KR20240011266A - Pharmaceutical composition for preventing or treating metabolic liver disease comprising culture product of stem cells overexpressing PGC-1α as an active ingredient - Google Patents

Abstract

The present invention relates to a novel use of a stem cell culture overexpressing PGC-1α, and more specifically, to the prevention, improvement, and/or use of a stem cell culture overexpressing PGC-1α as an active ingredient. It relates to therapeutic compositions (pharmaceutical compositions or food compositions). Stem cell cultures overexpressing PGC-1α not only have high safety and a significant therapeutic effect for metabolic liver diseases, but the secretome secreted by the stem cells does not cause immunological rejection, is easy to store, and is durable. It has many advantages such as mass production, immediate application when needed by patients, and exclusion of the risk of malignancy, making it an excellent treatment for metabolic liver disease.

Description

Pharmaceutical composition for preventing or treating metabolic liver disease comprising culture product of stem cells overexpressing PGC-1α as an active ingredient ingredient}

The present invention relates to a novel use of a stem cell culture overexpressing PGC-1α, and more specifically, to the prevention, improvement, and/or use of a stem cell culture overexpressing PGC-1α as an active ingredient. It relates to therapeutic compositions (pharmaceutical compositions or food compositions). The present invention also relates to the use of stem cells overexpressing PGC-1α to prevent, improve, or/and treat metabolic liver disease.

Recently, the number of patients with metabolic liver disease, a complex condition caused by various metabolic abnormalities such as obesity, diabetes, hyperlipidemia, and insulin resistance, has been increasing. While the prevalence of metabolic liver disease has increased in modern society due to obesity and diabetes due to unbalanced diet, high calorie intake, and lack of exercise, there are no officially approved drugs for metabolic liver disease. Metabolic liver disease is a disease caused by metabolic dysregulation in the liver that is not related to alcohol consumption. In particular, non-alcoholic fatty liver disease (NAFLD), a representative metabolic liver disease, occurs even in people of normal weight. It is reported that this happens. It is known that even with normal weight, fatty liver can occur due to abdominal obesity caused by excessive carbohydrate intake and visceral fat. PGC-1α (peroxisome proliferator-activated receptor gamma co-activator-1 α) is a factor that mediates the expression of lipogenesis markers and lipid accumulation in the liver (Non-patent Document 1), and the level of PGC-1α in the liver of NAFLD patients It has been reported that this has increased. When fat accumulates in the liver, PGC-1α expression increases, which promotes Acetyl CoA carboxylase-1 (ACC) and Fatty acid synthase (FAS) gene expression, and increases the conversion of glucose to Acetyl-CoA, leading to fat accumulation in the liver. It has been reported that this is accelerating (Non-patent Document 1). There is much controversy regarding the in vivo function of PGC-1α.

Because metabolic liver disease has the characteristics of a metabolic disease, its characteristics are very different from general liver diseases. Therefore, in the treatment of this disease, there are quite a few studies reporting that the application of previously known liver disease treatment drugs is ineffective in metabolic liver diseases such as non-alcoholic fatty liver disease. This is also the reason why there are currently no officially approved drugs for metabolic liver disease. For example, when UDCA (Ursodeoxycholic acid), which is known to have hepatocyte protection and liver cell regeneration effects, was administered to patients with non-alcoholic steatohepatitis, steatosis, necroinflammation, and It was reported that there was no therapeutic effect on liver tissue such as fibrosis (Non-patent Document 2).

Meanwhile, studies using stem cells as a treatment strategy for various diseases have been reported, but stem cells have a risk of immunological rejection when transplanted and require a sufficient amount of stem cells for treatment, resulting in high costs. In addition, there is instability of genes related to aging, the survival period of cells after transplantation may be limited, and there is a risk of malignancy (tumorization).

Min-Jung Park et al., Thioredoxin-interacting protein mediates hepatic lipogenesis and inflammation via PRMT1 and PGC-1α regulation in vitro and in vivo, J Hepatol. 2014 Nov;61(5):1151-7. Keith D Lindor et al., Ursodeoxycholic acid for treatment of nonalcoholic steatohepatitis: results of a randomized trial, Hepatology. 2004 Mar;39(3):770-8.

Accordingly, while researching safer and more effective treatment strategies for metabolic liver disease, the present inventors newly discovered that stem cell cultures overexpressing PGC-1α have a significant therapeutic effect on metabolic liver disease, which is substantially effective in treating metabolic liver disease. This effect is due to the secretome secreted by stem cells overexpressing PGC-1α. The secretome does not cause immunological rejection, is easy to store, can be continuously produced in large quantities, can be applied immediately when needed by patients, and prevents malignant transformation. It was confirmed that it can function as an excellent treatment for metabolic liver disease with many advantages such as eliminating concerns, and the present invention was completed.

Therefore, the object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of metabolic liver disease containing a stem cell culture overexpressing PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) as an active ingredient. will be.

Another object of the present invention is to provide a food composition for preventing or improving metabolic liver disease, which contains a stem cell culture overexpressing PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of metabolic liver disease containing stem cells overexpressing PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above object, the present invention provides a pharmaceutical for the prevention or treatment of metabolic liver disease containing stem cell culture overexpressing PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) as an active ingredient. A composition is provided.

In order to achieve another object of the present invention, the present invention provides a food for preventing or improving metabolic liver disease containing stem cell culture overexpressing PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) as an active ingredient. A composition is provided.

In one embodiment of the present invention, the metabolic liver disease includes non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NASH). NAFL), hepatic steatosis, Acute Fatty Liver of Pregnancy (AFLP), NAFLD-related liver failure, NAFLD-related liver fibrosis, and NAFLD-related liver cirrhosis. It may be any one or more selected from the group consisting of, but is not limited thereto.

In one embodiment of the present invention, the stem cells overexpressing PGC-1α may be obtained by transforming stem cells with a recombinant expression vector containing a polynucleotide encoding PGC-1α, but are not limited thereto. .

In one embodiment of the present invention, the stem cells include mesenchymal stem cells, adult stem cells, pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, and adipose-derived stem cells (ASC). It may be any one or more selected from the group consisting of, but is not limited to this.

In one embodiment of the present invention, the culture is characterized in that it contains a secretome secreted from the stem cells.

In one embodiment of the present invention, the culture may be prepared by a production method including the following steps, but is not limited thereto:

(a) transforming isolated stem cells with a vector containing a polynucleotide encoding PGC-1α; and

(b) culturing the transformed stem cells in a serum-free medium to obtain a culture medium of the stem cells.

In one embodiment of the present invention, the culture may not contain stem cells overexpressing PGC-1α, but is not limited thereto.

The present invention also provides a method for preventing, improving and/or treating metabolic liver disease, comprising administering a stem cell culture overexpressing PGC-1α to an individual in need thereof.

In addition, the present invention provides a method for preventing, improving and/or treating metabolic liver disease, comprising administering to an individual in need a composition containing a stem cell culture overexpressing PGC-1α as an active ingredient. .

