JP7083141B2 - Compositions and Their Use to Improve Muscle Loss or Weakness - Google Patents

Description

The present specification relates to compositions and their uses that enhance or improve the quality or quantity of muscle.

Building muscle is important not only for maintaining and improving health, but also for a healthy and independent life. On the other hand, muscular atrophy, muscle mass and muscle strength may decrease due to aging or disease. In addition, muscle mass and strength may decrease due to lack of exercise or long-term rest. A decrease in muscle mass or strength also causes a decrease in motor function and, in turn, a decrease in physical function, which may have a great effect on the subsequent quality of life (QOL).

In addition, decreased motor and physical function may increase the risk of suffering from falls in daily life, osteoporosis, obesity, metabolic disorders, and mental illness. In diabetes, loss of muscle mass can increase insulin resistance and exacerbate diabetes itself.

Routine exercise and rehabilitation are effective in suppressing the decrease in muscle mass and strength. However, continuous implementation of exercise and the like may be difficult.

Therefore, various muscle-building means are being studied. For example, muscle-building agents such as lactic acid and catechins are known (Patent Documents 1 and 2).

On the other hand, it is known that a composition containing dental pulp stem cells and its culture supernatant and dental pulp stem cell culture supernatant is effective in treating damages in skin and bones and injuries in central nervous system diseases such as cerebral infarction. (Patent Documents 3 and 4). In addition, skeletal muscle regeneration treatment using bone marrow mesenchymal stem cells, peripheral blood CD133-positive cells, and adipocyte-derived mesenchymal stem cells is also known (Non-Patent Document 1).

Japanese Unexamined Patent Publication No. 2014-208637 JP-A-2015-12986 International release WO2009 / 072527 International release WO2011 / 118795

Stem Cell Research & Therapy 6: 156, 2015

However, the methods disclosed in Patent Documents 1 and 2 have problems such as the need for oral administration and exercise, and the inability to obtain a sufficient effect. Further, Patent Document 3 describes that dental pulp stem cells are effective for bone and skin regeneration, but does not mention muscle. Further, Patent Document 4 describes that a certain therapeutic effect was obtained by administering a culture supernatant of dental pulp stem cells to skin, bone, periodontal tissue, cerebral infarction, and spinal cord injury. Is not mentioned. According to Non-Patent Document 4, when stem cells derived from dental pulp are differentiated into myoblasts, differentiated into myoblasts, and then transplanted into a muscular dystrophy model mouse, dystrophin lacking in muscular dystrophy is produced and skeletal muscle is regenerated. It is stated that

The present specification provides more practical compositions and uses thereof for muscle building loss due to diabetes and the like.

The present inventors have been studying the treatment of diabetic neuropathy by transplantation of dental pulp stem cells based on the neuroprotective action and blood flow improving action of dental pulp stem cells. Then, it was confirmed that the pulp stem cells were effective in improving the decreased neuropathy in the lower limbs by transplanting them into the lower limbs of a diabetic model animal, and that the pulp stem cells were present in the muscle bundle gap. However, surprisingly, the present invention has been completed by newly discovering the enhancement of muscle strength and the increase of the muscle bundle area at the transplantation site of dental pulp stem cells. Based on these findings, the present specification provides the following means.

(1) A composition for strengthening muscle, which comprises a dental pulp stem cell and / or a culture supernatant of dental pulp stem cell.
(2) The composition according to (1), wherein the muscle is skeletal muscle.
(3) The composition according to (1) or (2), which is for injection into a muscle-building site.
(4) The composition according to any one of (1) to (3), which comprises dental pulp stem cells.
(5) The composition according to any one of (1) to (4), which comprises frozen dental pulp stem cells.
(6) A composition for treating or improving sarcopenia, which comprises dental pulp stem cells and / or a culture supernatant of dental pulp stem cells.
(7) A composition for treating or improving sarcopenia associated with diabetes, which comprises a pulp stem cell and / or a culture supernatant of dental pulp stem cells.
(8) A composition containing dental pulp stem cells and / or a culture supernatant of dental pulp stem cells for suppressing or ameliorating a decrease in muscle mass or a decrease in muscle strength associated with diabetes.
(9) A composition for increasing muscle bundle size, which comprises a pulp stem cell and / or a culture supernatant of dental pulp stem cells.
(10) A non-therapeutic muscle-building method for administering an effective amount of pulp stem cells and / or a culture supernatant of dental pulp stem cells.

