KR102385978B1 - A composition for promoting myogenic differentiation comprising flavonoid derivatives - Google Patents

Abstract

The present invention relates to isoramnetin, a flavonoid derivative, promoting muscle differentiation of mesenchymal stem cells. Since it has an effect of remarkably promoting the muscle differentiation of bone marrow-derived mesenchymal stem cells (eBM-MSC), it can be usefully used for the treatment and regeneration of muscle damage through the promotion of muscle differentiation, It can be clinically applied to regenerative medicine.

Description

A composition for promoting muscle differentiation comprising a flavonoid derivative as an active ingredient {A COMPOSITION FOR PROMOTING MYOGENIC DIFFERENTIATION COMPRISING FLAVONOID DERIVATIVES}

The present invention relates to a composition for promoting muscle differentiation comprising a flavonoid derivative as an active ingredient, and more particularly, to isoramnetin, a flavonoid derivative, promoting muscle differentiation of mesenchymal stem cells.

Stem cells are cells capable of differentiating into various cells constituting biological tissues, and collectively refer to undifferentiated cells in the pre-differentiation stage obtained from each tissue of the embryo, fetus, and adult. Stem cells are differentiated into specific cells by differentiation stimuli (environment), have the ability to produce cells identical to themselves (self-renewal) by cell division, and can differentiate into different cells according to differentiation stimuli. It is characterized by having plasticity that can be used. Stem cells can be divided into pluripotency, multipotency, and unipotency stem cells according to their differentiation capacity. Pluripotent stem cells are pluripotent cells with the potential to be differentiated into all cells, and some stem cells have the potential to be pluripotent or unipotent. Based on the differentiation ability of the stem cells, there is a possibility that they can be used as a cell therapy, and research and development related thereto is actively in progress. However, in the case of cell therapy using embryonic stem cells, ethical issues and tissue compatibility inconsistency issues are raised, and when dedifferentiated stem cells are used as cell therapies, there is a problem of the possibility of tumor occurrence. Accordingly, many studies using mesenchymal stem cells, which are known to have low differentiation capacity, but are relatively safe, are being conducted. Mesenchymal stem cells (MSCs) are multi-potential nonhematopoietic progenitor cells in the adult bone marrow, etc., and refer to cells that can differentiate into various types of cells such as fat, cartilage, bone, muscle, and skin. Clinical studies for the regeneration of various tissues using these mesenchymal stem cells are being conducted, and their applicability is also shown in the field of organ transplantation.

Meanwhile, mesenchymal stem cells, ie, pluripotent cells, have limited self-renewal potential, and these cells can be isolated from bone marrow, adipose tissue, and umbilical cord blood. MSCs have been reported to be useful in the regeneration of damaged tissues such as osteodystrophy, myocardial infarction, lung injury and cerebral infarction in several literatures. (Horwitz EM, et al. Blood 97:1227-31, 2001; Shake JG, et al. Ann Thorac Surg 73:1919-26, 2002; Rojas M, et al. Am J Respir Cell Mol Biol 33: 145-52, 2005; Li Y, et al Neurology 59:514-23, 2002). Mesenchymal stem cells (MSCs) can differentiate into various cells of the mesenchymal lineage, ie, bone, cartilage, adipose tissue, or muscle in vivo and in vitro (Prockop). DJ. Science 276:71-4, 1997; Pittenger MF, et al. Science 284:143-7, 1999). In particular, myocyte differentiation is regulated by various muscle regulatory factors, including MyoD, Myf5, Myogenin and MRF4. MyoD initiates the expression of specific genes in muscle and induces the differentiation of mesenchymal stem cells into the muscle lineage, and the induction of myogenin expression is regulated by MyoD activity, which leads to terminal differentiation and myosin heavy chain (MHC) and muscle creatine kinase. (MCK) plays an important role in the induction of muscle-specific genes. Myocyte enhancer factor 2 (MEF2) family of transcription factors can regulate muscle differentiation through a cooperative relationship with muscle electron factors of the bHLH family, such as MyoD and Myogenin. In this regard, research and development are in progress to treat related diseases using cell therapeutics differentiated from stem cells in muscle-related diseases caused by congenital or genetic defect or loss of muscle or tissue deletion due to trauma, tumor removal, etc. . In particular, many studies have been conducted to differentiate and transplant muscle satellite cells extracted from muscle into myoblasts, but there is a disadvantage in that it is difficult to quantitatively secure the cells because the cell culture period is short. In addition, there are some studies of inducing differentiation from adult stem cells into muscle cells using a medium for muscle cell differentiation, but their use is very limited in terms of differentiation efficiency. In particular, some mesenchymal stem cells do not have the characteristics of myosatellite cells, making it difficult to differentiate them into complete muscle cells, so research is needed to select a specific origin and control the differentiation ability from mesenchymal stem cells to muscle cells.