Additionally, the present invention provides the use of a composition containing a stem cell culture overexpressing PGC-1α as an active ingredient for preventing, improving, and/or treating metabolic liver disease.

In addition, the present invention provides the use of stem cell cultures overexpressing PGC-1α for preparing (producing) an agent for preventing, improving, and/or treating metabolic liver disease.

In order to achieve another object of the present invention, the present invention provides a pharmaceutical for the prevention or treatment of metabolic liver disease containing stem cells overexpressing PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) as an active ingredient. A composition is provided.

The present invention also provides a method for preventing, improving, and/or treating metabolic liver disease, comprising administering stem cells overexpressing PGC-1α to an individual in need thereof.

In addition, the present invention provides a method for preventing, improving and/or treating metabolic liver disease, comprising administering to an individual in need a composition containing stem cells overexpressing PGC-1α as an active ingredient.

In addition, the present invention provides the use of a composition containing stem cells overexpressing PGC-1α as an active ingredient for the prevention, improvement and/or treatment of metabolic liver disease.

Additionally, the present invention provides the use of stem cells overexpressing PGC-1α for manufacturing (producing) an agent for preventing, improving, and/or treating metabolic liver disease.

The stem cell culture overexpressing PGC-1α of the present invention not only has high safety and a significant therapeutic effect for metabolic liver disease, but also the secretome secreted by the stem cells is free from immunological rejection and can be stored well. It has many advantages such as ease of use (can be used immediately when needed after frozen storage), continuous mass production, applicability to patients immediately when needed, and exclusion of the risk of malignancy, making it an excellent treatment for metabolic liver disease.

Figure 1 is an image schematically showing the PCM manufacturing process of the present invention (PGC-ASC: stem cell overexpressing PGC-1α).
Figure 2 shows the results of confirming changes in PPAR-alpha, a key factor in lipid and sugar metabolism, following PCM administration of the present invention through IHC staining in a representative NASH disease model (model made with the MCD diet) among metabolic liver diseases. This is an image showing (Normal: normal control, MCD: NASH disease model created with the MCD diet).
Figure 3 shows the results of IHC staining confirming changes in PPAR-alpha, a key factor in lipid and sugar metabolism, following PCM administration of the present invention in a representative NASH disease model (model made with HFHC diet) among metabolic liver diseases. This is the image shown (Normal: normal control group, HFHC: NASH disease model created with HFHC diet).
Figure 4 is an image showing the results of confirming changes in damaged liver tissue following PCM administration of the present invention through H&E staining in a NASH disease model produced with the MCD diet (Ct: control (normal control), MCD: MCD diet created NASH disease model).
Figure 5 is an image showing the results of confirming changes in damaged liver tissue following PCM administration of the present invention through H&E staining in a NASH disease model produced with a HFHC diet (Ct: control (normal control), HFHC: with HFHC diet created NASH disease model).
Figure 6 is an image showing the results of confirming changes in fatty liver tissue fibrosis according to PCM administration of the present invention through sirius red staining in the NASH disease model produced with the MCD diet (Normal: normal control, MCD: MCD diet produced NASH disease model).
Figure 7 is an image showing the results of confirming changes in fatty liver tissue fibrosis following PCM administration of the present invention through sirius red staining in a NASH disease model produced with a HFHC diet (Normal: normal control, HFHC: produced with an HFHC diet NASH disease model).
Figure 8 is an image showing the results of confirming changes in liver collagen I (Collagen I) according to PCM administration of the present invention through IHC staining in a NASH disease model produced with the MCD diet (Normal: normal control, MCD: MCD diet NASH disease model created with ).
Figure 9 is an image showing the results of confirming changes in liver collagen I (Collagen I) according to PCM administration of the present invention through IHC staining in a NASH disease model produced with a HFHC diet (Normal: normal control, HFHC: HFHC diet NASH disease model created with ).
Figure 10 is a graph showing the results of quantitatively confirming changes in mitochondrial ROS (reactive oxygen species) levels according to PCM administration of the present invention using the FACS method in an in vitro cirrhosis and liver fibrosis model (control: normal control, TAA : in vitro liver cirrhosis and liver fibrosis model).
Figure 11 is a graph showing the results of quantitative confirmation using the MTT assay method that the proliferation and activation of hepatic stellate cells were significantly inhibited by administration of the PCM of the present invention in an in vitro cirrhosis and liver fibrosis model (control: normal control, TAA: in vitro cirrhosis and liver fibrosis model).

Justice

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

In the present invention, 'metabolic liver disease' refers to a disease caused by metabolic dysregulation in the liver unrelated to alcohol consumption. In the present invention, the metabolic liver disease is not particularly limited as long as it is a metabolic liver disease known in the art, but preferably refers to a liver disease caused by abnormal carbohydrate or lipid metabolism. In the present invention, the metabolic liver disease is not limited thereto, but examples include non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (non-alcoholic fatty liver disease). alcoholic fatty liver (NAFL), hepatic steatosis, acute fatty liver of pregnancy (AFLP), NAFLD-related liver failure, NAFLD-related liver fibrosis, and NAFLD-related cirrhosis. It may be any one or more selected from the group consisting of (liver cirrhosis).

In the present invention, the term 'expression' refers to the production of proteins or nucleic acids in cells.

In the present invention, the term 'overexpression' refers to artificially expressing a gene containing a sequence encoding a polypeptide (protein) in a modified cell to exceed the expression level of the same polypeptide in the precursor host cell. It refers to the process of creating a level of expression. Accordingly, when the term is typically used in conjunction with a gene (or polynucleotide), the term 'overexpression' may be used to refer to increased levels of a protein resulting from overexpression of the gene encoding it. In some embodiments, overexpression of a gene encoding a protein is achieved by increasing the number of copies of the gene encoding the protein. In another embodiment, overexpression of a gene encoding a protein is achieved by increasing the avidity of the promoter region and/or ribosome binding site in a manner that increases transcription and/or translation of the gene encoding the protein. In other embodiments, overexpression can be achieved by increasing the copy number of the gene and increasing the avidity of the promoter region and/or ribosome binding site. In some embodiments, overexpression of a gene encoding a protein results from expression of one or more copies of the corresponding encoding polynucleotide present on a multiple copy plasmid introduced into the genome of a host cell. In another embodiment, overexpression of a gene encoding a protein results from expression of two or more copies of the corresponding encoding polynucleotide that are integrated into the genome of the host cell.

In the present invention, the term 'host cell' includes heterologous DNA introduced into the cell by any means (e.g., electric shock method, calcium phosphatase precipitation method, microinjection method, transformation method, viral infection, etc.) refers to a prokaryotic or eukaryotic cell.

In the present invention, the term 'protein' can be used interchangeably with 'polypeptide' and refers to, for example, a polymer of amino acid residues as is generally found in proteins in their natural state.