It is a figure which shows the measurement result of the muscle strength of the hind limb of a normal and diabetic rat which administered the dental pulp stem cell to the hind limb. It is a figure which shows the measurement result of the gastrocnemius muscle mass of the hind limb of a normal and diabetic rat which administered the dental pulp stem cell to the hind limb. It is a figure which shows the gastrocnemius muscle histology image of the hind limb of a normal and diabetic rat to which dental pulp stem cell was administered to the hind limb, and the muscle bundle size measurement result per unit area of the gastrocnemius muscle. It is a figure which shows the gene expression analysis result of the angiogenesis factor and the neurotrophic factor in the gastrocnemius muscle of the hind limb of normal and diabetic rats to which dental pulp stem cell was administered to the hind limb.

The disclosure herein relates to compositions for muscle building and their use. The composition for building muscle disclosed herein (hereinafter, simply referred to as the present composition) can contain dental pulp stem cells and / or a culture supernatant thereof as an active ingredient. Dental pulp stem cells are known to be effective for bone and skin regeneration (Patent Document 3), and the culture supernatant of dental pulp stem cells can be administered to skin, bone, periodontal tissue, cerebral infarction, and spinal cord injury. It is also known that a certain therapeutic effect was obtained for the injury. Furthermore, it has been reported that dental pulp stem cells are effective for alveolar bone regeneration and muscle dystrophin regeneration. However, against such a background, whether or not dental pulp stem cells and their culture supernatants can be used for muscle strengthening in diabetes and the like could not be predicted at all by those skilled in the art.

Muscle regeneration and recovery are generally associated with injury and inflammatory reactions and are not always apparent. According to the findings of the present inventors, it has been confirmed that the expression of myosynthetic genes is enhanced and the expression of muscular atrophic genes is decreased under diabetic conditions by using dental pulp stem cells and their culture supernatants. It is considered that as a result of promotion of myofibrils and the like by remodeling of myofibrils and fusion of myoblasts, an increase in muscle mass and an increase in muscle strength occur.

The term "muscle" in the present specification is not particularly limited, but is mainly skeletal muscle (voluntary muscle). The muscles herein can extend throughout the body from the head to the lower limbs (specifically, the head, neck, chest, abdomen, back, upper limbs, lower limbs). For example, the muscles in the present specification include, for example, antigravity muscles (erector spinae muscle, rectus abdominis muscle, gluteus maximus muscle, quadriceps muscle, etc.). Also, the muscles of the lower limbs can be mentioned. Examples of the muscles of the lower limbs include lower limb band muscles, thigh muscles, lower leg muscles, foot muscles, and the like, and among these, the lower leg muscles are preferable. Examples of the lower leg muscle include the muscle of the lower leg extensor muscle group, the fibula muscle, the muscle of the lower leg flexor muscle group, and the like, and among them, the muscle of the lower leg extensor muscle group and the muscle of the lower leg flexor muscle group are preferable. Examples of the muscles of the lower leg extensor muscle group include the anterior tibial muscle, the extensor digitorum longus (extensor digitorum longus), the third peroneal muscle, and the extensor hallucis longus. Examples of the muscles of the lower leg flexor muscles include plantaris muscle (plantaris flexor muscle), patellar muscle, lower leg triceps muscle (peroneal abdominal muscle, soleus muscle, etc.), long toe flexor muscle, posterior cervical flexor muscle, long mother toe flexor muscle, and the like.

Further, the "strengthening" of "muscle" is not particularly limited as long as the muscle is strengthened as a result, but can mainly mean an increase in muscle mass or an increase in muscle strength. Furthermore, it can also mean an increase in muscle bundle size. Therefore, the present composition can also be used, for example, for increasing muscle mass, increasing muscle strength, and increasing muscle bundle size. Further uses of the composition will be described in detail later.

Hereinafter, various embodiments of the composition for strengthening muscles disclosed in the present specification will be described in detail.

(Composition for strengthening muscle: this composition)
The composition can include pulp stem cells and / or culture supernatants of pulp stem cells. According to this composition, in addition to increasing muscle mass and / or increasing muscle strength, it is possible to obtain the effect of increasing muscle bundle size. This makes it possible to increase muscle strength.

The present composition may contain dental pulp stem cells as an active ingredient, a culture supernatant of dental pulp stem cells as an active ingredient, or both dental pulp stem cells and the culture supernatant thereof as an active ingredient. can.