On the other hand, flavonoids, a kind of natural product, exist extensively in fruits and vegetables. It has various functions such as antioxidant, antibacterial, antiviral, antifungal and anti-inflammatory. it's awsome In recent years, studies on important biological activities of flavonols, such as antioxidants and anticancer agents, have been actively conducted.

In the present invention, it is an object of the present invention to confirm the effect of isoramnetin, a flavonoid derivative, on the differentiation of mesenchymal stem cells into muscle cells, and to use it for muscle differentiation, muscle regeneration, and treatment of muscle-related diseases.

In order to achieve the above object, the present invention provides a composition for promoting muscle differentiation, comprising isoramnetine, a flavonoid derivative represented by Formula 1, or a pharmaceutically acceptable salt thereof, as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for promoting muscle regeneration, comprising isoramnetine or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a composition for promoting muscle tissue formation or muscle regeneration in equine animals, comprising Equine Bone Marrow-derived Mesenchymal stem cells (eBM-MSC) and isoramnetin as active ingredients provides

In addition, the present invention provides a pharmaceutical composition for the treatment of muscle damage and muscle-related diseases, containing isoramnetine as an active ingredient.

In addition, the present invention provides a method for differentiating mesenchymal stem cells into muscle cells.

According to the present invention, when isorhamnetin, a flavonoid derivative compound, is treated with a muscle differentiation medium, horse Since it has the effect of significantly promoting muscle differentiation by increasing the expression of muscle differentiation markers in bone marrow-derived mesenchymal stem cells (eBM-MSC) and upregulating muscle-specific genes, isoramnetine can be usefully used for the treatment and regeneration of muscle damage by promoting muscle differentiation, and can be clinically applied to regenerative medicine.

1 is a diagram illustrating the cell morphology of eBM-MSCs.
2 is a view confirming the expression of cell surface markers and stem cell markers.
3 is a diagram confirming the degree of proliferation of eBM-MSCs with time.
4 is a view confirming the colony formation, self-proliferation and proliferation ability of eBM-MSCs.
5 is a diagram confirming the osteogenic differentiation, adipogenic differentiation and cartilage differentiation ability of eBM-MSCs.
Figure 6 is a diagram confirming muscle differentiation by confirming the expression of myogenin (myogenin), a marker for muscle formation:
Undifferentiated: undifferentiated cells;
Myogenic Medium: myogenic medium; and
Myogenic Medium+Isorhamnetin: Muscle differentiation medium containing isorhamnetin.
7 is a diagram confirming the expression of muscle markers MyoD and MyHC2:
Undifferentiated: undifferentiated cells;
Myogenic Medium: myogenic medium; and
Myogenic Medium+Isorhamnetin: Muscle differentiation medium containing isorhamnetin.

Hereinafter, the present invention will be described in detail by way of embodiments of the present invention. However, the following embodiments are presented as examples for the present invention, and the present invention is not limited thereto, and the present invention is capable of various modifications and applications within the scope of equivalents interpreted and interpreted from the following claims. .