In the present invention, the 'PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha)' protein is a PGC-1α protein or polypeptide known in the art, and its specific origin and sequence (amino acid sequence composition) are particularly limited. However, as an example, in the present invention, PGC-1α may be PGC-1α derived from human ( homo sapiens ) and may include the amino acid sequence shown in SEQ ID NO: 1, preferably a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. (It may be NCBI (Genbank) Accession No. NP_001317680.1. In the present invention, PGC-1α includes functional equivalents thereof.

The functional equivalent refers to a sequence that is at least 70% or more, preferably 80% or more, and more preferably 90% or more of the amino acid sequence constituting the PGC-1α protein (a preferred example is the amino acid sequence shown in SEQ ID NO: 1). Refers to a polypeptide that has homology (i.e., identity). For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology It refers to a polypeptide that exhibits substantially the same physiological activity as the parent PGC-1α protein (a preferred example is the polypeptide shown in SEQ ID NO: 1). Here, ‘substantially homogeneous physiological activity’ refers to a secretome that exhibits a preventive, ameliorating, or/and therapeutic effect on metabolic liver disease in stem cells when introduced into and expressed in stem cells using a common transformation method known in the art. It means secreting secretome. Preferably, the functional equivalent of PGC-1α in the present invention may be produced as a result of addition, substitution, or deletion of part of the amino acid sequence of SEQ ID NO: 1. In the above, the substitution of amino acids is preferably a conservative substitution. Examples of conservative substitutions of naturally occurring amino acids are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn) ) and sulfur-containing amino acids (Cys, Met). In addition, the functional equivalent of PGC-1α also includes variants in which some amino acids are deleted in the amino acid sequence of the PGC-1α protein. The deletion or substitution of the amino acid is preferably located in a region not directly related to the physiological activity of PGC-1α. In addition, variants in which several amino acids are added to both ends of the amino acid sequence of PGC-1α or within the sequence are also included. In addition, the scope of functional equivalents of the present invention also includes polypeptide derivatives in which some chemical structures of the polypeptide are modified while maintaining the basic skeleton of PGC-1α and its physiological activity. For example, this includes structural changes to change the stability, storage, volatility or solubility of the protein.

In the present specification, sequence homology and homology are defined by aligning the candidate sequence with the original sequence (SEQ ID NO: 1 as a preferred example for an amino acid sequence, or SEQ ID NO: 2 as a preferred example for a nucleic acid sequence) and introducing gaps. , defined as the percentage of identical matching residues (amino acid residues or bases) of the candidate sequence with respect to the original sequence. When necessary, conservative substitutions are not considered as part of sequence identity to obtain maximum percent sequence identity. Additionally, in the case of determining protein sequence homology or homology, the N-terminus, C-terminus, or internal extension, deletion, or insertion of the PGC-1α amino acid sequence is not interpreted as a sequence that affects sequence identity or homology. Additionally, the sequence identity can be determined by general standard methods used to compare similar portions of the amino acid sequences of two polypeptides. Computer programs such as BLAST or FASTA align two polypeptides such that each amino acid is optimally matched (either along the full length of one or both sequences or along a predicted portion of one or both sequences). The program provides a default opening penalty and a default gap penalty and can be used in conjunction with a computer program called PAM250 (standard scoring matrix; Dayhoff et al., in Atlas of Protein). It provides a scoring matrix such as Sequence and Structure, vol 5, supp 3, 1978). For example, percent homogeneity can be calculated as follows: Multiply the total number of identical matches by 100 and then the sum of the length of the longer sequence in the matched span and the number of gaps introduced into the longer sequence to align the two sequences. Share.

In the present invention, the terms 'nucleic acid', 'DNA sequence', 'RNA sequence' or 'polynucleotide' refer to deoxyribonucleotides or ribonucleotides in single- or double-stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included.

In the present invention, the term 'coding' or 'encoding' refers to the case where it can be transcribed and/or translated to produce mRNA for a polypeptide and/or fragment thereof, either in its natural state or when manipulated by methods well known to those skilled in the art; Refers to a polynucleotide that is said to 'code' or 'encode' a polypeptide.

In the present invention, 'polynucleotide encoding PGC-1α, or PGC-1α mRNA' is, for example, a base (nucleic acid) encoding an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence homology thereto. There can be a hierarchy. The nucleic acids include both DNA, cDNA and RNA sequences. That is, the polynucleotide may have a base sequence encoding the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70% homology thereto, or may have a base sequence complementary thereto. Preferably, it may include the base sequence shown in SEQ ID NO: 2, and more preferably, it may include the base sequence shown in SEQ ID NO: 2 (NCBI (Genbank) Accession No. NM_001330751.2).

In the present invention, polynucleotides encoding the proteins of interest for expression are amino acids of the protein expressed from the coding region due to codon degeneracy or in consideration of the preferred codon in the organism in which the protein is to be expressed. Various modifications may be made to the coding region within the scope of not changing the sequence, and various modifications may be made to parts other than the coding region within the scope of not affecting the expression of the gene, and such modified genes are also within the scope of the present invention. Those skilled in the art will be able to understand well that it is included in . That is, as long as the polynucleotide of the present invention encodes a protein with equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included within the scope of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of metabolic liver disease comprising a stem cell culture overexpressing PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) as an active ingredient.

Additionally, the present invention provides a food composition for preventing or improving metabolic liver disease, comprising a stem cell culture overexpressing PGC-1α as an active ingredient.

In the present invention, the 'stem cell' refers to a cell that has self-replication ability and the ability to differentiate into various types of specific cell types, as commonly known in the art, and the type is not particularly limited. , for example, preferably at least one selected from the group consisting of mesenchymal stem cells, adult stem cells, pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, and adipose-derived stem cells (ASC). It may be. In the present invention, the mesenchymal stem cells can be used without limitation in the present invention as long as they are known in the art. Specifically, in the present invention, the mesenchymal stem cells are not particularly limited in the specific tissue from which they are derived, and for example, they may be derived from tissues selected from the group consisting of umbilical cord, umbilical cord blood, placenta, bone marrow, adipose tissue, muscle, amniotic fluid, and amniotic membrane. You can.

As used herein, the stem cells mean provided or used in a state separated from the individual in vitro. In one embodiment of the present invention, the stem cells may be used ex vivo.

In the present invention, the stem cells can be isolated and obtained according to methods known in the art. These known methods include, but are not limited to, density gradient fractionation (especially density differential centrifugation, etc.), immunoselection, layer separation culture, and differential adhesion separation. etc.

In the present invention, the 'stem cells overexpressing PGC-1α' may be obtained by transforming stem cells with a recombinant expression vector containing a polynucleotide encoding PGC-1α.

In a specific embodiment of the present invention, the 'stem cells overexpressing PGC-1α' are obtained by transforming stem cells with a recombinant expression vector containing a promoter and a polynucleotide encoding PGC-1α operably linked thereto. It may be something that can be obtained.

In the present invention, the recombinant expression vector containing the polynucleotide may use a variety of vectors known in the art, and may include a promoter, terminator, and enhancer depending on the type of cell to be transformed. Expression control sequences such as ), sequences for membrane targeting or secretion, etc. can be appropriately selected and combined in various ways depending on the purpose.