When the present composition contains dental pulp stem cells as an active ingredient, it is preferable that the composition contains only dental pulp stem cells and does not contain the culture supernatant. However, it may be acceptable to include a culture supernatant of dental pulp stem cells or dental pulp cells as impurities. Dental pulp stem cells can be obtained, for example, by the methods shown below.

When the present composition contains a culture supernatant of dental pulp stem cells as an active component, it is preferable that the composition contains only the culture supernatant and does not contain cell components such as dental pulp stem cells in addition to dental pulp stem cells. However, it may be acceptable to include dental pulp stem cells and dental pulp cells as impurities. The culture supernatant of dental pulp stem cells can be obtained, for example, by the method shown below.

When the present composition contains dental pulp stem cells and a culture supernatant of dental pulp stem cells as active ingredients, it is preferable that the composition contains only dental pulp stem cells and the culture supernatant and does not contain dental pulp cells. However, it is acceptable to include dental pulp cells as contaminants. The dental pulp stem cells and the culture supernatant may be obtained separately and then mixed, or the dental pulp stem cell culture medium itself may be used as an active ingredient.

(Pulp stem cells)
The pulp stem cells are not particularly limited as long as they are stem cells derived from the pulp obtained from the pulp. Permanent dental pulp stem cells or deciduous dental pulp stem cells may be used, but preferably, from the viewpoint of cell proliferation ability, the deciduous tooth or the extracted tooth at a young age (extraction by orthodontic treatment or third molar extraction). ) Derived from dental pulp stem cells. In relation to individuals to which this composition is applied, in order to suppress or avoid rejection, dental pulp stem cells of the same species (human origin in the case of humans) are preferable, and even allogeneic dental pulp stem cells are used. It is good, but more preferably autologous dental pulp stem cells are used.

Dental pulp stem cells can be sorted as adhesive cells among dental pulp cells. Adhesive cells in pulp cells collected from shed deciduous teeth and permanent teeth can be used. For example, human dental pulp stem cells can be obtained by the following methods.

(1) Collection of pulp The naturally shed deciduous teeth (or extracted deciduous teeth or permanent teeth) are disinfected with a chlorohexidine or isodine solution, and then the crown is divided and the pulp tissue is collected with a dental reamer.
(2) Enzyme treatment The collected dental pulp tissue is suspended in a basal medium (10% bovine serum / antibiotic-containing modified Dulbecco Eagle's medium) and treated with 2 mg / ml collagenase and dispase at 37 ° C. for 1 hour. The dental pulp cells after the enzyme treatment are collected by centrifugation for 5 minutes (5000 rpm). In principle, cell selection by a cell strainer is not used because it reduces the recovery efficiency of the neural stem cell fraction of SHED and DPSC.
(3) Cell culture (selection of adhesive cells)
The cells are resuspended in 4 cc basal medium and seeded in a 6 cm diameter adherent cell culture dish. After culturing in an incubator adjusted to 5% CO ₂ and 37 ° C. for 3 days, the colonized adhesive cells are treated with 0.05% trypsin / EDTA for 5 minutes at 37 ° C. The dental pulp cells exfoliated from the dish are seeded in a dish for adhering cell culture having a diameter of 10 cm and expanded culture is performed. For example, when the cells reach the subconfluent (a state in which cells occupy about 70% of the surface of the culture vessel) or confluence by observing with the naked eye, the cells are separated from the culture vessel and collected, and the culture is filled with the culture medium again. Seed in a container. Subculture may be repeated. For example, subculture is performed 1 to 8 times to grow to the required number of cells (for example, about 1 × ¹⁰⁷ cells / ml). The cells can be detached from the culture vessel by a conventional method such as trypsin treatment. After the above culture, the cells may be collected and stored (preservation conditions are, for example, −198 ° C.). The following alternative methods can also be mentioned.

(Another method)
The cells are resuspended in 4 cc basal medium and seeded in a 6 cm diameter adherent cell culture dish. After adding the culture solution (for example, DMEM (Dulvecco's Modified Eagle's Medium) containing 10% FCS), incubate in an incubator adjusted to 5% CO ₂ and 37 ° C. for about 2 weeks. After removing the culture medium, wash the cells once or several times with PBS or the like. Instead of this operation (removal of culture medium and washing of cells), adhesive cells (dental pulp stem cells) that have formed colonies may be collected. In this case, for example, the cells are treated with 0.05% trypsin / EDTA for 5 minutes at 37 ° C. to detach the cells from the dish.

