WO2017142367A1

WO2017142367A1 - Pharmaceutical composition for prevention or treatment of muscle disease, comprising mesenchymal stem cell or xcl1

Info

Publication number: WO2017142367A1
Application number: PCT/KR2017/001807
Authority: WO
Inventors: 장종욱; 나덕렬; 이지은; 권수진
Original assignee: 사회복지법인 삼성생명공익재단
Priority date: 2016-02-19
Filing date: 2017-02-17
Publication date: 2017-08-24
Also published as: KR20170098046A; KR101833971B1

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating a muscle disease, the composition comprising mesenchymal stem cells, or an XCL1 protein or a derivative thereof, and a method for screening a muscle disease-treating agent. Effective in inhibiting the death of muscle cells or myoblasts, the mesenchymal stem cells or the XCL1 protein of the present invention can be usefully used for preventing or treating muscle diseases, and an inhibitory agent against cell death, containing an XCL1 protein can be used for cell death studies.

Description

Pharmaceutical composition for the prevention or treatment of muscle diseases, including mesenchymal stem cells or XCL1

The present invention mesenchymal stem cells; Or, it relates to a pharmaceutical composition for the prevention or treatment of muscle diseases, including XCL1 protein or derivatives thereof, and to a method for screening a therapeutic agent for muscle diseases.

Mesenchymal stem cells are stromal-derived cells that have the characteristics of self-renewal and can differentiate into bone, cartilage, adipose tissue, muscle, tendons, ligaments, and nerve tissues. It is attracting attention as a cell suitable for therapy. Currently, bone marrow is the most representative tissue from which mesenchymal stem cells can be obtained. However, mesenchymal stem cells present in bone marrow have limited application range due to limited differentiation and proliferative capacity. Due to limitations derived from bone marrow, several steps are required, and the procedure is complicated, It is usually accompanied by time, mental and physical pain. In addition, for bone marrow transplantation, there is a problem in that a donor does not show a graft-versus-host response because the antigenic phenotypes are matched through histocompatibility antigen comparison. The umbilical cord, unlike the bone marrow, can be obtained through a simple procedure in the delivery process, and contains a large number of hematopoietic stem cells and stem cells compared to the amount. In recent years, research has been actively conducted, as it is known that there are a large amount of stem cells in the umbilical cord, placenta, cord blood, and the like. However, it has not been reported that the factors secreted from mesenchymal stem cells or mesenchymal stem cells isolated and cultured from the umbilical cord inhibit muscle cell death.

XCL1 (chemokine (C motif) ligand 1) is one of the chemokine subfamily members. Chemokines are known to be involved in inflammatory and immune responses, and there is no known relationship to cell death.

Muscle, on the other hand, is an essential tissue that supports our body and maintains life. Muscle myoblasts differentiate and form multinucleated myotubes, form myocytes, and myoblasts and the death of muscle cells are causing various diseases. In addition, diseases in which muscle cells of neuromuscular junctions are lost through chronic inflammation through gene mutation or various causes, muscle loss through autoimmune diseases or degeneration of peripheral nerves have been reported. There is no cure. Currently, mesenchymal stem cells have been shown to be effective in acute and chronic bacterial inflammatory diseases, cystic fibrosis, and sepsis, and their usefulness, but their effects on muscle diseases are negligible. . Thus, there is an urgent need for a new treatment that effectively treats muscle diseases caused by apoptosis and does not cause resistance.

Meanwhile, zebrafish is a kind of tropical fish and is widely used as an experimental animal model. Zebrafish can replace animal models such as rats and rats as vertebrates, as well as shorter development time and transparent embryos, making them easier to observe.

In particular, since recombinant proteins using microinjection technology can be directly injected into zebrafish, they are useful for the study of observing and identifying the physiological functions of these proteins. Nevertheless, there are no reports of zebrafish and studies made on XCL1 protein.

In addition, for screening large amounts of therapeutic substances, zebrafish, which is economical and time-efficient, is much more convenient than animal breeding and maintenance costs and analysis-intensive and time-consuming animals. Therefore, there is a need to develop a disease animal model production and screening method capable of simulating in vivo functions and capable of screening on a large scale.

The present inventors confirmed that mesenchymal stem cells have an effect of inhibiting myocyte death, and completed the present invention by identifying the XCL1 protein, which is a water-soluble factor secreted from mesenchymal stem cells.

An object of the present invention to provide a pharmaceutical composition for the prevention or treatment of muscle diseases including mesenchymal stem cells or a stem cell therapy for the treatment of muscle diseases.

Still another object of the present invention is to provide a pharmaceutical composition or apoptosis inhibitor for preventing or treating muscle diseases comprising XCL1 (chemokine (C motif) ligand 1) or a derivative thereof as an active ingredient.

It is another object of the present invention to provide a method for screening a therapeutic agent for muscle diseases.

It is still another object of the present invention to provide a zebrafish into which a gene carrier comprising an XCL1 protein or a nucleotide sequence encoding XCL1 is introduced.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases, including mesenchymal stem cells.

The present invention also provides a pharmaceutical composition for preventing or treating muscle diseases, including XCL1 (chemokine (C motif) ligand 1) or a derivative thereof as an active ingredient.

The present invention also provides a stem cell therapeutic agent for treating muscle diseases, including mesenchymal stem cells.

The present invention also provides an apoptosis inhibitor comprising an XCL1 (chemokine (C motif) ligand 1) protein or a derivative thereof.

The present invention also provides a method for screening a therapeutic agent for muscle diseases, comprising the steps of: (a) contacting a test substance to be analyzed with a cell expressing XCL1; (b) analyzing the effect of the test substance on the expression of the XCL1, and when the test substance increases the expression of the XCL1, the test substance is determined as a prophylactic or therapeutic agent for muscle diseases.

The present invention also provides a method for screening a therapeutic agent for muscle diseases, comprising the steps of: (a) contacting a test substance with XCL1; (b) analyzing the effect of the test substance on the activity of the XCL1, when the test substance increases the activity of the XCL1 is determined as a prophylactic or therapeutic agent of muscle diseases.

The present invention also provides a zebrafish into which a gene carrier comprising an XCL1 protein or a nucleotide sequence encoding XCL1 is introduced.

In another aspect, the present invention provides a method for screening a candidate drug for XCL1 co-administration for the prevention or treatment of muscle diseases, comprising the steps of: (a) treating the zebra fish with a candidate drug for co-administration of XCL1; And (b) observing the development of the zebrafish muscle cells.

In another aspect, the present invention is to treat the mesenchymal stem cells, XCL1 (chemokine (C motif) ligand 1) or derivatives thereof to the subject; It provides a method of preventing or treating muscle diseases comprising a.

In another aspect, the present invention comprises the steps of treating the individual with XCL1 (chemokine (C motif) ligand 1) protein or derivatives thereof; It provides a method for inhibiting apoptosis comprising a.

Since the mesenchymal stem cell or XCL1 protein of the present invention has an effect of inhibiting the death of myocytes or myoblasts, it can be usefully used for the prevention or treatment of muscle diseases, and the apoptosis inhibitor including the XCL1 protein is used for cell death research. It can be utilized.

1 is a diagram showing a co-culture of mesenchymal stem cells and killed myoblasts.

Figure 2 is a diagram showing the results of co-culture of mesenchymal stem cells and myoblasts (WJ-MSC: umbilical cord-derived mesenchymal stem cells).

Figure 3 is a diagram showing the results of confirming the cleavage of the apoptosis marker PARP in the myoblasts co-cultured with mesenchymal stem cells by Western blotting and the numerical value thereof.

Figure 4 shows the cell appearance of myoblasts co-cultured with concentration-dependently treated XCL1 protein.

5 is a graph showing the results obtained by Western blotting and the numerical value of the cleavage of the apoptosis marker PARP in myoblasts co-cultured with concentration-dependently treated XCL1 protein.