As used herein, “recombinant expression vector” refers to a vector capable of expressing a target protein or target nucleic acid (RNA) in a suitable host cell, and is a genetic construct containing essential regulatory elements operably linked to express the polynucleotide (gene) insert. It says sacrifice.

The type of the recombinant expression vector is not particularly limited as long as it is a vector used in transformation methods known in the art. For example, in the present invention, the vector may use one or more selected from the group consisting of plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors, but is not limited thereto. Suitable vectors include expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose.

The gene delivery method using a plasmid expression vector is a method of directly delivering plasmid DNA to mammalian cells, and is a method that can be used in humans approved by the FDA. Unlike viral vectors, plasmid DNA has the advantage of being able to be purified homogeneously. As a plasmid expression vector that can be used in the present invention, a mammalian expression plasmid known in the art can be used. For example, but not limited to this, pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91/08291), and pVL1392 (PharMingen) are representative examples. The plasmid expression vector may be used by methods known in the art, including but not limited to transient transfection, microinjection, transduction, cell fusion, and calcium phosphate precipitation. , liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, It can be introduced into target cells by a gene gun and other known methods for introducing DNA into cells.

In addition, as a method applicable to the present invention, the viral expression vector containing the above nucleic acid is not limited thereto, but includes, but is not limited to, a retrovirus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, and a vexinia virus. These include vaccinia virus vectors, herpes virus vectors, avipox virus vectors, and lenti virus vectors. The retroviral vector is designed so that all viral genes are removed or altered so that non-viral proteins are produced in cells infected by the viral vector. The main advantages of retroviral vectors for gene therapy are that they deliver large amounts of genes into replicating cells, accurately integrate the transferred genes into cellular DNA, and do not cause continuous infection after gene transfection. The retroviral vector certified by the FDA was manufactured using PA317 amphotropic retrovirus packaging cells. Non-retroviral vectors include adenovirus as mentioned above. The main advantage of adenovirus is that it carries large amounts of DNA fragments (36 kb genome) and has the ability to infect non-replicating cells at very high titers. Additionally, the herpes virus can also be usefully used for human gene therapy. In addition, known suitable viral vectors can be used in the present invention.

The term 'promoter' refers to a DNA sequence that regulates the expression of an operably linked nucleic acid sequence in a specific host cell, and 'operably linked' means that one nucleic acid fragment is linked to another nucleic acid fragment. It means that its function or expression is affected by other nucleic acid fragments. In other words, it refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA to perform a general function. In other words, it means that the nucleic acid sequence encoding a protein or RNA is linked in a way that enables gene expression by an expression control sequence. For example, a promoter and a nucleic acid sequence encoding a protein or RNA must be operably linked to encode the protein. may affect the expression of nucleic acid sequences. Operational linkage with a recombinant vector can be made using genetic recombination techniques well known in the art, and site-specific DNA cutting and ligation can be done using enzymes generally known in the art.

In addition, in the present invention, the recombinant expression vector may further include any operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. As the promoter, a promoter that consistently induces expression of the target gene at all times (constitutive promoter) or a promoter that induces expression of the target gene at a specific location and time (inducible promoter) can be used. Examples include the SV40 promoter. , CMV promoter, CAG promoter, CaMV 35S promoter, Rsyn7 promoter (US Patent Application No. 08/991,601), rice actin promoter, ubiquitin promoter, ALS promoter (US Patent Application No. 08/409,297), etc. In addition, US Patent Nos. 5,608,149; All of the promoters disclosed in Nos. 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, and 5,608,142 can be used.

In the present invention, the vector may contain an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, It may include, but is not limited to, a resistance gene for one or more substances selected from the group consisting of neomycin, puromycin, and tetracycline.

In the present invention, the term 'transformation' or 'transfection' refers to the process of introducing a foreign nucleotide sequence into a cell. As a transformation method, known transfection methods can be used, for example, transfection, microinjection method (Capecchi, M.R., Cell 22, 479 (1980)), calcium phosphate precipitation method (Graham, F.L. et al., Virology 52, 456 (1973)), precipitation with polyethylenglycol, electroporation (Neumann, E. et al., EMBO J. 1, 841 (1982)), liposome-mediated Transfection method (Wong, T.K. et al., Gene, 10, 87 (1980)), DEAE-dextran treatment method (Gopal, Mol. Cell Biol. 5, 1188-1190 (1985)), particle gun bombardment method (particle gun bombardment) and gene bombardment (Yang et al., Proc. Natl. Acad. Sci. USA 87, 9568-9572 (1990)), but are not limited thereto. Additionally, in the present invention, transformation includes transduction. Methods for transducing genes into cells may include biochemical methods, physical methods, or virus-mediated transduction methods. Additionally, as a biochemical method, FuGene6 (Roche, USA), LipofectamineTM 2000, Invitrogen, USA, or ExGen 500 (MBI Fermentas International Inc. CANADA) can be used. Additionally, a lipid-mediated method using lipofectamine can be used.

In the present invention, the step of selecting transformed cells can be easily performed using the phenotype expressed by the selection marker of the vector described above. For example, when the selection marker is a specific antibiotic resistance gene, transformed cells can be easily selected by culturing the transformant in a medium containing the antibiotic.

In the present invention, the culture is characterized in that it contains a secretome secreted from stem cells overexpressing PGC-1α. The present inventors first discovered that the secretome secreted from stem cells overexpressing PGC-1α has a significant effect on preventing, improving, and/or treating metabolic liver disease. The present inventors believe that the culture containing the secretome unique to the present invention is involved in sugar (glucose) metabolism, lipid and lipoprotein metabolism, etc. and is a key pharmacological target for the development of agonists in metabolic diseases. It has been confirmed that it has a significant effect in restoring PPAR-alpha levels to normal levels. In addition, the present inventors found that the culture containing the secretome was significantly effective in reducing steatosis, inflammation, ballooning, fibrosis, and/or cirrhosis. was confirmed, and it was confirmed to have excellent therapeutic effects on non-alcoholic fatty liver disease. In one embodiment of the present invention, the effect of the secretome secreted from the stem cells overexpressing PGC-1α or the stem cell culture overexpressing PGC-1α on preventing, improving, or/and treating metabolic liver disease is increased. It is an effect (synergy effect). In the present invention, the term "synergy effect" means that the effect produced when each component is administered together is greater than the sum of the effects produced when each component is administered alone as a single component, as described in existing literature. do. In the present invention, each of the components refers to PGC-1α alone and stem cell secretome (stem cell culture) alone.

In the present invention, the term 'secretome' is generally a collection of molecules secreted by stem cells or present on the surface, and refers to the general term for stem cell secretions. The secretome may contain bioactive peptides such as cytokines, chemokines, growth factors, etc., and is included in the culture medium through functions such as autocrine, paracrine, and endocrine. Can affect other cells.

In the present invention, the term 'culture' refers to a product obtained after culturing the stem cells according to a known stem cell culture method, etc., and its specific form, such as solid or liquid, is not particularly limited. In one embodiment of the present invention, the culture may be used in the form of a liquid culture medium.