(4) Recovery of cells Next, the cells are recovered. The cells can be recovered by exfoliating the cells from the culture vessel by trypsin treatment or the like and then subjecting them to centrifugation. The present composition can be prepared using the dental pulp stem cells thus recovered.

The dental pulp stem cells may be in a frozen state, that is, frozen dental pulp stem cells. By thawing and re-culturing at the time of use, it is possible to retain the same function as dental pulp stem cells that have not undergone freezing. Freezing and thawing of dental pulp stem cells can be performed according to a method for cryopreserving cells known to those skilled in the art. For example, 1 ml of a cell suspension adjusted to a predetermined concentration in a commercially available freezing medium is dispensed into an ampol, and the temperature is about -1 to 2 ° C./3 minutes. It freezes at a cooling rate and can eventually be stored in liquid nitrogen. For melting, for example, an ampoule is put into hot water at 37 ° C. so that the ampoule is melted quickly. Then, by appropriately separating the cells by centrifugation or the like, thawed dental pulp stem cells can be obtained.

(Culture supernatant of dental pulp stem cells)
The culture supernatant of dental pulp stem cells is a supernatant of a cell culture medium obtained by culturing dental pulp stem cells. That is, it is a humoral component that does not substantially contain a cellular component (pulp stem cell or dental pulp cell). The cultured dental pulp stem cells are removed from the humoral components of the culture medium by separating and removing the cell components after culturing. Separation of cellular components from the culture medium is possible by methods well known to those skilled in the art. Further, it was decided to use a culture supernatant obtained by appropriately subjecting the culture solution to various treatments (for example, centrifugation, concentration, solvent substitution, dialysis, freezing, drying, freeze-drying, dilution, desalting, storage, etc.). May be good.

For culturing dental pulp stem cells, a basal medium or a basal medium supplemented with serum or the like can be used. As the basic medium, in addition to DMEM, Iskoff-modified Dulbecco medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12) (SIGMA, GIBCO, etc.), RPMI1640 medium and the like can be used. Two or more kinds of basal media may be used in combination. As an example of the mixed medium, a medium in which IMDM and HamF12 are mixed in equal amounts (for example, commercially available as a trade name: IMDM / HamF12 (GIBCO)) can be mentioned. Examples of components that can be added to the medium include serum (fetal bovine serum, human serum, sheep serum, etc.), serum replacement (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, and various types. Examples include vitamins and various minerals.

It is preferable that the culture supernatant of dental pulp stem cells does not contain serum. The absence of serum enhances the safety of the composition. For example, by culturing dental pulp stem cells in a serum-free medium (serum-free medium), a serum-free culture supernatant can be prepared. A serum-free culture supernatant can also be obtained by performing one or a plurality of subcultures and culturing the last or several subcultures in a serum-free medium. On the other hand, serum-free culture supernatant can also be obtained by removing serum from the collected culture supernatant by using dialysis, solvent substitution with a column, or the like.

(Acquisition of culture supernatant of dental pulp stem cells)
For the culture of dental pulp stem cells, the conditions normally used for stem cells can be applied as they are or can be appropriately modified. A person skilled in the art can appropriately produce the pulp stem cell culture supernatant. For example, the culture supernatant may be obtained by the following operation.

First, as described above, the adhesive cells (pulp stem cells) selected from the dental pulp are cultured in the above-mentioned medium. For example, cells are seeded in an adherent cell culture dish and cultured in an incubator adjusted to 5% CO ₂ and 37 ° C. Perform subculture if necessary. For example, when the cells reach the subconfluent (a state in which cells occupy about 70% of the surface of the culture vessel) or confluence by observing with the naked eye, the cells are separated from the culture vessel and collected, and the culture is filled with the culture medium again. Seed in a container. Subculture may be repeated. For example, subculture is performed 1 to 8 times to grow to the required number of cells (for example, about 1 × ¹⁰⁷ cells / ml). The cells can be detached from the culture vessel by a conventional method such as trypsin treatment. After the above culture, the cells may be collected and stored (preservation conditions are, for example, −198 ° C.).

Next, the culture supernatant of the selected and cultured dental pulp stem cells is collected. For example, the culture solution can be sucked and collected with a dropper or a pipette. The recovered culture supernatant is used as it is or after undergoing one or more treatments as an active ingredient of the composition of the present invention. Examples of the treatment here include centrifugation, concentration, solvent replacement, dialysis, freezing, drying, freeze-drying, dilution, desalting, and storage (eg, 4 ° C., −80 ° C.).