Figure 6 is a graph showing the results obtained by Western blotting and the numerical value of the cleavage of PARP, apoptosis marker in myoblasts co-cultured with XCL1 protein treated in a time-dependent manner.

7 is a diagram showing the results of comparing the apoptosis inhibitory effect of the PAN inhibitor and XCL1 protein.

Figure 8 shows the morphology of mouse hippocampal neurons treated with XCL1 protein after apoptosis-inducing factor treatment.

9 is a graph showing the results obtained by Western blotting of the apoptosis marker PARP in mouse hippocampal neurons after treatment with apoptosis-inducing factor and XCL1 protein and a numerical value thereof.

10 is a diagram showing the morphology of rat Schwann cells treated with the XCL1 protein after the endoplasmic reticulum stress derivative treatment.

FIG. 11 is a graph showing results obtained by Western blotting of PARP, an apoptosis marker in rat Schwann cells treated with XCL1 protein after endoplasmic reticulum stress derivative treatment, and a numerical value thereof.

12 is a diagram showing the morphology of mouse myotube cells treated with XCL1 protein after lovastatin treatment.

FIG. 13 is a graph showing results obtained by Western blotting of PARP, which is an apoptosis marker, in mouse myoblasts and myotubes treated with XCL1 protein after lovastatin treatment and quantification thereof. FIG.

FIG. 14 is a graph showing results obtained by Western blotting and quantifying MHC in mouse myoblasts treated with XCL1 protein after lovastatin treatment. FIG.

Figure 15 shows the morphology of myoblasts co-cultured with mesenchymal stem cells treated with siRNA that inhibits XCL1 synthesis.

FIG. 16 is a graph showing the results obtained by Western blotting of the apoptosis marker PARP in myoblasts co-cultured with mesenchymal stem cells treated with siRNA that inhibits XCL1 synthesis and a numerical value thereof. FIG.

17 is a schematic diagram of micro-implantation for preparing a human XCL1 expression zebrafish model.

Figure 18 shows the phenotype of Day 3 after the birth of the human XCL1 expressing zebrafish model.

19 is a diagram illustrating the results of observing zebrafish after injecting XCL1 protein into zebrafish simulating muscle disease symptoms.

20 is a diagram showing a specific region of the body selected to observe the degree of muscle cell activation of zebrafish.

21 is a diagram showing the results of observing the degree of muscle cell activation of zebrafish with a confocal microscope.

22 is a diagram showing the results of quantifying the normal and abnormal embryos after treatment with zebrafish XCL1 protein at 0, 30, 50μg / mL.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating muscle diseases including mesenchymal stem cells.

Mesenchymal stem cells of the present invention comprises XCL1 (chemokine (C motif) ligand 1). XCL1 is a soluble factor secreted from mesenchymal stem cells and is one of the chemokine subfamily members (NCBI GenBank Accession number: NP — 002986).

XCL1 protein of the present invention is a protein consisting of the amino acid sequence of SEQ ID NO: 1.

The composition of the present invention contains the XCL1 protein itself, as well as pharmaceutically acceptable salts, hydrates or solvates thereof as an active ingredient. The term "pharmaceutically acceptable salts" denotes salts of said XCL1 protein with the desired pharmacological effect, ie the prophylactic or therapeutic effect of muscle disease. Such salts include inorganic acids such as hydrochloride, hydrobromide and hydroiodide, acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, p-toluenesulfonates, bisulfates, sulfamate, sulfates, Naphthylate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, hep Formed with organic acids such as tanoate, hexanoate, 2-hydroxyethanesulfate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, tosylate and undecanoate do. The term "pharmaceutically acceptable hydrate" refers to the hydrate of the XCL1 protein with the desired pharmacological effect. The term "pharmaceutically acceptable solvate" refers to a solvate of said XCL1 protein with the desired pharmacological effect. The hydrates and solvates may also be prepared using the acids described above.

In addition, the pharmaceutical composition of the present invention may include a pharmaceutically effective amount of a gene carrier comprising the nucleotide sequence encoding XCL1 as an active ingredient. The nucleotide encoding the XCL1 in the gene carrier comprising the nucleotide sequence encoding the XCL1 consists of the sequence of SEQ ID NO: 2.

The nucleotide sequence encoding the XCL1 may be applied to all gene carriers used for conventional gene therapy, and preferably, plasmids, adenoviruses, Adeno-associated viruses (AAV), retroviruses, lentiviruses, It can be applied to herpes simplex virus, basinia virus, liposomes or niosomes.

Adenovirus

Adenoviruses are widely used as gene transfer vectors because of their medium genome size, ease of manipulation, high titers, wide range of target cells and excellent infectivity. Both ends of the genome contain 100-200 bp of Inverted Terminal Repeat (ITR), which is an essential cis element for DNA replication and packaging. The genome El region (E1A and E1B) encodes proteins that regulate transcription and transcription of host cell genes. The E2 regions (E2A and E2B) encode proteins that are involved in viral DNA replication.

Among the adenovirus vectors currently developed, non-replicating adenoviruses lacking the E1 region are widely used.

On the other hand, the E3 region is removed from a typical adenovirus vector to provide a site for insertion of a foreign gene. Therefore, the XCL1 gene of the present invention is preferably inserted into the deleted E1 region (E1A region and / or E1B region, preferably E1B region) or E3 region, more preferably the deleted E3 region. On the other hand, the target nucleotide sequence to be carried into the cell is preferably inserted into the deleted E1 region (E1A region and / or E1B region, preferably E1B region) or E3 region, more preferably inserted into the deleted E1 region do.

The insertion sequences can also be inserted into the deleted E4 region. As used herein, the term "deletion" as used in connection with a viral genome sequence has the meaning including not only that sequence in its entirety, but also partially deleted.

The adenovirus gene carrier of the present invention has a structure in which the "promoter-purpose nucleotide sequence-poly A sequence" and the "promoter-XCL1 gene-poly A sequence" are linked, and the "promoter-purpose nucleotide sequence-poly A sequence" Inserted into the deleted E1 region (E1A region and / or E1B region, preferably E1B region) or E3 region, preferably deleted E1 region, wherein the "promoter-XCL1 gene-poly A sequence" is deleted E1 It is inserted into a region (E1A region and / or E1B region, preferably E1B region) or an E3 region, preferably a deleted E3 region. It is also expressed by a bicistronic expression system in which the target nucleotide and the XCL1 gene are linked by an internal ribosome entry site (IRES), such as "promoter-purpose nucleotide sequence-poly A sequence-IRES-XCL1 gene-poly A sequence". Can be.

In addition, because adenovirus can pack up to about 105% of the wild-type genome, about 2 kb can be additionally packaged. Thus, the above-described foreign sequence inserted into the adenovirus may additionally bind to the genome of the adenovirus.

Adenoviruses have 42 different serotypes and subgroups of A-F. Of these, adenovirus type 5 belonging to subgroup C is the most preferred starting material for obtaining the adenovirus vector of the present invention. Biochemical and genetic information for adenovirus type 5 is well known.

Foreign genes carried by adenoviruses replicate in the same way as episomes, with very low genetic toxicity to host cells. Therefore, gene therapy using the adenovirus gene carrier of the present invention is considered to be very safe.

2. Retrovirus

Retroviruses are widely used as gene transfer vectors because they insert their genes into the host genome, carry large amounts of foreign genetic material, and have a broad spectrum of cells that can infect them.

To construct the retroviral vector, the XCL1 gene and the target nucleotide sequence to be carried are inserted into the retroviral genome instead of the sequence of the retrovirus to produce a nonreplicating virus. To produce the virion, a packaging cell line is constructed that contains the gag, pol, and env genes but does not have a long terminal repeat (LTR) and sequence. When the recombinant plasmid comprising the XCL1 gene, the target nucleotide sequence to be carried, LTR and Ψ sequence is introduced into the cell line, the Ψ sequence enables the production of the RNA transcript of the recombinant plasmid, which is packaged into a virus , Virus is discharged into the medium. The medium containing the recombinant retrovirus is collected, concentrated and used as a gene carrier.