In one embodiment of the present invention, the culture may preferably not contain stem cells overexpressing PGC-1α. That is, in the present invention, the culture may be a conditioned medium in which stem cells overexpressing PGC-1α are cultured. Herein, the conditioned medium refers to a conventional basic medium known in the art for growing stem cells overexpressing PGC-1α (preferably a medium used for mammalian cell culture, more preferably a medium known in the art). It contains the secretome secreted by the stem cells by culturing them in a typical stem cell culture medium), meaning that the stem cells themselves overexpressing PGC-1α are removed and only the medium is collected.

In the present invention, the 'basic medium' is a mixture containing essential sugars, amino acids, water, etc. necessary for cells to live. The basic medium of the present invention can be artificially synthesized and manufactured or a commercially manufactured medium can be used. there is. Commercially prepared media include, for example, DMEM (Dulbecco's Modified Eagle's Medium), DMEM with low glucose concentration, MEM (Minimal Essential Medium), MEM-alpha medium, BME (Basal Medium Eagle), IMEM, K-SFM, EGM- 2, RPMI 1640, F-10, F-12, Ham's F-12 medium, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), McCoys 5A medium, Eagle's basal medium, CMRL medium , Glasgow minimum essential medium, Liebovitz' L-15 medium, KSB-3 basal medium, and Iscove's Modified Dulbecco's Medium, but are not limited thereto. Additionally, the culture medium of the present invention may additionally include a nutrient mixture. The nutrient mixture is a mixture containing various amino acids, vitamins, mineral salts, etc. commonly used in cell culture. It can be prepared by mixing the amino acids, vitamins, mineral salts, etc., or a commercially prepared nutrient mixture can be used. Commercially produced nutrient mixtures include, for example, F12, M199, MCDB110, MCDB131, MCDB202, MCDB302, etc. Preferably, F12 Nutrient Mixture, M199, MCDB media, etc. can be used.

In the present invention, the type of medium is not particularly limited as long as it is a medium known in the art as a medium used for mammalian cell culture, more preferably a typical stem cell culture medium known in the art, but for example, DMEM It may be selected from the group consisting of medium, M199/F12 medium, MEM-alpha medium, DMEM medium containing low concentration glucose, MCDB131 medium, IMEM medium, K-SFM medium, DMEM/F12 medium, and EGM-2 medium. In the present invention, the media may preferably be used in a serum-free state (i.e., serum-free, serum-free).

In the present invention, the culture may be prepared by a production method comprising the following steps:

(a) transforming isolated stem cells with a vector containing a polynucleotide encoding PGC-1α; and

(b) culturing the transformed stem cells in a serum-free medium to obtain (collect) a culture medium of the stem cells.

Collection of the culture medium can be performed by methods known in the art, for example, by centrifugation, but is not limited thereto.

In the culture preparation method of the present invention, an additional concentration (high concentration) step may be performed in order to contain a high concentration of the secretome secreted by stem cells overexpressing PGC-1α. In the present invention, the concentration means removing moisture (water) in the culture. The concentrated culture solution can be divided and stored frozen, and if necessary, it can be thawed and diluted to the desired concentration. The concentration can be performed by methods known in the art, for example, ultrafiltration, vacuum evaporation, ion chromatography, size exclusion chromatography, etc., but is not limited thereto. . The concentration can be used by those skilled in the art by adjusting the concentration temperature and concentration method as needed.

As used herein, the term 'ultrafiltration' refers to a membrane-based separation process that separates along a semi-permeable membrane by a pressure or concentration gradient. Ultrafiltration membranes have pore sizes with a constant cutoff value. In one embodiment of the present invention, the ultrafiltration used in the present invention may have a cut-off value of 0.001 to 100 kDa, and more specifically, a cut-off value of 0.01 to 50 kDa. It may have the following cutoff values: 0.01 kDa, 0.018 kDa, 0.02 kDa, 0.03 kDa, 0.04 kDa, 0.05 kDa, 0.06 kDa, 0.07 kDa, 0.08 kDa, 0.09 kDa, 0.1 kDa, 0.15 kDa, 0.2 kDa, 0.25 kDa, 0.3 kDa, 0.35 kDa, 0.4 kDa, 0.45 kDa, 0.5 kDa, 0.55 kDa, 0.6 kDa, 0.65 kDa, 0.7 kDa, 0.75 kDa, 0.8 kDa, 0.85 kDa, 0.9 kDa, 0.95 kDa, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa , 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, 31 kDa, 32 kDa, 33 kDa, 34 kDa, 35 kDa, 36 kDa, 37 kDa, 38 kDa, 39 kDa, 40 kDa, 41 kDa, 42 kDa, 43 kDa, 44 kDa, 45 kDa, 46 kDa, 47 kDa, 48 kDa, 49 kDa, 50 kDa. Preferably, the ultrafiltration used in the present invention may be characterized as having a cut-off value of 0.01 to 20 kDa. More preferably, the ultrafiltration used in the present invention may be characterized as having a cut-off value of 0.01 to 10 kDa. In one embodiment of the present application, the present inventors have performed ultrafiltration with a molecular weight cut-off value of 3 kDa, for example.

In this specification, various aspects or features of the invention may be presented in range format. In this specification, the description of a range value includes the corresponding boundary value, that is, it includes all values above the lower limit and below the upper limit, unless otherwise specified. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, statements of ranges should be considered as specifically disclosing not only the individual numerical values within the range, but also all possible subranges. For example, the statement of a range such as 1 to 6 refers to the individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6, as well as 1 to 3, 1 to 4, Subranges such as 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. should be considered as specifically disclosed. This applies regardless of the width of the scope.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating metabolic liver disease containing stem cells overexpressing PGC-1α as an active ingredient. As previously mentioned, in the present invention, stem cells overexpressing PGC-1α secrete a secretome that is effective in preventing, improving, and/or treating metabolic liver disease, and thus can be used in individuals for the purpose of treating the disease. It can be transplanted. In one embodiment of the present invention, the effect of stem cells overexpressing PGC-1α on preventing, improving, or/and treating metabolic liver disease is a synergistic effect.

In one embodiment of the present invention, the stem cells overexpressing PGC-1α in the composition may be used in combination with a substance that increases the expression or activity of PGC-1α. In this specification, the term 'increasing agent' is meant to include all terms commonly used in the art, such as increaser, enhancer, synergist, and activator.

The stem cell culture overexpressing PGC-1α (secretome secreted from stem cells overexpressing PGC-1α) or stem cells overexpressing PGC-1α according to the present invention are prepared by methods known in the art until use. It can be stored by, for example, ultra-low temperature refrigeration (freezing). In addition, the frozen (freezing) storage can be subjected to cryoprotection treatment before freezing (freezing), and a person skilled in the art can use it by optimizing other conditions, such as the specific type of cryoprotectant and treatment concentration, depending on the purpose.