In one aspect, the present invention relates to a composition for promoting muscle differentiation, comprising as an active ingredient a flavonoid derivative represented by the following formula (1), or a pharmaceutically acceptable salt thereof:

In one embodiment, the flavonoid derivative of Formula 1 is isorhamnetin, and can promote the differentiation of mesenchymal stem cells into muscle cells.

The isoramnetine of the present invention is a methylated flavonoid, having two benzene rings (rings A and B) linked by a 3 carbon chain forming a closed pyran ring (ring C) with a variable distribution of hydroxyl and methyl groups, There is (Table 1).

In one embodiment, the mesenchymal stem cells may be Equine Bone Marrow-derived Mesenchymal stem cells (eBM-MSCs).

In one embodiment, the horse bone marrow-derived mesenchymal stem cells are immunologically negative for the cell surface antigens CD19, CD34 and CD45 (PTPRC); positive immunological characteristics for the cell surface antigens CD73 (NT5E), CD90 (THY1) and CD105 (ENG); and positive immunological properties for Nanog, Sox2 and Oct4, which are undifferentiated stem cell marker proteins.

In one aspect, the present invention relates to a pharmaceutical composition for promoting muscle regeneration, comprising a flavonoid derivative represented by the following formula (1) as an active ingredient:

[Formula 1]

.

.

In one embodiment, the pharmaceutical composition may be for promoting muscle injury regeneration in horses and animals.

In one aspect, the present invention is horse bone marrow-derived mesenchymal stem cells (Equine Bone Marrow-derived Mesenchymal stem cell, eBM-MSC) and a flavonoid derivative represented by the following formula (1) containing as an active ingredient, equine muscle tissue It relates to a composition for promoting formation or muscle regeneration:

[Formula 1]

.

.

In one embodiment, the present invention comprises administering to a treatment area of a subject a therapeutically effective amount of the aforementioned composition of the present invention, wherein the treatment area is adjacent to the damaged area and to the damaged area of the subject or subject. Regeneration of adjacent muscles or tendons is promoted.

In one embodiment, the subject may suffer from a muscle injury, muscle waste, muscular dystrophy, amyotrophic lateral sclerosis, tendon injury, tissue ischemia, cerebral ischemia, lower extremity arterial disease or myocardial infarction, which includes the muscle or tendon of the injured area. cause damage

In one aspect, the present invention relates to a pharmaceutical composition for treating muscle damage and muscle-related diseases, containing a flavonoid derivative represented by the following formula (1) as an active ingredient:

[Formula 1]

.

.

In one embodiment, the muscle-related disease may be selected from the group consisting of degenerative muscle disease, muscular dystrophy, muscular atrophy, X-linked spinal-bulbar muscular atrophy (SBMA), cachexia and sarcopenia, wherein The amyotrophic disease may be muscular atrophy, myasthenia gravis, muscular dystrophy, myos stiffness, hypotonia, muscle weakness, muscular dystrophy, muscular dystrophy, amyotrophic lateral sclerosis or myasthenia gravis.

As used herein, the term “treatment” refers to any action that improves or beneficially changes muscle damage or muscle-related disease, or symptoms resulting therefrom, by administration of the composition of the present invention. Those of ordinary skill in the art to which the present invention pertains, with reference to the data presented by the Korean Medical Association, etc., know the exact standard of the disease for which the composition of the present application is effective, and can determine the degree of improvement, improvement and treatment will be.

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, external preparations, suppositories, sterile injection solutions and sprays.

The therapeutically effective amount of the composition of the present invention may vary depending on several factors, for example, the administration method, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. A pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compound described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable dosage form such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In addition, the composition of the present invention may contain one or more active ingredients exhibiting the same or similar function. The composition of the present invention comprises 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, stearic acid Calcium, sucrose, dextrose, sorbitol and talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally according to a desired method, and the dosage may vary depending on the patient's weight, age, sex, health status, The range varies depending on the diet, administration time, administration method, excretion rate, and the severity of the disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to divide and administer once to several times a day.

Liquid formulations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives and the like may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

In one aspect, the present invention relates to a method for differentiating mesenchymal stem cells into muscle cells, comprising culturing the mesenchymal stem cells in a muscle differentiation medium containing a flavonoid derivative of Formula 1 below:

[Formula 1]

.