3. AAV Vector

Adeno-associated virus (AAV) is suitable as the gene carrier of the present invention because it can infect non-dividing cells and has the ability to infect various kinds of cells. Details of the preparation and use of AAV vectors are disclosed in detail in US Pat. Nos. 5,139,941 and 4,797,368.

Studies on AAV as a gene carrier are described in LaFace et al, Viology, 162: 483486 (1988), Zhou et al., Exp. Hematol. (NY), 21: 928-933 (1993), Walsh et al, J. Clin. Invest., 94: 1440-1448 (1994) and Flotte et al., Gene Therapy, 2: 29-37 (1995).

Typically, an AAV virus is an expression plasmid comprising a plasmid comprising the gene sequence of interest (XCL1 gene and the desired nucleotide sequence to be transported) with two AAV terminal repeats flanked and a wild type AAV coding sequence without terminal repeats. Prepared by cotransformation.

4. Other Virus Vectors

Other viral vectors can also be used as the gene carrier of the present invention. Vectors derived from Basinia virus, lentivirus or herpes simplex virus can also be used as a delivery system capable of intracellularly carrying the XCL1 gene and the desired nucleotide sequence to be transported.

5. Liposomes

Liposomes are automatically formed by phospholipids dispersed in the aqueous phase. Examples of successfully delivering foreign DNA molecules into liposomes into cells include Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190 (1982) and Nicolau et al., Methods Enzymol., 149: 157-176 (1987). On the other hand, Lipofectamine (Gibco BRL) is the most used reagent for the transformation of animal cells using liposomes. Liposomes Containing the XCL1 Gene and the Desired Nucleotide Sequence Interact with cells through mechanisms such as endocytosis, adsorption to the cell surface, or fusion with plasma cell membranes, thereby incorporating the XCL1 gene and the desired nucleotide into the cell Carries the sequence.

In the present invention, when the gene carrier is produced based on the viral vector, the method of administering the pharmaceutical composition of the present invention is carried out according to a virus infection method known in the art. Infection of host cells with viral vectors is described in the references cited above.

In the present invention, when the gene delivery system is naked recombinant DNA molecules or plasmids, micro-injection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) can be introduced into the cell by the method.

In the present invention, the term "mesenchymal stem cell" refers to pluripotent progenitor cells before they are differentiated into cells of specific organs such as bone, cartilage, fat, tendon, nerve tissue, fibroblast and muscle cells. In the present invention, the mesenchymal stem cells are contained in the composition in an undifferentiated state. The mesenchymal stem cells of the present invention are human backpack (embryonic yolk sac), placenta, umbilical cord, umbilical cord blood, skin, peripheral blood, bone marrow, adipose tissue, muscle, liver, nerve tissue, periosteum, fetal membrane, synovial membrane, synovial fluid, amnion Meniscus, meniscus, anterior cruciate ligament, articular chondrocytes, teeth, perivascular cells, striatum, subpatellar mass, spleen and thymus, etc., but is preferably derived from the umbilical cord. In addition, the mesenchymal stem cells are preferably derived from humans, but may be derived from a fetus or a mammal except humans. Mammals other than humans may be more preferably canine, feline, ape, animal, cow, sheep, pig, horse, rat, mouse or guinea pig, and the like, without limitation.

Methods of isolating the mesenchymal stem cells are known in the art. For example, human backpack (embryonic yolk sac), placenta, umbilical cord, cord blood, skin, peripheral blood, bone marrow, adipose tissue, muscle, liver, nerve tissue, periosteum, fetal membrane, synovial membrane, synovial fluid, amniotic membrane, meniscus, precross It can be isolated and purified from ligaments, articular chondrocytes, teeth, perivascular cells, striatum, subpatellar mass, spleen and thymus, and the isolated mesenchymal stem cells can be cultured as necessary.

Mesenchymal stem cells of the present invention can be injected into the patient's living body in the cell alone or cultured in the incubator, for example, may be used clinical methods published by Lindbal et al. Or Douglas Kondziolka. . The preparation may include a pharmaceutically acceptable conventional carrier in addition to the mesenchymal stem cells, and in the case of injections, preservatives, analgesics, solubilizers or stabilizers, in the case of topical administration, bases, excipients, lubricants or Preservatives and the like.

In the present invention, "muscle disease" includes both diseases related to muscle wasting or diseases caused by muscle damage. The term "muscle wasting related disease" means a disease or condition involving symptoms such as gradual loss of muscle mass. The muscle wasting is genetic predisposition; Age-related diseases such as hypertension, impaired glucose tolerance, diabetes, obesity, dyslipidemia, atherosclerosis and cardiovascular disease; Diseases such as cancer, autoimmune diseases, infectious diseases, AIDS, chronic inflammatory diseases, arthritis, malnutrition, kidney disease, chronic obstructive pulmonary disease, emphysema, rickets, chronic lower spinal pain, peripheral nerve damage, central nerve damage and chemical damage Chronic diseases such as; Conditions such as organ fixation, conditions such as helplessness such as fractures or traumas, post-operative bed care; And progressive progression of skeletal muscle mass and strength according to the aging process. Muscle wasting related diseases can lead to a weakened physical condition that can lead to deterioration of health status or incapacity of physical performance. The term “muscle damage” refers to any muscle tissue damage, which may result from physical trauma to the muscle tissue resulting from accidents, motor injuries, endocrine gland disorders, diseases, wounds or surgical operations.

The muscle diseases include sprains, strains, convulsions, tendinitis, muscle cancer, myositis, rhabdomyolysis, muscular atrophy, atony, muscular dystrophy, muscle degeneration Syndrome, myasthenia, dystrophinopathy, myopathy, and sacopenia, and at least one selected from the group consisting of, preferably caused by the death of muscle cells or myoblasts. Disease. The muscular dystrophy is a phenomenon in which the amount of muscle is partially reduced or completely lost, and skeletal muscle atrophy caused by denervation due to denervation due to trauma, immobilization due to stabilization or joint fixation (pulmonary disuse atrophy) ), And soluble skeletal muscle atrophy is increasing in an aging society. Skeletal muscle atrophy is associated with reduced blood supply to muscles caused by various diseases, or consequent movement and immobilization, persistent weight loss, malnutrition or starvation, denervation to muscles, and cancer , AIDS, congestive heart failure, chronic obstructive pulmonary disease, renal failure, severe burns, and the like. The muscular dystrophy refers to a gradual decrease in skeletal muscle mass due to aging, which directly leads to a decrease in muscle strength, and as a result, a condition in which various physical functions may be reduced and impaired. The composition of the present invention has the effect of preventing or treating muscle diseases by inhibiting the death of myocytes or myoblasts, and muscles are not limited in kind.

Mesenchymal stem cell or mesenchymal stem cell-derived XCL of the present invention is characterized in that it inhibits myocyte or myoblast death and functions to protect cells.

In addition, the composition may further comprise a pharmaceutically acceptable carrier, the carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, know Nate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like However, the present invention is not limited thereto. The composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.

The compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a multi-dose container. The formulations may then be in the form of solutions, suspensions or emulsions in oil or aqueous media, and may further comprise dispersants or stabilizers.

The composition of the present invention can be administered parenterally, intravenous, subcutaneous, intraperitoneal or topical application, preferably administered directly to the site where the lesion has occurred. Compositions for parenteral administration (e.g. injections) of the compositions according to the invention are infused and dispersed in a pharmaceutically acceptable carrier, e.g., sterile purified water, a buffer of about pH 7, or physiological saline. And, if necessary, include conventional additives such as preservatives, stabilizers and the like.