The main culture supernatant can be appropriately concentrated. That is, the present culture supernatant may be contained as a concentrate. As the concentration method, a person skilled in the art can appropriately select and use from known methods. For example, a concentrate of the culture supernatant can be obtained by a spin column concentration method or an ethanol precipitation concentration method. The main culture supernatant may be freeze-treated or freeze-dried. That is, the present culture supernatant may be a frozen product or a freeze-dried product. Freezing or freeze-drying and thawing of the culture supernatant can be carried out according to the freezing method and thawing method known to those skilled in the art for solutions containing proteins and organic substances.

The present composition containing dental pulp stem cells and / or a culture supernatant of dental pulp stem cells can contain other components in addition to the active ingredient. For example, it can contain various media including the above-mentioned various basal media used for culturing dental pulp stem cells, and various components that can be added to such a medium. In addition, bioabsorbable materials such as hyaluronic acid, collagen and fibrinogen (eg, Borheel®) can also be included. Furthermore, gelling materials such as hyaluronic acid, collagen or fibrin glue can be included. The collagen used is preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.). Further, it can contain one or more selected from various amino acids including branched chain amino acids such as leucine, isoleucine and valine. This is because such amino acids are useful for skeletal muscle regeneration.
Further, it can be used not only for that purpose but also for suppressing (preventing) muscle attenuation or for suppressing (preventing) reduction of muscle mass from the viewpoint of maintaining muscle mass. In addition, the muscle-building agent of the present invention can also be used to recover muscles (muscle mass, muscle strength, etc.) that have been reduced or attenuated due to muscle atrophy (for example, disuse muscle atrophy, etc.).

The composition may also contain serum (fetal bovine serum, human serum, sheep serum, etc.), serum replacement (Knockout serum replacement (KSR), etc.), and bovine serum albumin (BSA).

(Aspects of the present composition)
The present composition containing dental pulp stem cells and / or a culture supernatant of dental pulp stem cells can take various embodiments depending on the type and morphology of the active ingredient. The composition may take the form of a liquid (liquid, gel, etc.) and a solid (powder, fine granules, granules, etc.). In addition, the present composition may take various known pharmaceutical forms depending on the administration method and dose described later.

The composition can be prepared as a preparation containing the active ingredient of the composition and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier generally refers to an inert, non-toxic, solid or liquid, bulking agent, diluent, encapsulating material, etc. that does not react with the active ingredient, such as water. , Ethanol, polyols (eg, propylene glycol, butylene glycol, glycerin, polyethylene glycol, etc.), suitable mixtures thereof, solvents such as vegetable oils or dispersion media and the like.

The composition is administered orally, parenterally, for example, intraorally, subcutaneously, subcutaneously, mucosally, intravenously, intraarterally, intramuscularly, intraperitoneally. Examples of the orally administered preparation include tablets, granules, fine granules, powders, capsules, pellets, syrups, liquids, suspensions and the like. Examples of parenteral preparations include infusions, injections, ointments, creams, gels and the like. In addition, this composition may contain a conventional additive as appropriate according to the pharmaceutical form. Such additives can be used as needed.

The target of application of this composition is preferably a warm-blooded vertebrate, and more preferably a mammal. As used herein, mammals include humans and non-human mammals such as monkeys, mice, rats, rabbits, dogs, cats, cows, horses and pigs. As an application target, humans are preferable from the viewpoint of improving QOL.

(Use of this composition)
The composition can be used to build muscle. Further, from the viewpoint of strengthening muscles, it can be used as a medicine for strengthening muscles, that is, as a pharmaceutical composition. In this case, the composition can be treated as a prophylactic or therapeutic agent for diseases or symptoms associated with muscle loss or weakness. Such diseases and symptoms (syndromes) include, for example, sarcopenia, sarcopenia that may be associated with diabetes, inflammatory myopathy, myopathy associated with medical disorders, muscular dystrophy, congenital myopathy, mitochondrial encephalopathy, glycogenosis, and tension. Examples include atrophy, muscular atrophy, sarcopenia, muscular degeneration, and myopathy. In addition, muscular atrophy means that the amount of muscle protein decreases due to the rate of decomposition of muscle protein exceeding the rate of synthesis, the number of muscle cells decreases or shrinks, and the amount of muscle or muscle strength decreases. Muscle atrophy includes disused muscular atrophy due to reduced gravity exposure due to inactivity and progressive muscular atrophy due to diseases such as amyotrophic lateral sclerosis.