.

In one embodiment, the mesenchymal stem cells may be Equine Bone Marrow-derived Mesenchymal stem cells (eBM-MSCs).

In one embodiment, the horse bone marrow-derived mesenchymal stem cells are immunologically negative for the cell surface antigens CD19, CD34 and CD45 (PTPRC); positive immunological characteristics for the cell surface antigens CD73 (NT5E), CD90 (THY1) and CD105 (ENG); and positive immunological properties for Nanog, Sox2 and Oct4, which are undifferentiated stem cell marker proteins.

In one embodiment, differentiation into muscle cells may be achieved by inducing upregulation of myogenin, a myogenic regulator.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1. Characterization of horse bone marrow-derived mesenchymal stem cells (eBM-MSC)

1-1. Check cell morphology

Equine Bone Marrow-derived Mesenchymal stem cell (eBM-MSC), a horse bone marrow-derived mesenchymal stem cell, was prepared in 10% fetal bovine serum, 50U/mL penicillin and 50μg/mL streptomycin (Invitrogen, CA, USA) solution. After being suspended in the added DMEM-HG (Dulbecco'modified Eagles medium high glucose) culture solution, 5% CO ₂ , and cultured at 37°C. Then, as a result of confirming its morphology with a microscope (EVOS XL Core Cell Imaging System, Electron microscopy sciences, USA), eBM-MSC had a long spindle-shaped morphology similar to fibroblast cells (FIG. 1).

1-2. Confirmation of expression of cell surface markers and stem cell markers in eBM-MSCs

Total RNA was extracted from eBM-MSC, a mesenchymal stem cell derived from horse bone marrow cultured as in Example 1-1, using Labo Pass Kit, TRIzol (Cosmogenetech, Seoul, Korea). The concentration of total RNA was measured with a Nanodrop (ND1000) spectrophotometer (Nanodrop Technologies Inc., Wilmington DE, USA), and cDNA was synthesized according to the manufacturer's method using 2 μg of total RNA and M-MLV reverse transcriptase (Promega). did To quantify the change in the expression level of the target gene, cDNA and primers were mixed with SYBR Green master mix (Elpis Biotech, South Korea) according to the manufacturer's method, and then gene expression was measured with an Applied Biosystem 7500 real-time PCR system. The expression level of the target gene was calculated based on GAPDH. Cell surface markers CD19, CD34, CD45 (PTPRC), CD73 (NT5E), CD90 (THY1) and CD105 (ENG) were used as target genes, and stem cell markers Nanog, Sox2 and Oct4 were used. Each primer for the gene is listed in Table 2 below.

gene name primer order SEQ ID NO: CD19 Forward CAT CCC GAG AAG ACT GCC TC One Reverse AGT CTC CAT CAG CCA ATG CC 2 CD34 Forward CAG AAA TTC CCA GCA AGC TC 3 Reverse ATA GCA AAT GAG GCC CAA GA 4 CD45 (PTPRC) Forward TGA TGA TTT CTG GAG GAT GAT CTG 5 Reverse CAC TTG TTC CTA TTT CCT TCT TCA CA 6 CD73 (NT5E) Forward GGG ATT GTT GGA TAC ACT TCA AAA G 7 Reverse GCT GCA ACG CAG TGA TTT CA 8 CD90 (THY1) Forward AGA ATA CCA CCG CCA CA 9 Reverse GGA TAA GTA GAG GAC CTT GAT G 10 CD105 (ENG) Forward TGA CGA CCA CCT CAT TAC TG 11 Reverse AAG AGC TCA TCT CGA GTC TG 12 Nanog Forward TAC CTC AGC CTC CAG CAG AT 13 Reverse CGT TCC CAG CAG TGT TCA 14 Sox2 Forward CAC CCA CAG CAA ATG ACA GC 15 Reverse TTT CTG CAA AGC TCC TAC CG 16 Oct4 Forward ACT TCA CCT TCC CTC CAA CC 17 Reverse GTC CTC ACT TCA CTA CGC TGT 18 GAPDH Forward GGC AAG TTC CAT GGC ACA GT 19 Reverse CAC AAC ATA TTC AGC ACC AGC AT 20

As a result, CD19, CD34 and CD45 were negatively expressed, and CD105, CD90 and CD73 were positively expressed in eBM-MSC, which is a horse bone marrow-derived mesenchymal stem cell. In addition, Nanog, Oct4 and Sox2 were positively expressed ( FIG. 2 ).