The formulation of the composition may vary depending on the method of use, but may be used as a warning agent (Plasters), granules (Granule), powders (Sowrups), syrups (Syrups), solutions (Fluidextracts I), emulsions (Emulsions) It can be prepared as Suspensions, Infusions, Tablets, Injections, Capsules and Pills. Proper formulation is dependent upon the route of administration chosen. Any of the known techniques, carriers and excipients can be used suitably and as understood in the art, for example in Remingston's Pharmaceutical Sciences described above.

In addition, the amount of mesenchymal stem cells injected in the present invention may be administered 10 ⁴ -10 ¹⁰ cells / cycle, preferably 10 ⁵ -10 ⁹ cells / cycle, most preferably 5x10 ⁷ cells / It may be administered in a circuit, but is not limited thereto. In addition, the XCL1 injected in the present invention may be administered 0.0001ng-500mg / time, and preferably 0.0001mg-200mg / time, but is not limited thereto. The dose may be prescribed in various ways, such as by the method of formulation, the mode of administration, the age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to the patient.

As used herein, the term "prevention" means any action that inhibits or delays progression of muscle disease by administration of a composition of the present invention.

As used herein, the term "treatment" refers to any action in which muscle disease improves or benefits altered by administration of a composition of the present invention.

The XCL1 (chemokine (C motif) ligand 1) may be naturally generated and derived from mesenchymal stem cells, and naturally generated XCL1 includes XCL1 having a wild-type amino acid sequence of XCL1 which is usually associated with an animal, but It is not limited. The naturally occurring XLC1 may include, but is not limited to, naturally occurring variants of XCL1, including allelic variants, polymorphic variants, and the like.

In addition, the XCL1 of the present invention may include, without limitation, proteins, analogs, derivatives, and mutants thereof recombined by a known method in the art having a bioactivity that achieves the effect of preventing or treating muscle diseases.

In the present invention, the mutation may be a mutation found in nature or an artificial mutation with or without the effect of replacing, deleting or inserting one or more amino acids in a nucleic acid sequence encoding XCL1.

The mutations may have conservative amino acid substitutions that do not affect protein expression and are 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence of the natural XCL1 protein. Or amino acid sequences with 100% identity.

In addition, the XCL1 protein of the present invention includes polypeptide variants by fragments, deletions, truncation, etc., and may be fragments in which amino acid residues are removed from natural XCL1 protein. Such fragments, deletions, truncations and the like have virtually no negative impact on the activity of the resulting polypeptides. In specific embodiments, the activation domain of XCL1 can be derived by mapping the protein domain of XCL1 or through truncation from the C-terminus, N-terminus, or both C-terminus and N-terminus end, such truncated polypeptides May have virtually no negative impact on activity or may indicate an increase in activity.

"Recombinant" in such a recombinant protein, when used in connection with a cell, typically indicates that the cell has been modified by introduction of a foreign nucleic acid sequence or the cell is derived from a cell so modified. For example, a recombinant cell may comprise a gene that is not found in the same form in the native (non-recombinant) form of the cell, or the recombinant cell may contain a native gene (found in the natural form of the cell). However, such genes have been modified and reintroduced into cells. Recombinant cells can include nucleic acids inherent in a modified cell without removal of the nucleic acid from the cell, and such modifications include those obtained by gene substitution, site-specific mutations, and related techniques known to those skilled in the art. Recombinant DNA technology includes techniques for producing recombinant DNA in vitro and delivering such recombinant DNA into cells that can be expressed or propagated to produce recombinant polypeptides. "Recombinant", "recombinant", and "recombinant" of a polynucleotide or nucleic acid generally refers to assembling or combining two or more nucleic acids or polynucleotide strands or fragments to produce a new polynucleotide or nucleic acid. Recombinant polynucleotides or nucleic acids are sometimes referred to as chimeras. A nucleic acid or polypeptide, when artificial or engineered, is a "recombinant" nucleic acid or polypeptide.

Recombinant XCL1 may include any member of the VWF family derived from, for example, primates, humans, monkeys, rabbits, pigs, rodents, mice, rats, hamsters, gerbils, dogs, cats, and biologically active derivatives thereof. Include. Mutant and variant XCL1 proteins with activity are included as well as functional fragments and fusion proteins of the XCL1 protein.

The "derivative" in the XCL1 (chemokine (C motif) ligand 1) or derivatives thereof is obtained by substituting a part of the structure of XCL1 (chemokine (C motif) ligand 1) with another atom group, a substituent, or the like, or with other biological substances Or one or more XLC1 polypeptides fused. Also included are proteins improved by methods of protein improvement known in the art.

One or more XLC1 polypeptides associated with the other biological material include antibodies, fragments of antibodies, immunoglobulins, peptides, enzymes, growth factors, cytokines, transcription factors, toxins, antigenic peptides, hormones, transporters Proteins, Motor Function Proteins, Receptors, Signaling Proteins, Storage Proteins, Membrane Proteins, Transmembrane Proteins, Internal Proteins, External Proteins, Secretory Proteins, Viral Proteins, Glycoproteins, Cleaved Proteins A protein, a protein complex, a chemically modified protein, or the like may be a fused XCL1 fusion protein. Such biological materials include, without limitation, various small peptides, other proteins, chemical means (known in the art as “tags”) useful for isolating or identifying bound polypeptides.

The biological material may be fused to the N terminus or C terminus of the XCL1 amino acid sequence, and may be prepared by methods known in the art.

Specifically, some fusion domains that can be fused with XCL1 are particularly useful for the isolation of fusion proteins by affinity chromatography. For the purpose of affinity purification, suitable matrices for affinity chromatography are used, such as glutathione-, amylase- and nickel- or cobalt-bonded resins. Many such matrices are available in "kit" form, such as the Pharmacia GST purification system and the (HIS6) fusion partner and the useful QLAexpressTM system (Qiagen). As another example, the fusion domain can be selected to facilitate the detection of the XCL1 protein. Examples of such detection domains include various epitope proteins (eg, GFP) as well as “epitope tags”, which are generally short peptide sequences in which specific antibodies are available. Known epitope tags readily available for certain monoclonal antibodies include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domain has a protease cleavage site, such as factor Xa or thrombin, that allows the appropriate protease to partially digest the fusion protein and thereby release the recombinant protein therefrom. The released protein can then be isolated from the fusion domain by subsequent chromatographic separation. The domain may also be, for example, polyarginine-tag, Strep-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain. (cellulose-binding domain), SBP-tag, chitin-binding domain, glutathione S-transferase-tag, maltose-binding protein binding protein), NusA, TrxA, DsbA, protein A, protein G, human albumin (human albumin) may be further included.

In certain embodiments, the XCL1 protein may be fused with a domain that stabilizes the XCL1 protein in vivo (“stabilizer” domain). "Stabilizing" means something that increases serum half-life, whether or not the increase in serum half-life is due to reduced destruction, reduced clearance by the kidney, or other pharmacokinetic effects. Fusion of immunoglobulins with the Fc portion is known to impart desirable pharmacokinetic properties to a wide range of proteins. Likewise, fusion to human serum albumin can impart desirable properties. Other types of fusion domains that may be selected include multimerization (eg dimerization, tetramerization) domains and functional domains (which confer additional biologic functions, if desired). Fusions can be constructed such that the heterologous peptide is fused at the amino terminus of the polypeptide of the invention and / or at the carboxy terminus of the polypeptide of the invention.

In some embodiments of the invention, the XCL1 amino acid sequence may be a recombinant polypeptide, natural polypeptide, or synthetic polypeptide comprising a fragment thereof. In some embodiments, the polypeptide is a multimer. In some embodiments, the polypeptide is a dimer. It will be appreciated in the art that some amino acid sequences of the binders described herein can be varied without significantly affecting the structure or function of the protein. Accordingly, the present invention further encompasses variations of polypeptides that exhibit substantial activity or comprise regions of fragments thereof. In some embodiments, amino acid sequence variations include deletions, insertions, inversions, repeats, and / or other types of substitutions.

