WO2023140524A1 - Antifibrotic inhibitory composition - Google Patents

Antifibrotic inhibitory composition Download PDF

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WO2023140524A1
WO2023140524A1 PCT/KR2022/021433 KR2022021433W WO2023140524A1 WO 2023140524 A1 WO2023140524 A1 WO 2023140524A1 KR 2022021433 W KR2022021433 W KR 2022021433W WO 2023140524 A1 WO2023140524 A1 WO 2023140524A1
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mir150
present
stem cells
transformed
cell culture
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PCT/KR2022/021433
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French (fr)
Korean (ko)
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김세준
김기환
김옥희
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가톨릭대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/204—Animal extracts
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a composition comprising a peptide derived from a stem cell culture medium of stem cells transformed with miRNA and a use thereof.
  • Liver fibrosis is a precursor to liver cirrhosis and is initiated by the action of various cytokines and growth factors as a result of severe liver damage that causes chronic liver disease.
  • liver fibrosis is reversible and consists of thin fibrils, and when there is no nodule formation and the cause of liver damage is temporary, normal recovery is possible due to the breakdown of extracellular matrix (ECM) increased by apoptosis and matrix metalloproteinases (MMP).
  • ECM extracellular matrix
  • MMP matrix metalloproteinases
  • liver cirrhosis is induced through the process of liver fibrosis in which abnormal extracellular matrix proteins, including collagen, are accumulated due to liver cell damage due to various inflammatory factors, it is important to control the accumulation of extracellular matrix to control the expression of liver cirrhosis.
  • liver cirrhosis is histologically caused by excessive accumulation of connective tissue in the liver tissue due to an imbalance in the metabolic process of collagen production and decomposition, resulting in liver fibrosis, accompanied by necrosis or inflammation. Since liver fibrosis and cirrhosis do not show pain or subjective symptoms in the early stages and are discovered only in the late stages, their treatment is not easy, and the mortality rate is high, causing social problems.
  • Liver fibrosis is a bioadaptive reaction that occurs in the process of repairing tissue damage, but inevitably deteriorates liver function in that the liver is replaced with fibrous tissue that cannot perform its own functions such as metabolism and bile secretion.
  • liver fibrosis When liver fibrosis is continuously repeated, it develops into cirrhosis and leads to death. Therefore, the development of an appropriate therapeutic agent has been performed as an important task in drug development. However, until now, since the mechanism itself of liver fibrosis has not been clearly identified, appropriate therapeutic drugs have not been developed.
  • miRNAs have been extensively studied and reported in various liver diseases including liver fibrosis and liver cirrhosis, and miRNAs are known to regulate various processes of liver fibrosis.
  • HSCs hepatic stellate cells
  • miRNAs plays an important role in liver disease treatment, studies on liver disease treatment using miRNA are insufficient.
  • stem cells are cells that have the ability to differentiate into various types of cells constituting the body, and recently, technologies for directly using stem cells as a treatment for liver diseases are being actively researched. These stem cells are largely classified into embryonic stem cells and adult stem cells. Embryonic stem cells have excellent differentiation ability, but have ethical problems and difficult to obtain in large quantities, and adult stem cells can obtain a large number of cells. However, there is a disadvantage in that the risk of infection or differentiation ability is relatively low when transplanted to others, and a lot of cost is required to collect and maintain stem cells.
  • the present inventors discovered peptides having anti-fibrotic function from a secretome containing substances secreted by adipose-derived stem cells and studied the effect of treating liver diseases. As a result, it was confirmed that peptides derived from adipose stem cell cultures transformed with miRNAs related to liver cirrhosis treatment had an inhibitory effect on liver fibrosis, thereby completing the present invention.
  • An object of the present invention is to provide a pharmaceutical composition for preventing or treating liver fibrosis comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
  • Another object of the present invention is to provide a method for preparing a stem cell culture transformed with miR150, comprising the following steps:
  • Another object of the present invention is to provide a method for separating and purifying PTX3 protein from a culture of stem cells transformed with miR150, comprising the following steps:
  • Another object of the present invention is to provide a food composition for improving liver fibrosis comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis, comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
  • the stem cell culture transformed with miR150 may contain PTX3 protein, but is not limited thereto.
  • the PTX3 protein may be isolated from a stem cell culture transformed with miR150, but is not limited thereto.
  • the PTX3 protein may include any one or more of the following characteristics, but is not limited thereto:
  • a 36 amino acid His Tag is fused to the N-terminus.
  • the stem cells may be adipose-derived stem cells (ADSC), but are not limited thereto.
  • ADSC adipose-derived stem cells
  • the liver fibrosis may be caused by liver toxin, but is not limited thereto.