1-3. Confirmation of proliferative capacity of eBM-MSC

To measure the proliferative capacity of eBM-MSCs in a time-dependent manner (days 0, 2, 4, 6, 8, and 10), cells were removed at each time point and stained with trypan blue. The number of stained cells was observed under a microscope and counted using a hemocytometer. As a result, it was found that eBM-MSCs proliferated in a time-dependent manner, and had remarkable proliferative ability up to 10 days ( FIG. 3 ).

In addition, in order to evaluate the colonization, self-proliferation and proliferation ability of eBM-MSCs through colonization analysis, eBM-MSCs were inoculated into a 35 mm ² culture vessel (Corning) with a low cell number (1 X 10 ³ ). Then, it was cultured in basal medium for 14 days. On the 14th day of culture, after washing twice with D-PBS, 0.3% crystal violet (Sigma-Aldrich) prepared in methanol was used for staining at room temperature for 30 minutes. After dyeing, the crystal violet was removed, the vessel was washed with D-PBS and distilled water, and then completely dried. After that, colonies were photographed, and only clusters having 50 or less cells were counted. As a result, eBM-MSCs self-proliferated to form many colonies, and it was seen that these colonies were stained purple ( FIG. 4 ).

1-4. Confirmation of multilineage differentiation of eBM-MSCs

The eBM-MSCs were attached to 24 wells at a density of 2x10 ⁴ /well, and when the density reached 80%, they were replaced with the respective differentiation media to induce adipogenic differentiation, osteogenic differentiation and chondrogenic differentiation. 10% FBS, 1% penicillin/streptomycin, 100 nM dexamethasone (Sigma-Aldrich, MO) in Dulbecco'modified Eagle'medium (DMEM)-low glucose (Invitrogen, CA, USA) as a differentiation medium for osteogenic differentiation , USA), 50 μg/ml ascorbate-2-phosphate (Sigma-Aldrich, MO, USA) and 10 mM β-glycerophosphate (Sigma-Aldrich, MO, USA) were added. As a differentiation medium for adipogenesis, 10% FBS, 1% penicillin/streptomycin, 500 μM IBMX (isobutylmethylxanthine), 1 μM dexamethasone, 200 μg/ml ascorpate-2-phosphate, 100 μM indomethacin and 10 μg in DMEM-low glucose as a differentiation medium for adipogenesis. /ml insulin (insulin) was added. As a medium for cartilage differentiation, DMEM-low glucose, 2% FBS, 1% penicillin/streptomycin, 50 μg/mL ascorbate-2-posphate, 100 μg/mL sodium pyruvate, 1% ITS-X (Insulin-Transferrin-Selenium-Ethanolamine) ) (Gibco), 100 nM dexamethasone, 40 μg/mL L-proline and 10 ng/mL TGF-β3 (Prospec, East Brunswick, NJ, USA) were added. Each differentiation medium was replaced every 2 days for 2 weeks. Differentiation was confirmed by staining each cell inducing differentiation. Specifically, for osteogenic differentiation, cells were fixed with 4% paraformaldehyde for 15 minutes at the end of differentiation, washed with sterile water, and stained using Alizarin Red S staining method. For adipogenic differentiation, at the time of differentiation, cells were fixed with 4% paraformaldehyde for 15 minutes, washed first with sterile water, washed second with 60% isopropanol, and then washed with 5% isopropanol (wt/vol) diluted with isopropanol (wt/vol). It was stained using Oil Red O. Cartilage differentiation was fixed with 4% paraformaldehyde for 15 minutes at the end of differentiation, washed with sterile water, and stained with Alcian blue, which stains acidic mucopolysaccharides such as glycosaminoglycan.