XCL1 proteins, polypeptides, derivatives, analogs and variants thereof may also be modified to contain additional chemical moieties that are not typically part of the polypeptide. Derivatization moieties can improve or otherwise modulate the solubility, biological half-life and / or absorption of a polypeptide. The moiety may also reduce or eliminate undesirable side effects of the polypeptides and variants. An overview of chemical moieties can be found in Remington: The Science and Practice of Pharmacy, 22st Edition, 2012, Pharmaceutical Press, London.

The proteins, polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to construction of DNA sequences encoding polypeptide sequences and expression of these sequences in a suitable host. In some embodiments, a DNA sequence is constructed by isolating or synthesizing a DNA sequence encoding a wild type protein of interest using recombinant techniques. Optionally, by mutating this sequence by site-specific mutagenesis, functional analogs thereof can be provided.

In some embodiments, DNA sequences encoding polypeptides of interest can be constructed by chemical synthesis using oligonucleotide synthesizers. Oligonucleotides can be designed based on the amino acid sequence of the polypeptide of interest, by selecting a codon that is advantageous in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize polynucleotide sequences encoding isolated polypeptides of interest. For example, a complete amino acid sequence can be used to construct a reverse translated gene. In addition, DNA oligomers containing nucleotide sequences encoding particular isolated polypeptides can be synthesized. For example, several small oligonucleotides encoding portions of the polypeptide of interest can be synthesized and then ligated. Individual oligonucleotides typically contain 5 'or 3' overhangs for complementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or by another method), the polynucleotide sequence encoding the particular polypeptide of interest is inserted into an expression vector and operated on an expression control sequence appropriate for expression of the protein in the desired host. Possibly connected. Proper assembly can be identified by nucleotide sequencing, restriction enzyme mapping and / or expression of a biologically active polypeptide in a suitable host.

In the present invention, "fusion" refers to the integration of two molecules having different or identical functions or structures, and fusion by any physical, chemical or biological method in which the peptide can bind to the protein, small molecule drug, nanoparticle or liposome. Can be. The fusion can be by linker peptides.

XCL1 of the present invention may be present in further association with a bioactive compound to enhance the therapeutic effect. The term "bioactive compound" refers to a compound that modifies a disease when applied to a mammal having the disease. Biologically active compounds may include antagonistic or agonistic properties, or may be proteinaceous bioactive compounds or non-proteinaceous bioactive compounds. Such proteinaceous bioactive compounds can be covalently attached to the binding domains of the invention, eg, by generating standard DNA cloning techniques, gene fusion polypeptides, and then their standard expression and purification. Such non-protein biologically active compounds, via chemical means, for example via maleimide linkers with cysteines coupled via a peptide linker to the N- or C-terminus of the binding domain, as described herein By coupling to a cysteine thiol, it can be covalently attached to, for example, the binding domain of the invention. Examples of proteinaceous bioactive compounds include pronounced target specificities (e.g., neutralizing growth factors by binding them), cytokines (e.g. interleukins), growth factors (e.g. human growth hormones), antibodies and fragments thereof Fragments, hormones (eg GLP-1) and any possible proteinaceous drug. Examples of non-proteinaceous bioactive compounds include toxins (eg, DM1 from ImmunoGen), small molecule targeting GPCRs, antibiotics and any possible non-protein drug.

The term "binding domain" encompasses protein domains representing the same "fold" (three-dimensional arrangement) as protein scaffold (c) and has predetermined properties. Such binding domains can be obtained by rational, or most often, combinatorial protein engineering techniques known in the art. For example, a binding domain comprising predetermined properties may comprise (a) providing the same fold as the protein scaffold as further defined below; And (b) selecting from the various collections to obtain the at least one protein domain comprising the various collection screenings and / or the predetermined properties. Various collections of protein domains may be provided by various methods depending on the screening and / or selection system used, and may include the use of methods well known to those skilled in the art, such as phage display or ribosomal display. Can be. Preferably, the binding domain is a recombinant binding domain. Also preferably, the binding domain is a repeat protein or designed repeat protein.

As described above, any recombinant XCL1 that binds the proteins or domains presented herein may be, for example, a bi-specific binder, a bioactive compound, a labeling moiety (e.g., a fluorescent label such as fluorescein or radiotracer), Moieties that allow protein purification (eg small peptide tags such as His- or strep-tags), moieties that provide effector functions for elevated therapeutic efficacy (eg antibodies that provide cell dependent cytotoxicity Fc portion of, toxic protein moiety such as Pseudomonas aeruginosa exotoxin A (ETA), or small molecule toxic agents such as maytansinoid or DNA alkylating agents) or other to generate moieties that provide improved pharmacokinetics It may be intended to covalently bond with one or more additional moieties that include a moiety that binds the target. Improved pharmacokinetics can be assessed according to perceived therapeutic needs. It is often desirable to improve the bioavailability and / or increase the dose interval time, perhaps by increasing the time that the protein remains available in the serum after dosing. In some instances, it is desirable to improve the persistence of the serum concentration of the protein over time (eg, to reduce the difference in serum concentration of the protein between the concentration immediately after administration and just before the next administration).

The XCL1 (chemokine (C motif) ligand 1) or derivatives thereof has an effect of inhibiting myocyte or myoblast death.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of muscle diseases.

The mesenchymal stem cell is characterized in that it comprises an XCL1 protein.

As used herein, the term "cell therapeutic agent" refers to the proliferation, selection, or in vitro of living autologous, allogenic, or xenogenic cells to restore the function of cells and tissues. Means a drug (Article 2 of 2008-78), which is a drug product used for the purpose of treatment, diagnosis and prevention through a series of behaviors that change its characteristics.

In the term "apoptosis inhibitor" of the present invention, apoptosis is a concept that is differentiated from necrosis or pathological death of cells in a form of dying controlled by genes. In addition, an inhibitor means a substance that exhibits an action of inhibiting a chemical reaction or a physiological action.

The XCL1 protein of the present invention includes the XCL1 protein as well as variants thereof. As used herein, the term “variant” refers to a protein in which the amino acid sequence of the XCL1 protein and one or more amino acid residues have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchange in proteins and fragments that do not alter the activity of the molecule as a whole is known in the art. XCL1 proteins or variants thereof may be extracted from nature or prepared by genetic recombination methods based on synthetic or DNA sequences.

When using the XCL1 protein or derivatives thereof according to the present invention has a feature that can inhibit apoptosis, it can be usefully used for the purpose of apoptosis research.

According to the method of the present invention, a test substance is first contacted with a cell expressing the XCL1 of the present invention. As used to refer to the screening methods of the present invention, the term "test material" refers to an unknown material used in screening to examine whether it affects the expression level or activity of XCL1 of the present invention or muscle.

In the present invention, the test substance includes various materials. For example, the test substance includes, but is not limited to, chemicals, proteins, peptides, antibodies, nucleic acids, and natural extracts. The test substance analyzed by the screening method of the present invention may be a single compound or a mixture of compounds (eg, a natural extract or a cell or tissue culture). The test substance can be obtained from a library of synthetic or natural compounds. Methods of obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA). ) And MycoSearch (United States). Samples can be obtained by a variety of combinatorial library methods known in the art, for example biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution required By a synthetic library method, a "1-bead 1-compound" library method, and a synthetic library method using affinity chromatography screening. Methods of synthesizing molecular libraries are variously disclosed in the art.

The test substance is contacted with cells expressing XCL1, and the expression level of XCL1 is measured in the cells treated with the test substance. The expression level can be measured as described below, and as a result of the measurement, when the expression of XCL1 is increased, the test substance can be determined as a prophylactic or therapeutic agent for muscle diseases.