In addition, this composition can be used for the following uses through a muscle-building action. For example, for rehabilitation after various surgical operations such as fracture joint injury, meat separation, sprain, or after medical surgery; for improving age-related (elderly) muscle weakness; for aging (elderly) ) For improving muscle weakness; it can also be used for improving the state of being asleep. Furthermore, this composition does not cause problems of exercise enthusiasts and athletes who desire improvement of muscle mass or strength, those who lack exercise, loss of muscle mass or strength, or hindrance to daily life, but maintain physical strength, muscle mass or strength. For muscle building for humans who desire improvement, and for humans who do not currently have the problem of muscle mass or muscle weakness but who desire to prevent muscle mass or muscle weakness due to aging or inactivity expected in the future. Can be used for.

The route of administration of this composition is not particularly limited. Various known administration forms can be adopted depending on the mode of the active ingredient contained in the present composition, the application site of the present composition, and the target disease. For example, parenteral administration may be systemic administration or topical administration. More specifically, injection (transplantation) into a muscle mass loss site and a muscle strength loss site can be mentioned. Intravenous administration, intraarterial administration, portal vein administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, oral administration and the like can be mentioned.

The dosage of the composition is not particularly limited. It can be set in consideration of the age, weight, pathological condition, etc. of the target of application of this composition. As the administration schedule, for example, once to several times a day, once every two days, once every three days, etc. can be adopted. In preparing the administration schedule, the gender, age, body weight, pathological condition, etc. of the target can be taken into consideration.

The dose of the present composition can be appropriately determined depending on the condition of the individual, body weight, sex, age, activity of the active ingredient, administration route, administration schedule, pharmaceutical form or other factors. For example, when dental pulp stem cells are administered as an active ingredient to muscles by injection or the like, the number of dental pulp stem cells is 104 or more and ¹⁰⁸ or less, preferably 105 or more and ¹⁰⁶ or less ^{per 10} cm ³ of muscle. Is.

When the culture supernatant of dental pulp stem cells is administered to muscle by injection or the like, the culture supernatant used for 104 or more and ¹⁰⁸ or less dental pulp stem cells per ¹⁰ cm ³ of muscle, preferably ¹⁰⁵ or more. 10 The culture supernatant used for about ⁶ or less is appropriately concentrated and used.

Further, when the dental pulp stem cells and the culture supernatant of the dental pulp stem cells are administered to the muscle by injection or the like, the respective dosages of the dental pulp stem cells and the culture supernatant can be combined with the above-mentioned doses. ..

The dose of the active ingredient of the present composition is appropriately determined by administering the active ingredient of the present composition at different doses and measuring the resulting muscle mass mass, muscle strength, and muscle bundle size. can do. Further, the administration or ingestion of the active ingredient may be systemic administration or local administration.

According to the present specification, there is also provided a method for strengthening the muscles of an individual by administering the composition to an individual such as a human. This method can also be carried out as a method for therapeutically or non-therapeutically strengthening the muscles of an individual, as well as a method for preventing, treating or ameliorating various diseases and symptoms, depending on the purpose of administration of the present composition.

In the present specification, "treatment" means to cure or ameliorate a disease or symptom in an individual. Further, "prevention" means preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of developing a disease or symptom in an individual. "Prevention" means preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of developing a disease or symptom in an individual. Further, as used herein, the term "improvement" means improvement of a disease, symptom or condition, prevention or delay of deterioration of the disease, symptom or condition, or suppression or delay of progression of the disease, symptom or condition. Further, as used herein, the term "non-therapeutic" means that it does not include medical practice, that is, treatment of the human body by treatment.

Hereinafter, specific examples embodying the present specification will be described, but the disclosure of the present specification is not limited to the following examples.

(1) Induction of diabetes For experimental animals, 6-week-old males, Sprague-Dawley (SD) rats (Chubu Kagaku Shizai, Nagoya, Japan) were fasted overnight, and then streptozotocin (STZ) (Sigma Chemical Co., MO, USA). ) Was intraperitoneally administered (60 mg / kg). One week after the administration of STZ, the blood glucose level was measured, and 14 mmol / l or more was regarded as diabetes. Animals were bred at room temperature (23 + 1.0 ° C.) and humidity (45 ± 10%) under artificial lighting with a 12-hour light-dark cycle and fed solid feed under free drinking water.