As a result, it was found that eBM-MSCs have multi-differentiation ability capable of bone differentiation, adipogenesis, and cartilage differentiation (FIG. 5).

Through this, it was possible to verify the differentiation potential of eBM-MSC cells into three lineages and their application to clinical trials.

Example 2. According to the addition of isorhamnetin Confirmation of muscle differentiation potential of eBM-MSCs

First, Equine Bone Marrow-derived Mesenchymal Stem Cell (eBM-MSC), a horse bone marrow-derived mesenchymal stem cell, was treated with 10% fetal bovine serum, 50U/mL penicillin and 50μg/mL streptomycin (Invitrogen, CA, USA). ) solution was suspended in DMEM-LG (Dulbecco'modified Eagles medium low glucose) culture medium, and then incubated at 5% CO ₂ , 37°C. Thereafter, for immunocytochemical staining, glass slides (18/18 mm, Superior-Marienfeld, Germany) were washed with 70% ethanol for 15 minutes, washed with distilled water, and dried. After placing the glass slide prepared above in the 12-well at the bottom of the well, eBM-MSCs were adhered to the glass slide at a density of 4 x 10 ⁴ cells/well and cultured. One day after attachment, myogenic medium (5% horse serum, 10% fetal bovine serum, 50U/mL penicillin, 50μg/mL streptomycin (Invitrogen, CA, USA) and 3uM DMEM-HG (Dulbeccomodified Eagles medium high glucose) with 5-Aza-2′-deoxycytidine (Sigma Chemical Co., St Louis, MO, USA) solution of Alternatively, it was replaced with a muscle differentiation medium supplemented with 20 μM isoramnetine and cultured for 2 weeks (replaced with fresh medium every 2 days). After incubation, the cells were fixed with 4% paraformaldehyde for 20 minutes and washed with PBS. Cells were infiltrated for 10 minutes using 0.3% Triton X-100, washed with PBS, and left at room temperature for 1 hour with 10% normal goat serum to block non-specific antibody binding. Cells were reacted with an anti-myogenin (Santa Cruz, USA) antibody as a primary antibody for 12 hours at 4°C, then the cells were washed with PBS and Dylight 549-conjugated goat anti-mouse antibody was used as a secondary antibody for 1 hour at room temperature. (Jackson Immuno Research Labs, PA, USA) was incubated at room temperature. Cell nuclei were stained with DAPI contained in the mounting solution. All images were taken and observed with an ultra-high-resolution confocal laser scanning microscope (LSM 800, Carl Zeiss, Germany).

As a result of observation, it was found that when the muscle differentiation medium was added, the expression of myogenin, a marker for muscle formation, increased, and thus muscle differentiation was significantly increased. It was found that the expression of myogenin was remarkably increased compared to that of the old cells (FIG. 6).

Example 3. Confirmation of specific expression of muscle-related markers of eBM-MSC by addition to isoramnetine

As in Example 2, eBM-MSC, which is a horse bone marrow-derived mesenchymal stem cell, was cultured and total RNA from eBM-MSC cultured in a muscle differentiation medium containing or without isoramnetine was collected using Labo Pass Kit, TRIzol (Cosmogenetech, Seoul, Korea) was used for extraction. The concentration of total RNA was measured with a Nanodrop (ND1000) spectrophotometer (Nanodrop Technologies Inc., Wilmington DE, USA), and cDNA was synthesized according to the manufacturer's method using 2 μg of total RNA and M-MLV reverse transcriptase (Promega). did In order to quantify the change in the expression level of the target gene, cDNA and the primers of SEQ ID NOs: 21 to 24 of Table 3 below were mixed with SYBR Green master mix (Elpis Biotech, South Korea) according to the manufacturer's method, respectively, followed by Applied Biosystem 7500 real time The expression of muscle-related marker genes, MyoD and MyHC, was measured using a PCR system. Their expression level was calculated based on GAPDH.