Measurement of the change in the expression level of the above-described nucleotide sequence can be carried out through various methods known in the art. For example, it can be carried out using hybridization reaction using RT-PCR, northern blotting, cDNA microarray or in situ hybridization reaction.

When performing according to the RT-PCR protocol, first, total RNA is isolated from cells treated with the test substance, and then, first-chain cDNA is prepared using oligo dT primers and reverse transcriptase. Subsequently, the first chain cDNA is used as a template, and a PCR reaction is performed using an expression material-coding nucleotide-specific primer set. The primer set described above is a sequence included in the XCL1-encoding nucleotide sequence to be used. The PCR amplification products are then electrophoresed and the formed bands are analyzed to determine changes in the amount of expression of the nucleotide sequence.

In addition, the change in the amount of XCL1 expression can be carried out through various immunoassay methods known in the art. For example, changes in XCL1 expression may include immunostaining, radioimmunoassay, radioimmunoprecipitation, western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, and sandwich assay. Including, but not limited to.

The immunoassay or method of immunostaining is described in Enzyme-linked immunosorbent assay (ELISA), in Techniques in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1987, and the like, which is incorporated herein by reference.

For example, when the method of the invention is carried out in accordance with radioimmunoassay methods, an antibody labeled with a radioisotope (eg, C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ) detects a marker molecule of the invention. It can be used to.

When the method of the present invention is carried out in an ELISA method, the present invention comprises the steps of: (a) coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; (b) reacting said cell lysate with an antibody to a target as a primary antibody; (c) reacting the resultant of step (b) with a secondary antibody to which an enzyme is bound; And (d) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

Enzymes bound to the secondary antibody include, but are not limited to, enzymes catalyzing color reaction, fluorescence, luminescence or infrared reaction, for example, alkaline phosphatase, β-galactosidase, hose Radish peroxidase, luciferase and cytochrome P450. When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) if colorimetric substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used -N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), TMB (tetramethylbenzidine), ABTS (2,2'-Azidine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS substrates such as phenzaine methosulfate can be used. The.

Measurement of the final enzyme activity or signal in the ELISA method and the capture-ELISA method can be carried out according to various methods known in the art. Detection of this signal allows for qualitative or quantitative analysis of the XCL1 of the present invention. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

The zebrafish may be introduced with a gene carrier comprising an XCL1 protein or a nucleotide sequence encoding XCL1. The introduction may proceed according to known methods, for example microinjection, electroporation, particle bombardment, sperm-mediated genetransfer, viral infection (viralinfection), direct muscle injection (direct muscle injection), insulators (insulator) and transposon (trnasposon), and the like, and preferably include, but not limited to microinjection.

In addition, the present invention provides a method for screening a candidate substance for XCL1 co-administration for preventing or treating a muscular disease comprising the following steps. (a) treating the zebrafish of claim 14 with an XCL1 co-administration candidate; And (b) observing the development of the zebrafish muscle cells.

In the present invention, the individual is preferably a mammal including a human, a patient in need of the treatment of muscle diseases, patients in the treatment of muscle diseases, patients with muscle diseases who have been treated with muscle diseases, muscle diseases need to be treated It can include all patients.

In the present invention, the muscle disease may be caused by the death of muscle cells or myoblasts, sprains, strains, convulsions, tendonitis, muscle cancer, myositis, rhabdomyolysis, muscular dystrophy ( 1 species selected from the group consisting of muscular atrophy, atony, muscular dystrophy, muscular degeneration, myasthenia, dystrophinopathy, myopathy and sacopenia It is preferable that it is above.

The mesenchymal stem cells, XCL1 (chemokine (C motif) ligand 1) or derivatives thereof may be delivered in a pharmaceutically effective amount to a site for the treatment of muscle disease in an individual, and the delivery is for mammalian administration to mammals. More preferably, it is delivered in form, preferably for human patient administration.

In addition, suitable dosages of the mesenchymal stem cells of the present invention, chemokine (C motif) ligand 1 (XCL1) or derivatives thereof may be formulated by the method of administration, mode of administration, age, weight, sex, morbidity, food, time of administration, administration of the patient. Various prescriptions can be made by factors such as pathway, excretion rate and response sensitivity.

In order to prevent or treat muscle diseases, other mesenchymal stem cells of the present invention, XCL1 (chemokine (C motif) ligand 1), or derivatives thereof may be administered in combination with other known muscle disease therapeutic agents, and such combination administration may be performed simultaneously or It can be administered sequentially.

In another aspect, the present invention comprises the steps of treating the individual with XCL1 (chemokine (C motif) ligand 1) protein or derivatives thereof; It provides a method for inhibiting apoptosis, including the cells may be muscle cells or myoblasts.

The XCL1 (chemokine (C motif) ligand 1) protein or derivative thereof of the present invention may inhibit the occurrence or progression of apoptosis in the subject when treated in the subject, particularly preferably in muscle cells and / or myoblasts. By effectively inhibiting cell death can achieve a cell protective effect, through which can prevent or treat muscle diseases.

Terms not defined otherwise in this specification are intended to have a meaning commonly used in the art to which the present invention pertains.

Hereinafter, the present invention will be described in detail by way of examples and preparation examples. However, the following Examples and Formulation Examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following Examples and Formulation Examples.

실시예 1: 인간 탯줄 중간엽 줄기세포의 근육아세포 사멸 억제 효과 확인Example 1 Confirmation of Myoblast Killing Inhibitory Effect of Human Umbilical Cord Mesenchymal Stem Cells

1.1 영양분 고갈을 통한 근육아세포 사멸의 유도Induction of Myoblast Death by Nutrient Depletion

In order to induce myoblast death, C2C12 mouse muscle myoblasts were cultured in serum-free medium for 12 to 24 hours to induce apoptosis.

1.2 인간 탯줄 중간엽 줄기세포의 근육아세포 사멸 억제 효과 확인1.2 Inhibitory Effect of Human Umbilical Cord Mesenchymal Stem Cells on Myoblast Death

To identify the myoblast killing effect of human umbilical cord mesenchymal stem cells, Wharton's Jelly-mesenchymal stem isolated from the dead myoblasts obtained in 1.1 and the umbilical cord discarded after childbirth. cell (WJ-MSC) was co-cultured using a transwell chamber and the schematic diagram is shown in FIG. 1, and the culture results are shown in FIG. 2. As a control group, a group without co-culture of human umbilical cord mesenchymal stem cells in a serum-free medium was used.

As shown in Figure 2, 12 and 24 hours incubation in a serum-free medium was confirmed that the death of myoblasts, apoptosis was reduced when co-culture with human umbilical cord mesenchymal stem cells.

In addition, in order to quantify the effect of inhibiting apoptosis of human umbilical cord stem cells, the cleavage of the apoptosis marker PARP was detected by Western blotting method known in the art, and the result and the result of quantification thereof are shown in FIG. 3. It was.

As shown in Figure 3, when the treatment of human umbilical cord mesenchymal stem cells was confirmed that the detection of 85kDa fragments by apoptosis is significantly reduced.

Therefore, it can be seen that human umbilical cord mesenchymal stem cells inhibit the death of myoblasts due to nutrient depletion. In addition, this myoblast killing effect was shown by the co-culture, and because the hole of the co-culture chamber is a size that the cells cannot move, it can be inferred that the water-soluble factor secreted by human umbilical cord mesenchymal stem cells inhibits myoblast death. have.