(2) Separation and culture of dental pulp stem cells Pentvalbital (1.5 mg / 1.5 mg /) in 6-week-old male SD rats and GFP rats (SD-Tg (CAG-EGFP) Cz-0040sb) (Japan SLC, Inc., Hamamatsu, Japan) kg) was intraperitoneally administered, and after slaughter, the upper and lower central incisors were extracted. Pulp tissue was collected from the extracted tooth, treated with an enzyme using 0.1% collagenase and 0.25% trypsin / EDTA solution, and then seeded on a plastic dish to obtain α-minimum essential medium (α-MEM) (α-MEM). GIBCO Lab Inc., Grand Island, NY) was added with 20% fetal bovine serum for ES cells (GIBCO) and 1% penicillin-streptomycin (G1BCO), and dental pulp stem cells were separated and cultured using the culture medium. rice field.

(3) Identification of stem cells Identification was performed using pulp stem cells (GFP-OPSCs) of the third cut, which were isolated and cultured from the dental pulp of a 6-week-old male GFP rat. As the antibody, PE-labeled hamster anti-rat C029 monoclonal antibody, PE-labeled mouse anti-rat CD90, and CD45 monoclonal antibody (Becton Dickinson, F ranklin Lakes, NJ) were used. As controls, PE-labeled hamster 19M monoclonal antibody and PE-labeled mouse 19G monoclonal antibody (Becton Dickinson) were used. Flow cytometry was performed using MACS (Miltenyi Biotec, Bergisch Gladbach, Germany), and MACSQuant (Miltenyi Biotec) was used as the analysis software.

(4) Evaluation of pluripotency The dental pulp stem cells were cultured in a medium for inducing differentiation of adipocytes, osteoblasts, and chondrocytes, and pluripotency was examined. As the adipocyte differentiation-inducing medium, 1% Adipogenic Supplement (R & D systems, Minneapolis, MN, USA) was added to α-MEM medium containing 10% FBS. After culturing for 14 days, oil red 0 (Polysciences, Warrington, PA) staining and fatty acid-binding protein-4 (FABP-4) immunostaining were performed. As the osteoblast differentiation-inducing medium, 5% Osteogenic Supplement (R & D systems) was added to α-MEM medium containing 10% FBS. After culturing for 21 days, 1% Alizarin red S (Merck, Darmstadt, Germany) staining and osteocalcin (R & D systems) immunostaining were performed. As the chondrocyte differentiation-inducing medium, Dulbecco's Modified Eagle's Medium (D-MEM) / F-12 medium (GIBCO) containing 1% ITS Supplement (R & D systems) was used. After culturing for 21 days, frozen sections of 5 μm were prepared and subjected to Aggrecan (R & D systems) immunostaining.

(5) Pulp stem cell transplantation and evaluation after dental pulp stem cell transplantation 8 weeks after the induction of diabetes, 10 GFP-DPSCs (lx10 ⁶ cells / rat) collected from 6-year-old male GFP rats were applied to normal and diabetic rat unilateral hindlimb skeletal muscles. It was transplanted in different places. Physiological saline was administered to the control side. The following measurements were made 4 weeks after transplantation.

(A) Measurement of muscle strength The muscle strength of the hind limbs of the rat was measured by a conventional method using a grip strength meter (Columbus). The results are shown in FIG. As shown in FIG. 1, in both normal rats and diabetic rats, administration of dental pulp stem cells increased muscle strength. The degree of increase in muscle strength after administration compared to before administration was greater in diabetic rats (about 1.2 times).

(B) Measurement of gastrocnemius muscle mass The gastrocnemius muscle wetness was measured. The results are shown in FIG. In normal rats, there was no change in muscle mass before and after administration, but in diabetic rats, an increase in muscle mass was observed.

(C) Histological evaluation The area per muscle bundle of the hind limb skeletal muscle was measured using ImageJ. The results are shown in FIG. As shown in FIG. 3, it was confirmed that the muscle bundle size was increased by the administration of dental pulp stem cells in both normal rats and diabetic rats. In diabetic rats, the muscle bundle size tended to be smaller by nature than in normal rats, but transplantation of dental pulp stem cells tended to increase muscle bundles of relatively large diameter. On the other hand, in diabetic rats, the proportion of small-sized muscle bundles of 1000 μm ² to 2000 μm ² accounted for about a quarter of the total, but the administration of dental pulp stem cells reduced the proportion of small-sized muscle bundles to about 10%. , 9000 μm ² to 10000 μm ² , and other muscle bundles in a muscle bundle size region that did not exist at all in the past could be observed.