  • the liver toxin may be thioacetamide, but is not limited thereto.
  • the PTX3 protein or the culture may increase the proliferation of hepatocytes and decrease the proliferation of hepatic stellate cells, but is not limited thereto.
  • HGF Hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • TRIP1 metallopeptidase inhibitor 1
  • ⁇ -SMA alpha-smooth muscle actin
  • MMP-2 matrix metalloproteinase-2
  • collagen I collagen I
  • MMP matrix metalloproteinase
  • the present invention provides a method for preparing a stem cell culture transformed with miR150 comprising the following steps:
  • the present invention provides a method for separating and purifying PTX3 protein from a culture of stem cells transformed with miR150, comprising the following steps:
  • the stem cells may be adipose-derived stem cells (ADSC), but are not limited thereto.
  • ADSC adipose-derived stem cells
  • the present invention provides a food composition for improving liver fibrosis comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
  • the food may be a health functional food, but is not limited thereto.
  • the present invention provides a method for preventing, improving or treating liver fibrosis, including a method of administering a stem cell culture transformed with PTX3 protein or miR150, or a composition containing at least one of the protein or the culture as an active ingredient to a subject in need thereof.
  • the present invention provides a stem cell culture transformed with PTX3 protein or miR150, or a composition comprising at least one of the protein or the culture as an active ingredient to prevent, improve, or treat liver fibrosis.
  • the present invention provides the use of a stem cell culture transformed with PTX3 protein or miR150, or a composition containing at least one of the protein or the culture as an active ingredient for preparing a preventive, ameliorative or therapeutic agent for liver fibrosis.
  • the present invention provides the use of miR150 or adipose-derived stem cells for preparing PTX3, a stem cell culture medium-derived peptide, or stem cell culture medium.
  • the pharmaceutical composition for preventing or treating liver fibrosis comprising the peptide derived from stem cell culture medium of stem cells transformed with miR150 of the present invention suppresses the expression of fibrotic factors that induce liver fibrosis and increases the expression of anti-fibrotic factors, thereby effectively inhibiting liver fibrosis, thereby effectively treating or preventing liver fibrosis. Therefore, the pharmaceutical composition can be applied to the field of liver fibrosis treatment, particularly clinical application technology.
  • a food composition containing peptides derived from stem cell culture medium of stem cells transformed with miR150 of the present invention can be used to improve liver fibrosis.
  • FIG. 1 shows the effect of the culture medium-derived peptide composition according to the present invention on increasing hepatocyte proliferation in a liver injury model.
  • Figure 2 shows the effect of reducing hepatic stellate cell proliferation in a hepatic stellate cell activation model of the culture medium-derived peptide composition according to the present invention.
  • Figure 3 shows the liver regeneration factor expression analysis of the culture medium-derived peptide composition according to the present invention.
  • Figure 4 shows the results of Western blot analysis of factors related to fibrosis of the culture medium-derived peptide composition according to the present invention in an animal model.
  • Figure 5 shows the results of RealtimePCR analysis of factors related to fibrosis of the culture medium-derived peptide composition according to the present invention in an animal model.
  • FIG. 6 shows the histological characteristics analysis results of the culture medium-derived peptide composition according to the present invention in an animal model.
  • the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis, comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
  • the stem cell culture transformed with miR150 may contain PTX3 protein, but is not limited thereto.
  • the PTX3 protein may be isolated from stem cell cultures transformed with miR150, but is not limited thereto.
  • the PTX3 protein may include any one or more of the following characteristics, but is not limited thereto:
  • a 36 amino acid His Tag is fused to the N-terminus.
  • miRNA is a single-stranded RNA (ssRNA) molecule with a length of 19 to 25 nucleotides and is generated by endogenous hairpin-shaped transcript. miRNAs complementarily bind to the 3' untranslated regions (UTRs) of target mRNAs, act as post-transcriptional gene suppressors, and suppress target genes by inducing translational repression and messengerRNA (mRNA) destabilization. miRNAs play important roles in various processes such as development, differentiation, proliferation, apoptosis and metabolism.
  • the miRNA may be synthesized into single-stranded RNA (shRNA, small interfering RNA) or double-stranded RNA (siRNA, small interfering RNA) having a hairpin structure composed of a loop and a stem by chemical synthesis, and a DNA sequence encoding the miRNA
  • shRNA small interfering RNA
  • siRNA small interfering RNA
  • a recombinant vector prepared by inserting into a virus or plasmid vector for expression may be provided in the form of a plasmid vector.
  • the expression vector includes a promoter efficient for siRNA expression.