실시예Example 2: 수용성 인자 2: water soluble factor XCL1XCL1 재조합 단백질의 Of recombinant protein 근육아세포Myoblasts 사멸 억제 효과 확인 Confirmation of killing effect

2.1 XCL1 단백질의 농도 의존적 근육아세포 사멸 억제 효과 확인2.1 Determination of Inhibitory Effect of XCL1 Protein on Concentration-dependent Myoblast Death

After obtaining the medium in the co-culture chamber of each experimental group, the antibody array was performed in the medium. As a result of the antibody array, XCL1 was identified as a water-soluble factor of human umbilical cord mesenchymal stem cells and selected as a potential candidate.

In order to confirm the myoblast killing effect of XCL1, the killing of myoblasts was induced in the same manner as in 1.1. After the treatment of killed myoblasts with XCL1 recombinant protein (R & D Systems, Catalog Number: 695-LT / CF) at 0, 10, and 50 ng / mL for 12 hours, the results were observed in FIG. 4. As a control group, a group which was not treated with XCL1 during incubation in a serum-free medium was used.

As shown in Figure 4, it was confirmed that the XCL1 recombinant protein inhibits the death of myoblasts in a concentration-dependent manner.

In order to further confirm the apoptosis inhibitory effect of the XCL1 recombinant protein, the results of detecting and quantifying the apoptosis marker PARP by Western blotting methods commonly used in the art are shown in FIG. 5. .

As shown in Western blotting of Figure 5, it was confirmed that the 85kDa fragments by apoptosis significantly reduced by the treatment of XCL1. In addition, this apoptosis inhibitory effect was confirmed to be dependent on the concentration of XCL1.

2.2 XCL1 단백질의 시간 의존적 근육아세포 사멸 억제 효과 확인2.2 Confirmation of the Time-dependent Myoblast Killing Effect of XCL1 Protein

In order to confirm the effect of inhibiting apoptosis with the treatment time of the XCL1 recombinant protein, the XCL1 recombinant protein was treated at 50ng / mL for 3, 6, 12 hours. 6 shows a result of detecting the cleavage of the apoptosis marker PARP by the conventional Western blotting method used in the art in the sample and a numerical value thereof.

As shown in Figure 6 it was confirmed that the XCL1 recombinant protein exhibits apoptosis inhibitory effect.

Therefore, it was confirmed that human umbilical cord mesenchymal stem cells or XCL1 can be effectively used for the prevention or treatment of muscle diseases caused by muscle cell death by effectively inhibiting the death of myoblasts in a concentration and time dependent manner.

2.3 PAN 저해제(PAN inhibitor)와 XCL1 단백질의 세포사멸 억제 효과 비교2.3 Comparison of apoptosis inhibitory effect between PAN inhibitor and XCL1 protein

In order to compare the myoblast killing effect of PCL inhibitor and XCL1, which are known as inhibitors of apoptosis due to depletion of nutrients, ie, caspase-3 mediated apoptosis, the apoptosis of myoblasts is reduced due to the depletion of nutrients. The cells were treated with 5, 10, 20, 50 μM PAN inhibitor and 0.5, 1, 2, 5 nM XCL1 protein, respectively. As a control group, a group which was not treated with PAN inhibitor or XCL1 protein during culture in serum-free medium was used. PARP cleavage was confirmed by Western blotting method and the result of quantification is shown in FIG. 7.

As shown in Fig. 7, PAN inhibitor significantly less progress in myoblast killing when treated with the XCL1 protein of the present invention, compared to the case of treatment with a PAN inhibitor which is well known as an inhibitor of apoptosis due to depletion of nutrients. It was confirmed that the cytoprotective effect of the XCL1 protein is better than. In addition, it was confirmed that 50μM treatment effect of PAN inhibitor was observed similar to 1nM treatment effect of XCL1 protein.

실시예 3: XCL1 단백질의 세포사멸 억제 효과의 특성 확인Example 3: Characterization of apoptosis inhibitory effect of XCL1 protein

XCL1 has the effect of inhibiting apoptosis of myoblasts C2C12, and accordingly, mouse hippocampal neurons HT22, rat Schwann cells, and S16 were used to confirm whether the effect is applied to other cells. In the Alzheimer's disease model, apoptosis was induced by treating amyloid beta (Amyloid-ß), a protease inhibitor, and protease inhibitor MG132, with mouse hippocampal neurons HT22. After inducing apoptosis, the result of treating the XCL1 protein is shown in FIG. 8.

In addition, the results of detecting the cleavage of the apoptosis marker PARP in the sample by the conventional Western blotting method in the sample and the result of quantification thereof are shown in FIG. 9. As a control group, the group not treated with amyloid beta, MG132, and XCL1 protein was used in culture in serum-free medium.

As shown in Figures 8 and 9, the cell morphology and Western blotting results in the mouse hippocampal neurons did not show a clear inhibitory effect of apoptosis.

In addition, the rat Schwann cell S16 cells were treated with Thapsigargin, an endoplasmic reticulum stress derivative, for 12 hours to confirm cell specificity as described above. The results are shown in FIGS. 10 and 11. As a control group, Thapsigargin and XCL1 protein were not treated in the serum-free medium.

As shown in Figure 10 and 11, the rat Schwann cells as a result, it was confirmed that there is no effect of inhibiting apoptosis by XCL1. Therefore, it was confirmed that the XCL1 protein has an apoptosis inhibitory effect on myoblasts.

In addition, Lovastatin, a cholesterol synthesis inhibitor, was used to determine whether the XCL1 protein had an effect of inhibiting apoptosis of myoblasts caused by other apoptosis inducing agents. Lovastatin is known to induce myoblasts atrophy, and Lovastatin was dose-dependently treated with C2C12 mouse myoblasts and C2C12 mouse myotubes induced differentiation for 7 days at 0.1, 0.5, 1, 2ng / mL. . The sample was cultured with or without treatment with XCL1 protein (50 ng / mL) to confirm the effect of inhibiting death of XCL1 protein by PARP cleavage, which is shown in FIGS. 12 and 13.

As shown in FIG. 12 and FIG. 13, it was confirmed that PARP cleavage induced by Lovastatin was completely inhibited by XCL1 protein. Therefore, it was confirmed that the XCL1 protein has an inhibitory effect on the death of myoblasts due to nutritional depletion as well as the death of myoblasts by other causes.

In addition, the muscle myotubes C2C12 induced differentiation for 7 days was treated with concentration-dependent Lovastatin at 1, 2, 5, and 10 μM to confirm the loss of myotubes. Thus, after treatment with XCL1, The result of confirming the MHC which is Myosin Heavy Chain by the conventional western blotting method and the numerical value thereof is shown in FIG. 14. As a control group, the group that was not treated with Lovastatin and XCL1 in the serum-free medium was used.

As shown in Figure 14, the apoptosis inhibitory effect of XCL1 can be inferred to have a specific effect on myoblasts and myotubes.

실시예Example 4: 4: siRNA를siRNA 통한 through XCL1XCL1 합성의 저해가 인간 탯줄 Inhibition of Synthetic Human Umbilical Cord 중간엽Mesenchyme 줄기세포의 세포사멸 억제효과를 상실시킴을 확인 Confirmed that the stem cell loss of cell death inhibition effect

XCL1-siRNA1, an siRNA that can inhibit the synthesis of XCL1 protein in apoptotic myoblasts induced by nutrient depletion, was confirmed to inhibit the synthesis of XCL1 protein by siRNA. And co-cultured human umbilical cord mesenchymal stem cells pretreated with XCL1-siRNA2, and confirmed the death of myoblasts by the same method as in Example. At this time, the positive control group was co-cultured with C2C12 cells and human umbilical cord stem cells cultured in serum-free medium, and the negative control group with control siRNA (CTRL-siRNA) that did not inhibit the synthesis of XCL1 protein. Cells treated with and co-cultured with C2C12 cells grown in serum-free medium were used, and the results are shown in FIGS. 15 and 16.