The term 'prevention' used in the present invention refers to any action that suppresses or delays the onset of the disease subject to the present invention by preemptively administering the pharmaceutical composition according to the present invention before the onset of the disease targeted by the present invention (metabolic liver disease). means action.

The term 'treatment' used in the present invention means improvement or beneficial change in the symptoms of the disease subject to the present invention by administration of the pharmaceutical composition according to the present invention after the onset of the disease subject to the present invention (in the present invention, metabolic liver disease). It means all actions that occur. In the present invention, the term 'improvement' refers to any action that reduces parameters related to the disease of interest in the present invention, for example, the degree of symptoms.

In the present invention, 'individual' refers to a subject in need of treatment for a disease, and more specifically, a human or non-human primate, mammal, mouse, rat, dog, cat, horse, and It may be a mammal such as a cow, but is not limited thereto.

The pharmaceutical composition according to the present invention may contain only the above active ingredients in a pharmaceutically effective amount, or may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The carrier includes all types of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.

The excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention can be prepared as powder, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, ellipsis, emulsions, suspensions, spirits, troches, perfumes, and limonadese according to conventional methods. , tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta preparations, sprays, inhalants, patches, sterilized injection solutions, or aerosols, and the external preparations include creams, gels, patches, sprays, ointments, and warning agents. , it may have a dosage form such as lotion, liniment, pasta, or cataplasma.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. These include phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition according to the present invention may be formulated as a preparation for parenteral administration. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As a sterilized aqueous solution, an appropriate buffer solution such as Hank's solution, Ringer's solution, or physically buffered saline can be used. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, olive oil, and Vegetable oils, such as injectable esters such as ethyl oleate, etc. can be used. If necessary, preservatives, stabilizers, wetting or emulsifying agents, salts and/or buffering agents for adjusting osmotic pressure may be used. Meanwhile, in the case of suppositories, common bases such as witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Additives to tablets, powders, granules, capsules, pills, and troches according to the present invention include corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, and phosphoric acid. Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl. Excipients such as cellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin. , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. binders can be used, Hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicic anhydride, 1-hydroxy Propylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cocoa fat, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, 6000, liquid paraffin, hydrogenated soybean oil (Lubri) wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, Macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, Lubricants such as sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.

Additives to the liquid according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

A solution of white sugar, other sugars, or sweeteners may be used in the syrup according to the present invention, and, if necessary, flavoring agents, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc. may be used.

Purified water can be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as needed.

The suspension according to the present invention may include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, and HPMC 2910. Surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV solution, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV solution, ethanol, propylene glycol, non-volatile oil - sesame oil. solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristic acid, and benzene benzoate; Solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nicotinic acid amide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and buffering agents such as gums; Isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; Sulfurizing agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; Analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; It may contain suspending agents such as CM sodium, sodium alginate, Tween 80, and aluminum monostearate.

Suppositories according to the present invention include cacao oil, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, lecithin, Lanet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75(S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Massaupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppositories type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), Tegestor triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention. The route of administration may be oral or parenteral. All modes of administration may be expected of those skilled in the art, including, but not limited to, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration. It can be administered by mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the "pharmaceutically effective amount" means an amount sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, and activity of the patient's disease. , sensitivity to the drug, administration time, administration route and excretion rate, patient's age, weight, health status, gender, treatment period, factors including concurrently used drugs, and other factors well known in the medical field can be determined by a person skilled in the art. there is. For example, when administered to a human patient, the total daily usage amount of the composition according to the present invention can be determined by treatment within the scope of medical judgment. The specific therapeutically effective amount for a specific patient depends on the type and degree of response to be achieved, whether other agents are used as the case may be, the patient's age, weight, general health, gender, diet, administration time, administration route, and composition. It is desirable to apply it differently depending on various factors including secretion rate, treatment period, and similar factors well known in the medical field.

When using the culture of the present invention as a food additive, the culture can be added as is or used together with other food compositions or food ingredients, and can be appropriately used according to conventional methods known in the food industry. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, the culture medium may be added in an amount of 15% by weight or less, or 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There are no special restrictions on the types of foods above. Examples of foods to which the active ingredient of the present invention (culture of the present invention) can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. It includes dairy products, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and includes all health functional foods in the conventional sense.

The health drink composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional drinks. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may contain carbonating agents used in carbonated drinks. In addition, the composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

Below, preferred examples and experimental examples are presented to aid understanding of the present invention. However, the following examples and experimental examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples and experimental examples.

Example 1. Production of PGC-1α overexpressing stem cells and obtaining PCM of the present invention

1-1. cell culture

Human adipose-derived stem cells (ASC) were obtained from Hyulim Biocell Co., Ltd. and used in the present invention. ASCs were grown in low-glucose supplemented with 10% FBS (Thermo Fisher Scientic), 1% P/S (100 U/mL penicillin (Thermo Fisher Scientic) and 0.1 mg/mL streptomycin (Thermo Fisher Scientic)). -glucose) was plated on a culture flask using DMEM medium (Thermo Fisher Scientific) and cultured under conditions of 95% relative humidity, 37°C and 5% CO ₂ .

1-2. Obtaining secretome from stem cells overexpressing PGC-1α (obtaining PCM of the present invention)

The series of processes for producing PGC-1α (SEQ ID NO: 1) overexpressing stem cells and obtaining secretome therefrom are schematically shown in Figure 1.

First, a plasmid expressing PGC-1α (pcDNA4 mycPGC-1alpha plasmid) was purchased from addgene.

ASCs were cultured in 100 mm cell dishes (Corning Glass Works). After reaching 70-80% confluence, ASCs were transiently transformed with 1 μg of pcDNA4 mycPGC-1alpha plasmid using lipofectamine (invitrogen) according to the manufacturer's protocol. After 24 hours, 1.0 x 10 ⁶ ASCs were cultured in 5 mL serum-free low-glucose DMEM for 24 hours. Afterwards, the culture medium was centrifuged at 1000 rpm to remove ASC, and conditioned media was obtained. The conditioned medium was concentrated 25-fold using ultrafiltration units (Amicon Ultra-PL 3; Millipore) with a 3-kDa molecular weight cut-off to obtain 0.2 mL of secretome preparation. The secretome preparation thus obtained is referred to as 'PCM' of the present invention. Then, in subsequent experiments, 0.1 mL (100 μl) of secretome was injected per mouse. This means that the secretome obtained from 5 x 10 ⁵ ASC is injected into one mouse.

The secretome preparation obtained above was divided into small portions, stored in an ultra-low temperature freezer (frozen) in a deep freezer, and used in experiments.

Comparative Example 1. Production of NCM (normal secretome) from stem cells

A normal secretome (herein referred to as NCM) was obtained in the same manner as in Example 1-2 except for transforming ASC with an empty vector, and used as a comparative material in the experiment. did.

Example 2. Confirmation of therapeutic effect of PGC-1α overexpressing stem cell culture medium on metabolic liver disease

As a representative example of metabolic liver disease, the treatment effect on non-alcoholic steatohepatitis is demonstrated as follows.

