttc acaaagctta 4320 gatctttaac cttgcagctg ccccacagtg cttgacctct gagtcattgg ttatacagtg 4380 tagtcccaag cacataaact aggaagagaa tgtatttgta ggagcgctac cacctgtttt 4440 caagagaaca tagaactcca acgtaaccgt catttcaaag atttactgta tgtatagttg 4500 attttgtgga ctgaatttaa tgcttccaaa tgtttgcagt taccaaacat tgttatgcaa 4560 gaaatcataa aatgaagact tataccattg tgtttaagct gtattgaatt atctgtggaa 4620 tgcattgtga actgtaaagc aaagtatcaa taaagcttat agacttacct ttgtgtttag 4680 tgttttagtt tcatgaatgc acagcaaaaa cacggtggta ggcttagaga gtggacacat 4740 ggtaacatgc ttttagaaag gttttagttc atgaaacagc ttaagaacaa agaatatatt 4800 tacatagtga gatttatttg actcataaca aaaggtttta aattatttta tactttgaaa 4860 ataaattcat gcaccaatat tttaacagaa tacagtgcaa gatttatgaa tatacataaa 4920 attacaccat ataaatttta caaataagac tttcaaagtc tttataacag acactattgc 4980 tcttcaaata tatacatata tcattgatta gtcagttgtt catccacatg gttacttaat 5040 gcaagatctg tctgaatgaa atgtcagtag tacaagacag gcagacacag tgatcactca 5100 gcatcaccag gtacagaaaa cagaatcagg gctgcatagg gctctactga ggacccagca 5160 acctgctagt gggttgatgt aaggcattaa taagttggcg tgtaaaatag cttaatgtgt 5220 aatctaattc ttttagaatg ctgaagcact tctgtggtaa atgttgataa tctattcttt 5280 aactgaaaat gcttatttca acctttctct aaaatggcaa cttcatataa ctagaaactc 5340 aaggtctaga attttagtgc acagactgga aggactcggt ggtgtgtgta ctcacgactc 5400 caactcccat cagccttctt aactaatagt cgtcaagtca cattctgtcc gacaactgac 5460 tggagaaact caaatactcc ttacagtggg gaatatgttc aagaggtttt taaaaatctg 5520 aatttaccct gcattaatca tctgaaatga gcagagccaa gccagtccta cccaagaggg 5580 tgtaaactaa acagcaagac agctcactcg tcacactgga gctttccctg ctttccgtgt 5640 tgctactttc tgtggagctg gactcttctt tgctgctcac cttataactg ctttcctaga 5700 gcaggacata gtggtgaatt tgctaatccc atagccctcc tcagttttga agttttagca 5760 ccatgtgaag gaagaagacg acatgggagg tgaggcagca gcccacacaa ctgggaactt 5820 tgaaggcact taatggatat aaaactgcaa atgaaacttt taaaaattaa cattttaaaa 5880 cttgtttata catggctcat ttagttttga aagctaaatg tggcaaacag ggtatttctg 5940 tacttaagtg cttccaactt acattgtgtc cagtgaacat tcttaaaata catagaaaca 6000 gaatagcaaa acacctttgt aaaagtctta atgcaaatta aacgctacat attggttagg 6060 gtaattgagg atgtaaattt tatctgtggg ctacatcatc ccaattttgc cgttagtgaa 6120 gttacaataa gtataggttt tagtctccag aacagaagca aacag 6165 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NogoA F <400> 3 caagctcccg gaggacgac 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NogoA R <400> 4 gccctctctg gcaattctct 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh F <400> 5 actcacggaa aattcaacgg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh R <400> 6 accagtggat gcagggatga 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> qNogoA F <400> 7 ctcagtggat gagacccttt ttgc 24 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> qNogoA R <400> 8 cagtgttacc tggctgctcc t 21

Claims (20)

A composition for confirming the degree of differentiation of muscle precursor cells into muscle cells, comprising an agent for measuring an expression level of mRNA or protein of Nogo-A gene. The composition of claim 1, wherein the gene encodes a polypeptide of SEQ ID NO: 1. The composition of claim 1, wherein the agent is a primer pair that specifically binds to the gene, a probe, or an antibody that specifically binds to the protein. A kit for confirming the degree of differentiation of muscle precursor cells into muscle cells, comprising the composition of any one of claims 1 to 3. 5. The kit according to claim 4, wherein the kit is a reverse transcription polymerase chain reaction kit, a DNA chip kit, an ELISA kit, or a protein chip kit. A method for identifying the degree of differentiation of muscle precursor cells into muscle cells, comprising measuring the expression level of Noga-A in cells that are differentiating from muscle precursor cells to muscle cells. 7. The method of claim 6, wherein the measurement of the level of expression utilizes an antibody that specifically binds to a primer pair, probe, or Nogo-id protein that specifically binds Nogo-Ag gene. The method according to claim 6, wherein when the expression level of Nogo-A in the differentiating cell is higher than the expression level of Nogo-A in the control group, the differentiating cell is determined to be a cell that is more differentiated than the control, - &lt; / RTI &gt; expression level of the &lt; RTI ID = 0.0 &gt; A &lt; / RTI &gt; is lower than that of the control. delete delete delete delete delete delete delete delete delete delete delete delete

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