As shown in Figure 15 and Figure 16, when treated with XCL1-siRNA1 and XCL1-siRNA2, it was confirmed that the myoblast killing effect of human umbilical cord mesenchymal stem cells is lost. Therefore, it was confirmed that the active ingredient for inhibiting apoptosis of human umbilical cord mesenchymal stem cells is XCL1 protein.

실시예Example 5: 인간 5: human XCL1XCL1 발현 Expression 제브라피쉬Zebrafish 모델 제조 및 인간 Model manufacture and human XCL1XCL1 단백질이 도입된 후 3일 지난 제브라피쉬의 발생 정도 관찰 Observe the development of zebrafish three days after the protein was introduced.

5.15.1 제브라피쉬의 사육 및 발생배의 준비Breeding of zebrafish and preparation of embryos

Wild type zebrafish were bred under conditions (temperature: 28-28.5 ° C., contrast: lighted from 9 am to 8 pm, other times off, food: brine shrimp). The embryos were divided into two partitions each day by zebrafish before mating, and then brightened the next morning to remove mating between females and males. The zebrafish eggs obtained through the mating were transferred to a mold made of agar gel.

5.2 인간 5.2 Human XCL1XCL1 발현 Expression 제브라피쉬Zebrafish 모델 제조 및 인간 Model manufacture and human XCL1XCL1 단백질이 도입된 후 3일 지난 제브라피쉬의 발생 정도 관찰 Observe the development of zebrafish three days after the protein was introduced.

To prepare a human XCL1 expressing zebrafish model, recombinant XCL1 protein was microinjected 12 hours after zebrafish fertilization. As shown in FIG. 17, zebrafish was transferred to a mold made of agarose gel, and the protein was microinjected with a micropipette mounted in a micromanipulator (World Precision Instruments Inc., Sarasota, FL, USA).

The phenotype of the zebrafish model prepared as above was observed 3 days after birth and the results are shown in FIG. 18.

As shown in FIG. 18, when comparing the XCL1 protein with the micro-injected control group, it was confirmed that the zebrafish to which the human XCL1 protein was introduced generally occurred normally without any abnormality.

실시예 6: 인간 XCL1이 도입된 제브라피쉬의 원위근육계 조사Example 6: Distal Muscle Examination of Zebrafish with Human XCL1

In order to confirm how the introduction of human XCL1 protein affects zebrafish, after injecting human XCL1 protein into zebrafish simulating muscle disease symptoms, zebrafish was observed and the results are shown in FIG. 19.

As shown in FIG. 19, it was observed that the overall degree of movement and occurrence of zebrafish were alleviated.

In addition, in order to confirm the development of muscle cells using zebrafish embryos, laminin and MHC were identified as markers and fluorescent staining was performed. Thereafter, to observe the degree of activation of muscle cells, specific regions of the body were selected as shown in FIG. 20, and muscle cells were observed through a confocal microscope. The results are shown in FIG. In addition, zebrafish embryos were treated with 0, 30, 50 μg / mL of XCL1 protein, and normal and abnormal embryos were observed and quantified.

As shown in FIGS. 21 and 22, the zebrafish of the muscle disease model without the XCL1 protein injection showed abnormal expression of both markers, whereas the zebrafish to which the human XCL1 protein was introduced was normally expressed in both markers. . In addition, it was confirmed that the muscle disease of zebrafish normalized concentration-dependently on the treated XCL1 protein.

Therefore, by confirming the efficacy of XCL1 in preventing or treating muscle diseases in the zebrafish model, it was confirmed that XCL1 could be operated in vivo . In addition, zebrafish can be usefully used as a model for muscle diseases, and it was confirmed that it would be useful for screening for the prevention or treatment of muscle diseases such as XCL1 protein.

Although the present invention has been described as the preferred embodiment mentioned above, it is possible to make various modifications or variations without departing from the spirit and scope of the invention. The appended claims also cover such modifications and variations as fall within the spirit of the invention.

제제예 - 의약품의 제조Formulation Example-Preparation of Pharmaceuticals

산제의 제조Manufacture of powder

Mesenchymal stem cells 5x10 ⁸ cells or XCL1 100 mg

Lactose 100mg

10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

주사제의 제조Preparation of Injectables

Mesenchymal stem cells 5x10 ⁸ cells or XCL1 100 mg

Appropriate sterile distilled water for injection

Moderator

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

액제의 제조Preparation of liquid

Mesenchymal stem cells 5x10 ⁸ cells or XCL1 100 mg

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Purified water was added to adjust the total volume to 100 ml. According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Claims

Pharmaceutical composition for the prevention or treatment of muscle diseases, including mesenchymal stem cells.
The pharmaceutical composition of claim 1, wherein the mesenchymal stem cells comprise XCL1 (chemokine (C motif) ligand 1).
According to claim 1, wherein the mesenchymal stem cells, characterized in that derived from human umbilical cord, pharmaceutical composition for the prevention or treatment of muscle diseases.
According to claim 1, wherein the muscle disease is caused by the death of muscle cells or myoblasts, pharmaceutical composition for the prevention or treatment of muscle diseases.
The method of claim 1, wherein the muscle disease is sprain, strain, spasms, tendonitis, muscle cancer, myositis, rhabdomyolysis, muscular atrophy, atony, muscular dystrophy A pharmaceutical composition for the prophylaxis or treatment of muscle diseases, which is at least one member selected from the group consisting of muscular dystrophy, muscular degeneration, myasthenia, dystrophinopathy, myopathy and sacopenia.
XCL1 (chemokine (C motif) ligand 1) or a derivative thereof comprising as an active ingredient, a pharmaceutical composition for preventing or treating muscle diseases.
Stem cell therapy for treating muscular diseases, including mesenchymal stem cells.
According to claim 7, wherein the mesenchymal stem cells, XCL1 (chemokine (C motif) ligand, characterized in that the ligand 1), characterized in that for treating stem disease therapeutic muscle cells.
Apoptosis inhibitor including XCL1 (chemokine (C motif) ligand 1) protein or derivatives thereof.
10. The apoptosis inhibitor of claim 9, wherein the cells are myocytes or myoblasts.
Screening method for the treatment of muscle diseases comprising the following steps:

(a) contacting a test substance to be analyzed with a cell expressing XCL1;

(b) analyzing the effect of the test substance on the expression of the XCL1, and when the test substance increases the expression of the XCL1, the test substance is determined as a prophylactic or therapeutic agent for muscle diseases.
Screening method for the treatment of muscle diseases comprising the following steps:

(a) contacting the test substance with XCL1;

(b) analyzing the effect of the test substance on the activity of the XCL1, when the test substance increases the activity of the XCL1 is determined as a prophylactic or therapeutic agent of muscle diseases.
The method of claim 11 or 12, wherein the test substance is selected from the group consisting of chemicals, proteins, peptides, and natural extracts.
A zebrafish into which a gene carrier comprising an XCL1 protein or a nucleotide sequence encoding XCL1 is introduced.
A method for screening a candidate drug for concomitant use of XCL1 for the prevention or treatment of muscle disease, comprising the following steps:

(a) treating the zebrafish of claim 14 with an XCL1 co-administration candidate; And

(b) monitoring the development of the zebrafish muscle cells.
Treating the mesenchymal stem cells, XCL1 (chemokine (C motif) ligand 1) or derivatives thereof with the subject; Prevention or treatment method of muscle disease comprising a.
The method of claim 16, wherein the muscle disease is sprain, strain, spasms, tendonitis, muscle cancer, myositis, rhabdomyolysis, muscle atrophy, atony, muscular dystrophy A method for the prevention or treatment of muscular diseases, which are at least one member selected from the group consisting of muscular dystrophy, muscular degeneration, myasthenia, dystrophinopathy, myopathy and sacopenia.
Treating the subject with an XCL1 (chemokine (C motif) ligand 1) protein or derivative thereof; Apoptosis inhibition method comprising a.
The method of claim 18, wherein the cells are myocytes or myoblasts.