(D) Measurement of intramuscular blood flow and intramuscular blood vessel count Hindlimb skeletal muscle blood flow was measured using a laser blood flow meter (OMEGAFLO, OMEGAWAVE, Inc, Japan). That is, normal rats and diabetic rats were deeply anesthetized using pentparbital, the hind limbs were incised, a sensor was applied to the incision, and the measured values were read. In addition, sections of the incision site were prepared by a conventional method, and the number of blood vessels per muscle bundle was measured by immunostaining. As a result, it was found that transplantation of dental pulp stem cells increases muscle blood flow at the transplant site. It was also found that transplantation of dental pulp stem cells increased the number of capillaries in the transplanted skeletal muscle. It was also found that the degree of increase in blood flow and the number of blood vessels was greater in diabetic rats than in normal rats.

(E) Gene expression analysis 1
Gene expression of growth and nutrition factors (basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) in hindlimb skeletal muscle by real-time PCR method The results are shown in FIG. 4. That is, after homogenizing the skeletal muscle, RNA was recovered by a conventional method, and real-time PCR was performed using primers and probes corresponding to these growth factors. As a result, normal results were obtained. Increased expression of bFGF was observed in both rats and diabetic rats, but in VEGF, NGF and NT-3, increased expression of growth and trophic factors could be confirmed only in diabetic rats.

(F) Localization of transplanted DPSCs The presence of transplanted dental pulp stem cells in the hind limb skeletal muscle was confirmed by fluorescent immunostaining. As a result, it was found that dental pulp stem cells exist between the muscle bundles of the hind limb skeletal muscle.

(G) Gene expression analysis 2
Gene expression of muscle atrophy markers (Atrogin-1, MuRF-1) in hindlimb skeletal muscle was analyzed by real-time PCR method. That is, after homogenizing skeletal muscle, RNA was recovered by a conventional method, and real-time PCR was performed using primers and probes corresponding to these growth factors. As a result, in diabetic rats, Atrogin-1 and MuRF-1 were significantly reduced by transplantation of dental pulp stem cells. In contrast, in normal rats, there was no significant change before and after dental pulp stem cell transplantation.

(6) Effect of addition of culture supernatant of dental pulp stem cells to myotube cells Using L6 myoblasts derived from rat skeletal muscle, cultured in DMEM medium containing 2% horse serum for 7 days to differentiate into L6 myoblast cells. I let you. Then, the cells were washed with PBS and then replaced with serum-free culture medium (usually glucose DMEM and high glucose (25 mM) DMEM). The culture supernatant of dental pulp stem cells cultured for 24 hours was collected, added to L6 myotube cells, RNA was collected 24 hours later, and gene expression of muscle synthesis markers (PGC-1α, PPARα, PPARδ, UCP-3) was performed. Analysis was performed. As a result, at normal glucose concentration, even with the addition of the culture supernatant of dental pulp stem cells, only PGC-1α increased, but the others were equivalent to or decreased with no addition of the culture supernatant. On the other hand, at high glucose concentration, PGC-1α, PPARδ, and UCP-3 were increased by the addition of the culture supernatant.

In addition, after exchanging L6 myoblasts with serum-free culture medium (normal glucose DMEM and high glucose (25 mM) DMEM) in the same procedure as above, the culture supernatant of dental pulp stem cells cultured for 24 hours was collected, and L6 muscle was collected. It was added to blast cells. After several hours, RNA was recovered and gene expression analysis of muscle differentiation markers (Myogenin, MyoD1) was performed. As a result, in the normal glucose medium, only Myogenin increased by the addition of the culture supernatant of dental pulp stem cells, but in the high glucose medium, MyoD1 increased together with Myogenin.

Claims (7)

A composition comprising dental pulp stem cells and / or a culture supernatant of the dental pulp stem cells for improving the decrease in skeletal muscle mass or the decrease in skeletal muscle strength due to sarcopenia. The composition according to claim 1, wherein the sarcopenia is sarcopenia associated with diabetes. The composition according to claim 1 or 2, wherein the decrease in the amount of the skeletal muscle or the decrease in the strength of the skeletal muscle is improved by increasing the muscle bundle size of the skeletal muscle. The composition according to any one of claims 1 to 3, which is for injection into skeletal muscle. The composition according to any one of claims 1 to 4, which comprises the dental pulp stem cells. The composition according to any one of claims 1 to 5, which comprises the frozen dental pulp stem cells. The composition according to any one of claims 1 to 6, which comprises the culture supernatant of the dental pulp stem cells.

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