2-1. Method for constructing an in vivo NASH (non-alcoholic steatohepatitis) disease mouse model and verifying the therapeutic effect of the present invention

A total of two types of NASH mouse models were produced: the MCD diet-NASH disease model and the HFHC diet-NASH disease model. An MCD diet-NASH mouse model was created by feeding MCD diet to 5-week-old C57BL/6J mice (about 20-25g) for 4 weeks. The HFHC Diet-NASH mouse model was created by feeding HFHC Diet to 5-week-old C57BL/6J mice (about 20-25g) for 8 weeks.

The NCM (normal secretome) prepared in Comparative Example 1 and the PCM (secretome from stem cells overexpressing PGC-1α) of the present invention prepared in Example 1-2 were administered to the NASH mouse model once every two weeks. It was administered intraperitoneally. 0.1 mL of secretome was injected per mouse.

As a negative control, 0.1 mL of cell culture medium (medium of Example 1-1) was administered to the NASH mouse model in the same manner, and as a normal control, normal C57BL/6J mice were administered cell culture medium (Example 1). -1 medium) 0.1 mL was administered in the same manner.

2-2. Confirmation of the effect of PCM on PPAR-alpha, a key factor in fat metabolism disease (MCD Diet-NASH model)

After administering the test substances to the MCD Diet-NASH model by the method described in Example 2-1 above, the mice were sacrificed to obtain liver tissue. To evaluate the effect on PPAR-alpha, a key factor in lipid and sugar metabolism, IHC was performed using the VECTOR VECTASTAIN ABC kit (PK-6102, PK-6101) and the VECTOR ImmPACT NovaRED Kit (SK-4805) according to the manufacturer's protocol. It was stained and analyzed under a light microscope.

Energy homeostasis refers to a precise balance between energy absorbed and energy expended. When this balance is broken due to excessive energy intake, it causes metabolic diseases such as obesity, arteriosclerosis, and type 2 diabetes. Over the years, PPAR (peroxisome proliferator-activated receptor), an orphan receptor, has been an important factor in controlling energy balance. It has been revealed that In particular, PPAR-alpha is involved in sugar (glucose) metabolism, lipid and lipoprotein metabolism, and is known to be a key pharmacological target for the development of agonists in metabolic diseases.

As shown in Figure 2, the experimental results showed that the PPAR-alpha level was very low in the negative control group, whereas in the PCM-administered group of the present invention, the PPAR-alpha level was significantly increased to a level almost similar to that of the normal control group. I was able to confirm. In the group injected with NCM, the PPAR-alpha level was shown to be slightly improved compared to the negative control group, but the effect was found to be significantly lower when compared to the PCM of the present invention.

2-3. Confirmation of the effect of PCM on PPAR-alpha, a key factor in fat metabolism disease (HFHC Diet-NASH model)

After administering the test substances to the HFHC Diet-NASH model by the method described in Example 2-1 above, the mice were sacrificed to obtain liver tissue. To evaluate the effect on PPAR-alpha, a key factor in lipid and sugar metabolism, IHC was performed using the VECTOR VECTASTAIN ABC kit (PK-6102, PK-6101) and the VECTOR ImmPACT NovaRED Kit (SK-4805) according to the manufacturer's protocol. It was stained and analyzed under a light microscope.

As shown in FIG. 3, the level of PPAR-alpha was confirmed to be very low in the negative control group, whereas the level of PPAR-alpha was significantly increased in the PCM-administered group of the present invention to a level almost similar to that of the normal control group. I was able to confirm. In the group injected with NCM, it was confirmed that the effect of improving PPAR-alpha levels was very low compared to the negative control group.

2-4. In fatty liver tissue of the MCD Diet-NASH model, the effect of PCM on damaged tissue was confirmed.

After administering the test substances to the MCD Diet-NASH model by the method described in Example 2-1 above, the mice were sacrificed to obtain liver tissue. H&E staining was performed on the liver tissue using a conventional method, and changes in the tissue were confirmed using an optical microscope.

As shown in FIG. 4, it was confirmed that serious tissue damage occurred in the negative control group, whereas in the PCM-administered group of the present invention, liver tissue damage was significantly improved to a level almost similar to that of the normal control group. . In the group injected with NCM, it was confirmed that the tissue improvement effect was very low compared to the negative control group.

2-5. In fatty liver tissue of the HFHC Diet-NASH model, the effect of PCM on damaged tissue was confirmed.

After administering the test substances to the HFHC Diet-NASH model by the method described in Example 2-1 above, the mice were sacrificed to obtain liver tissue. H&E staining was performed on the liver tissue using a conventional method, and changes in the tissue were confirmed using an optical microscope.

As shown in FIG. 5, it was confirmed that serious tissue damage occurred in the negative control group, whereas in the PCM-administered group of the present invention, liver tissue damage was significantly improved to a level almost similar to that of the normal control group. . In the group injected with NCM, it was confirmed that the tissue improvement effect was very low compared to the negative control group.

2-6. Determination of the effect of PCM on tissue fibrosis in fatty liver tissue of the MCD Diet-NASH model

After administering the test substances to the MCD Diet-NASH model by the method described in Example 2-1 above, the mice were sacrificed to obtain liver tissue. To analyze the fibrosis status of liver tissue, the tissue was stained with Sirius Red using the Picrosirius Red Stain Kit (Polyscience, #24901) according to the manufacturer's protocol and then analyzed under an optical microscope.

As shown in Figure 6, in the negative control group, it was confirmed that the degree of fibrosis and ballooning (hepatocyte ballooning) was very severe, whereas in the PCM-administered group of the present invention, the tissue was almost similar to the normal control group (normal). It was confirmed that fibrosis and ballooning (hepatocyte ballooning) were significantly reduced. In other words, it was confirmed that the PCM of the present invention has a significant therapeutic effect on fatty liver cirrhosis. In the group injected with NCM, it was confirmed that the effect of improving tissue fibrosis and ballooning (hepatocyte ballooning) was very low compared to the negative control group.

2-7. Confirmation of the effect of PCM on tissue fibrosis in fatty liver tissue of the HFHC Diet-NASH model

After administering the test substances to the HFHC Diet-NASH model by the method described in Example 2-1 above, the mice were sacrificed to obtain liver tissue. To analyze the fibrosis status of liver tissue, the tissue was stained with Sirius Red using the Picrosirius Red Stain Kit (Polyscience, #24901) according to the manufacturer's protocol and then analyzed under an optical microscope.

As shown in FIG. 7, it was confirmed that the negative control group had very severe fibrosis and ballooning, whereas in the PCM-administered group of the present invention, tissue tissue was almost similar to that of the normal control group (normal). It was confirmed that fibrosis and ballooning (hepatocyte ballooning) were significantly reduced. In other words, it was confirmed that the PCM of the present invention has a significant therapeutic effect on fatty liver cirrhosis. In the group injected with NCM, it was confirmed that the effect of improving tissue fibrosis and ballooning (hepatocyte ballooning) was very low compared to the negative control group.