  • the promoter is preferably recognized by RNA polymerase II or RNA polymerase III, more preferably recognized by RNA polymerase III, and most preferably a U6 promoter or H1 promoter.
  • siRNA expression vectors include pSilencer (Ambion, Inc.), pSiEx (Novagen, Inc.), siXpress (Takara Bio, Inc.), pBLOCK-iT TM (Invitrogen, Inc.), and SilenCircle TM (Allele), but are not limited thereto.
  • miR122, miR150, and miR214 according to the present invention were purchased from Samchully Pharmaceuticals and used as microRNAs exhibiting liver cirrhosis inhibitory effects.
  • the stem cells may be adipose-derived stem cells (ADSC), but are not limited thereto.
  • ADSC adipose-derived stem cells
  • stem cells refer to cells of various origins as pluripotent cells capable of differentiating into various tissue cells when appropriate conditions are met in an undifferentiated state.
  • the “stem cells” may be bone marrow-derived stem cells, cord blood-derived stem cells, or adipose-derived stem cells.
  • the bone marrow-derived stem cells, cord blood-derived stem cells, or adipose-derived stem cells may be human or animal-derived stem cells.
  • Adipose-derived stem cells are stem cells isolated from adipose tissue capable of differentiating into most mesenchymal cells, such as adipocytes, osteoblasts, chondroblasts, and myofibroblasts.
  • the adipose-derived stem cells may be derived from mammals including, but not limited to, pigs, cattle, primates, and humans that can be transplanted into humans. Meanwhile, since mesenchymal stem cells represent the typical adult stem cells, the adipose-derived stem cells in the present invention are generally used as adipose-derived mesenchymal stem cells.
  • Mesenchymal stem cells are routinely isolated from bone marrow (BM) aspirates and validated as pluripotent cell populations that can be induced to express fat, bone and cartilage markers.
  • the "stem cell marker” in the present invention is a substance that can discriminate between stem cells before differentiation proceeds and undifferentiated stem cells, and includes proteins or nucleic acids showing an increased or decreased pattern in stem cells that have initiated, progressed, or completed differentiation, organic biomolecules such as lipids, glycolipids, and glycoproteins.
  • CD90 and CD105 are surface markers of mesenchymal stem cells, and their expression is reduced when differentiation proceeds. Decreased expression of CD90 and CD105 may be a common feature for the differentiation of adipose-derived stem cells (ADSC) and mesenchymal stem cells (MSC).
  • ADSC adipose-derived stem cells
  • MSC mesenchymal stem cells
  • culture medium in the present invention refers to a substance containing components contained in a medium obtained by culturing stem cells.
  • the stem cell culture medium-derived peptide refers to a peptide with increased expression among peptides obtained by performing LC/MS on the culture medium of miR150-transformed adipose-derived stem cells.
  • the culture medium-derived peptide may be (Recombinant) PTX3.
  • conditioned medium refers to a medium in which only the culture medium is collected by replacing the cells with a serum-free medium when the cells reach the logarithmic growth phase, which is the peak of cell division, and then re-culturing. It means a medium containing growth factors, cytokines, etc.
  • secretome is generally a collection of molecules secreted from or present on the surface of stem cells, and refers to a generic term for stem cell secretions.
  • the secretome may contain bioactive peptides such as cytokines, chemokines, and growth factors, and may affect cells through autocrine, paracrine, and endocrine functions when included in the culture medium.
  • transformation is a generic term for changes in the genetic properties of an organism by an injected nucleic acid, that is, DNA or RNA
  • transgenic organism is a molecular genetic method. It may mean a living organism or material produced by injecting an external gene, but is not limited thereto.
  • the living organism may be included without limitation as long as it is a living organism such as a microorganism, eukaryotic cell, insect, animal, plant, etc., preferably E. coli, salmonella, bacillus, yeast, animal cells, mice, rats, dogs, monkeys, pigs, horses, cows, Agrobacterium tumefaciens, plants, etc., but is not limited thereto.
  • the material means any cell into which the nucleic acid can be injected, and in the present invention, it may be an adipose-derived stem cell, but is not limited thereto.
  • the transformants are transformation, transfection, Agrobacterium-mediated transformation method, particle gun bombardment, sonication, microinjection, electroporation, PEG (Polyethylen glycol)-mediated transformation method, viral infection method, direct muscle injection method, insulin It may be prepared by methods such as insulator and transposon, but is not limited thereto.
  • G-fectin, Mirus TrasIT-TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticles, cationic polymers, cationic micelles, cationic emulsions, or liposomes may be introduced into cells together with delivery reagents, or biocompatible polymers such as polyethylene glycol may be conjugated to increase uptake into cells, but is not limited thereto.