gene name primer order SEQ ID NO: MyoD Forward GTC GAG GAC AGT CGG GTG TA 21 Reverse AAG TCG TCC GCT GTA GCA AA 22 MyHC2 Forward TGA GTC CCA GGT CAA CAA GC 23 Reverse TCC TTT GCA GTA GGG TGG AA 24 GAPDH Forward GGC AAG TTC CAT GGC ACA GT 19 Reverse CAC AAC ATA TTC AGC ACC AGC AT 20

As a result, when a muscle cell differentiation medium was added to eBM-MSC, which is a horse bone marrow-derived mesenchymal stem cell, the expression of muscle markers MyoD and MyHC was increased, which was significantly increased by the addition of isoramnetin (Fig. 7).

<110> Konkuk University Industrial Cooperation Corp <120> A COMPOSITION FOR PROMOTING MYOGENIC DIFFERENTIATION COMPRISING FLAVONOID DERIVATIVES <130> PN1712-448 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD19 forward primer <400> 1 catcccgaga agactgcctc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD19 reverse primer <400> 2 agtctccatc agccaatgcc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD34 forward primer <400> 3 cagaaattcc cagcaagctc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD34 Reverse primer <400> 4 atagcaaatg aggcccaaga 20 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CD45 forward primer <400> 5 tgatgatttc tggaggatga tctg 24 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CD45 Reverse primer <400> 6 cacttgttcc tatttccttc ttcaca 26 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CD73 forward primer <400> 7 gggattgttg gatacacttc aaaag 25 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD73 reverse primer <400> 8 gctgcaacgc agtgatttca 20 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> CD90 forward primer <400> 9 agaataccac cgccaca 17 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CD90 reverse primer <400> 10 ggataagtag aggaccttga tg 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD105 forward primer <400> 11 tgacgaccac ctcattactg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD105 reverse primer <400> 12 aagagctcat ctcgagtctg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nanog forward primer <400> 13 tacctcagcc tccagcagat 20 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Nanog reverse primer <400> 14 cgttcccagc agtgttca 18 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox2 forward primer <400> 15 cacccacagc aaatgacagc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sox2 reverse primer <400> 16 tttctgcaaa gctcctaccg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oct4 forward primer <400> 17 acttcacctt ccctccaacc 20 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Oct4 reverse primer <400> 18 gtcctcactt cactacgctg t 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 19 ggcaagttcc atggcacagt 20 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 20 cacaacatat tcagcaccag cat 23 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MyoD forward primer <400> 21 gtcgaggaca gtcgggtgta 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MyoD reverse primer <400> 22 aagtcgtccg ctgtagcaaa 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MyHC2 forward primer <400> 23 tgagtcccag gtcaacaagc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MyHC2 reverse primer <400> 24 tcctttgcag tagggtggaa 20

Claims (10)

Horse bone marrow-derived mesenchymal stem containing isorhamnetin represented by the following Chemical Formula 1 as an active ingredient, wherein the isorhamnetin induces upregulation of myogenin A medium composition for promoting differentiation of cells (Equine Bone Marrow-derived Mesenchymal stem cells, eBM-MSCs) into muscle cells:
[Formula 1]

. delete delete The method of claim 1, wherein the horse bone marrow-derived mesenchymal stem cells are immunologically negative for the cell surface antigens CD19, CD34 and CD45 (PTPRC); positive immunological characteristics for the cell surface antigens CD73 (NT5E), CD90 (THY1) and CD105 (ENG); and mesenchymal stem cells having positive immunological properties for the undifferentiated stem cell marker proteins Nanog, Sox2 and Oct4, the medium composition. delete delete delete delete delete A method comprising culturing horse bone marrow-derived mesenchymal stem cells in a muscle differentiation medium comprising the medium composition of claim 1, Equine Bone Marrow-derived Mesenchymal stem cells (eBM-MSC) muscle How to differentiate into cells.

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