2-8. In fatty liver tissue of the MCD Diet-NASH model, the effect of PCM on fibrosis-related collagen (Collagen I) was confirmed.

After administering the test substances to the MCD Diet-NASH model by the method described in Example 2-1 above, the mice were sacrificed to obtain liver tissue. To evaluate the effect on Collage I, a major factor related to tissue fibrosis, IHC staining was performed using the VECTOR VECTASTAIN ABC kit (PK-6102, PK-6101) and the VECTOR ImmPACT NovaRED Kit (SK-4805) according to the manufacturer's protocol. and analyzed with an optical microscope.

As shown in Figure 8, the level of Collagen I was confirmed to be significantly increased in the negative control group, while the level of Collagen I was significantly reduced in the PCM-administered group of the present invention to a level almost similar to that of the normal control group (normal). could be confirmed. In the group injected with NCM, Collagen I levels were found to be slightly reduced compared to the negative control group, but the effect was found to be significantly lower when compared to the PCM of the present invention.

2-9. In fatty liver tissue of the HFHC Diet-NASH model, the effect of PCM on fibrosis-related collagen (Collagen I) was confirmed.

After administering the test substances to the HFHC Diet-NASH model by the method described in Example 2-1 above, the mice were sacrificed to obtain liver tissue. To evaluate the effect on Collage I, a major factor related to tissue fibrosis, IHC staining was performed using the VECTOR VECTASTAIN ABC kit (PK-6102, PK-6101) and the VECTOR ImmPACT NovaRED Kit (SK-4805) according to the manufacturer's protocol. and analyzed with an optical microscope.

As shown in Figure 9, the level of Collagen I was confirmed to be significantly increased in the negative control group, while the level of Collagen I was significantly reduced in the PCM-administered group of the present invention to a level almost similar to that of the normal control group (normal). could be confirmed. In the group injected with NCM, Collagen I levels were found to be slightly reduced compared to the negative control group, but the effect was found to be significantly lower when compared to the PCM of the present invention.

Example 3. Confirmation of therapeutic effect of PGC-1α overexpressing stem cell culture medium in the progression and worsening of liver metabolic abnormalities

Nonalcoholic steatohepatitis subsequently progresses to liver cirrhosis (cirrhosis). Accordingly, the therapeutic effect of the PCM of the present invention on liver cirrhosis was confirmed.

First, an in vitro cirrhosis and liver fibrosis model was created by treating LX2 cells, which are human hepatic stellate cells (HSCs), with 5mM TAA (thioacetamide). LX2 human hepatic stellate cell line (donated from Professor Mira Cho's laboratory at Catholic University) was prepared in DMEM. maintained in medium. The cells were cultured under conditions of 37°C in a humidified chamber containing 5% CO ₂ .

An in vitro cirrhosis and liver fibrosis model was treated with NCM (normal secretome) and PCM (secretome from stem cells overexpressing PGC-1α) of the present invention for 48 hours. The NCM and the PCM of the present invention were obtained in the same manner as described in Comparative Example 1 and Example 1-2, except that 5 x 10 ⁴ ASC was used.

First, in order to confirm the antioxidant effect of the PCM of the present invention for each experimental group and control group, it was stained with mitoSOX and the effect on mitochondrial ROS (reactive oxygen species) was analyzed by FACS (Fluorescence-activated cell sorting). First, LX2 cells were cultured on Lab-Tek chamber slides (Thermo Fisher Scientific). Cells were treated with NCM and PCM, respectively, for 24 hours. Afterwards, LX2 cells were stained with 10 μM MitoSOX reagent at 37°C for 10 minutes. Mitochondrial ROS levels were determined using a fluorescence imaging system (EVOS U5000; Invitrogen). MitoSOX-based flow cytometry (FACS) was performed to quantify mitochondrial ROS levels in cells. After incubating the cells with MitoSOX reagent for 10 min in the dark at 25°C, the fluorescence intensity of the cells was analyzed using an Attune NxT acoustic focusing cytometer (Thermo fisher Scientific).

As shown in Figure 10, the experimental results confirmed that ROS, which was significantly increased in the in vitro liver cirrhosis and liver fibrosis model, was significantly suppressed by the PCM treatment of the present invention. However, in the NCM treatment group, the improvement effect was very minimal compared to the negative control group.

Then, for each experimental group and control group, MTT assay was performed using the EZ-Cytox Cell Proliferation Assay kit (Itsbio, Seoul, Republic of Korea) according to the manufacturer's protocol.

As shown in Figure 11, the experimental results confirmed that the proliferation and activation of hepatic stellate cells were significantly inhibited by the PCM treatment of the present invention in an in vitro liver cirrhosis and liver fibrosis model. As a result, it was verified that the PCM of the present invention prevents liver fibrosis and has a therapeutic effect on cirrhosis.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

Claims (12)

A pharmaceutical composition for the prevention or treatment of metabolic liver disease comprising a stem cell culture overexpressing PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) as an active ingredient.
The method of claim 1, wherein the metabolic liver disease is
Non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), hepatic steatosis, acute pregnancy A composition characterized in that it is at least one selected from the group consisting of Acute Fatty Liver of Pregnancy (AFLP), NAFLD-related liver failure, NAFLD-related liver fibrosis, and NAFLD-related cirrhosis.
The method of claim 1, wherein the stem cells overexpressing PGC-1α are
A composition obtained by transforming stem cells with a recombinant expression vector containing a polynucleotide encoding PGC-1α.
The method of claim 1, wherein the stem cells are
A composition comprising at least one selected from the group consisting of mesenchymal stem cells, adult stem cells, pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, and adipose-derived stem cells (ASC). .
The method of claim 1, wherein the culture
A composition comprising a secretome secreted from the stem cells.
The composition according to claim 1, wherein the culture is prepared by a production method comprising the following steps:
(a) transforming isolated stem cells with a vector containing a polynucleotide encoding PGC-1α; and
(b) culturing the transformed stem cells in a serum-free medium to obtain a culture medium of the stem cells.
The method of claim 1, wherein the culture
A composition characterized in that it does not contain stem cells overexpressing PGC-1α.
A food composition for preventing or improving metabolic liver disease, comprising a stem cell culture overexpressing PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) as an active ingredient.
A pharmaceutical composition for the prevention or treatment of metabolic liver disease containing stem cells overexpressing PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) as an active ingredient.
The method of claim 9, wherein the metabolic liver disease is
Non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), hepatic steatosis, acute pregnancy Characterized by at least one selected from the group consisting of fatty liver (Acute Fatty Liver of Pregnancy (AFLP)), NAFLD-related liver failure, NAFLD-related liver fibrosis, and NAFLD-related liver cirrhosis composition.
The method of claim 9, wherein the stem cells overexpressing PGC-1α are
A composition obtained by transforming stem cells with a recombinant expression vector containing a polynucleotide encoding PGC-1α.
The method of claim 9, wherein the stem cells are
A composition comprising at least one selected from the group consisting of mesenchymal stem cells, adult stem cells, pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, and adipose-derived stem cells (ASC). .