  • yeast Sacharomyce cerevisiae
  • insect cells e.g., human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells may be used, but are not limited thereto.
  • human cells e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines
  • plant cells may be used, but are not limited thereto.
  • the transfer method into the host cell when the host cell is a prokaryotic cell, may be carried out by the CaCl 2 method, the Hanhan method (Hanahan, D, J Mol Biol, 166:557-580 (1983)), and the electroporation method.
  • the vector when the host cell is a eukaryotic cell, the vector may be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment, but is not limited thereto.
  • a “primer” is a nucleic acid sequence having a short free 3-terminal hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying a template strand. It means a short nucleic acid sequence.
  • the primer may initiate DNA synthesis in the presence of a reagent (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphosphates (dNTPs) for polymerization at an appropriate buffer solution and temperature.
  • a reagent DNA polymerase or reverse transcriptase
  • dNTPs deoxynucleoside triphosphates
  • PCR Polymerase Chain Reaction
  • RT-PCR reverse transcription polymerase reaction
  • measurement of expression level means measuring the expression level of biological molecules such as polypeptide (or protein) or nucleic acid (or mRNA) that is increased or decreased in cells. In the present invention, it includes, but is not limited to, measuring the DNA, mRNA, or protein expression levels of BIS (or BAG3), SOX2, and SOX4 in glioblastoma.
  • the “mRNA expression level measurement” is a process of identifying the presence and expression of the mRNA of the desired genes.
  • the analysis method is the reverse transcriptional polymerase reaction (RT-PCR), the competitive reverse transcriptional polymerase reaction (Competitive RT-PCR), the real-time polymerase RT-PC R), RNase protection assay (RPA), Northern Blotting or DNA chip, but are not limited thereto.
  • the above “measurement of protein expression level” is a process of confirming the presence and expression level of a protein expressed from a gene of interest in a biological sample, preferably using an antibody that binds specifically to the protein of the gene of interest. Analysis methods for this include Western Blotting, ELISA (Enzyme Linked Immunosorbent Assay), Radioimmunoassay (RIA: Rodioimmunoassay), Radioimmunodiffusion, Ouchterolony immunodiffusion method, Rochet immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay (Complement Fixation Assay), flow cytometry (Fluorescence Activated Cell Sorter, FACS), protein chip (Protein Chip), etc., but are not limited thereto.
  • ELISA Enzyme Linked Immunosorbent Assay
  • Radioimmunoassay RIA: Rodioimmunoassay
  • Radioimmunodiffusion Ouchterolony
  • the liver fibrosis may be caused by liver toxins, but is not limited thereto.
  • Liver fibrosis refers to a phenomenon in which liver tissue damage and infiltration of inflammatory cells as a result of repeated liver disease activates hepatic stellate cells (HSCs), which secrete collagen, a major material for fibrosis, to form a thick fibrous septa in the liver.
  • HSCs hepatic stellate cells
  • Hepatic stellate cells (hereinafter referred to as HSCs) reside in the space called Disse between hepatocytes and sinusoidal endothelial cells during the quiescent period in the liver, and function to store 70% of retinol (vitamin A) required by the body and supply it as needed.
  • retinol vitamin A
  • Hepatocyte in the present invention is a parenchyma cell that functions in the liver, and damage and death of hepatocytes may activate hepatic stellate cells, which are cells that promote liver fibrosis, and abnormally accumulate fibrin, but is not limited thereto.
  • the peptide or the composition may increase the proliferation of hepatocytes in a liver injury model and decrease the proliferation of hepatic stellate cells that induce liver fibrosis, but is not limited thereto.
  • liver cirrhosis is a phenomenon in which liver fibrosis further progresses and liver function decreases.
  • the causes of liver cirrhosis include HBV, HCV, alcoholic liver disease, toxic liver disease, and the like, and when liver cirrhosis becomes more severe, liver cancer develops.
  • liver cirrhosis causes complications such as ascites (ADSCites), hepatitis, hepatic coma, and variceal bleeding.
  • Liver toxin in the present invention is a substance that causes damage or disease to liver tissue, and is any one selected from the group consisting of carbon tetrachloride (CCl 4 ), tert-butyl hydroperoxide (tBHP), acetaminophen, thioacetamide, tacrine, rubratoxin B, and hydrogen peroxide (H 2 O 2 ), but is not limited thereto.
  • the liver toxin may be thioacetamide, but is not limited thereto.
  • factors related to fibrosis may include all factors related to improvement or aggravation of fibrosis, which may be classified into fibrotic factors and anti-fibrotic factors.
  • fibrosis factor is a concept including a fibrosis-inducing factor, and at this time, the fibrosis-inducing factor may include all substances that induce liver fibrosis or increase when liver fibrosis is induced, and may include all factors related to growth, progression, exacerbation, and complications caused by fibrosis in addition to causing fibrosis.
  • anti-fibrotic factor may mean all factors that are markers of treatment, improvement, and alleviation of the induction and progression of fibrosis, aggravation of symptoms due to fibrosis, and occurrence of complications, and the like.
  • ⁇ -SMA is a protein encoded by the human ACTA2 gene and is a key component of the cell contraction system. Expression of ⁇ -SMA may be a key marker of hepatic stellate cell activity, and in the present invention, MMP-2 may play a role in promoting fibrosis.
  • the peptide of the present invention can reduce the expression of fibrotic factors and increase the expression of anti-fibrotic factors.
  • HGF Hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • TRIP1 metallopeptidase inhibitor 1
  • ⁇ -SMA alpha-smooth muscle actin
  • MMP-2 matrix metalloproteinase-2
  • collagen I collagen I
  • MMP matrix metalloproteinase
  • PTX3 used in one embodiment of the present invention may be represented by SEQ ID NO: 1, but is not limited thereto.
  • PTX3 of the present invention may include a variant of the above amino acid sequence.
  • the amino acid sequence of PTX3 represented by SEQ ID NO: 1 of the present invention may include an amino acid sequence having 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more sequence homology with the amino acid sequence.
  • percent identity can refer to, but are not limited to, a measure of the degree of similarity of two sequences based on an alignment of the sequences that maximizes similarity between aligned amino acid residues or nucleotides and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment.
  • the pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • the excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.
  • compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, solutions, eye drops, elsilic agents, emulsions, suspensions, spirits, troches, perfumes, limonadese, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, and soft drinks, respectively, according to conventional methods. It can be formulated and used in the form of external preparations such as extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, warning agents, lotions, pastes, sprays, inhalants, patches, sterilized injection solutions, or aerosols.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, and propylhydroxy. hydroxybenzoate, talc, magnesium stearate and mineral oil.
  • diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • Additives of the liquid formulation 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, carr boxymethylcellulose sodium and the like can be used.
  • a solution of white sugar, other sugars, or a sweetener may be used, and aromatics, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. may be used as necessary.
  • Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.
  • Acacia, tragacantha, methylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, HPMC 2910, etc. may be used in the suspension agent according to the present invention, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as necessary.
  • the injection according to the present invention includes injected distilled water, 0.9%sodium chloride liquid, ring gel injection, dextros+sodium chloride injection, fiji (PEG), lactated ring gel injection, ethanol, propylene glycol Sorghum oil, oleic acid, mirist isopropyl, and sabbatic hyangsan benzene; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twins, nijuntinamide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, and gums; tonicity agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO 3
  • the suppository according to the present invention includes cacao butter, lanolin, witapsol, 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, twin or span, Imhausen, monolen (monosteol) Propylene Glycol Phosphate), Glycerin, Adeps Solidus, Buytyrum Tego-G, Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrocote (Hydr okote) 25, Hydrocote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the extract with at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like.
  • excipients for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like.
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc.
  • various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
  • composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level may be determined according to the type of patient's disease, severity, drug activity, drug sensitivity, administration time, administration route and discharge rate, treatment period, factors including concurrently used drugs, and other factors well known in the medical field.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention belongs.
  • the pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.
  • routes can be envisaged, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.
  • the pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient together with various related factors such as the disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient.
  • “individual” means a subject in need of treatment of a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow. It means.
  • administration means providing a given composition of the present invention to a subject by any suitable method.
  • prevention means any action that inhibits or delays the onset of a desired disease
  • treatment means any action that improves or beneficially changes a desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention
  • improvement means any action that reduces a parameter related to a desired disease, for example, the severity of symptoms, by administration of the composition according to the present invention.
  • the present invention provides a method for preparing a stem cell culture transformed with miR150 comprising the following steps:
  • the present invention provides a method for separating and purifying the PTX3 protein from a culture of stem cells transformed with miR150, comprising the following steps:
  • the present invention provides a food composition for improving liver fibrosis comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
  • food means a natural product or processed product containing one or more nutrients, preferably means a state that can be directly eaten through a certain degree of processing, and as a general meaning, health functional food, It means to include all beverages, food additives and beverage additives.
  • the term "acceptable salt in food science” includes salts derived from organic acids, inorganic acids, or bases acceptable in food science.
  • the food may be a health functional food, but is not limited thereto.
  • the "functional food” is the same term as food for special health use (FoSHU), and means a food with high medical and medical effects processed to efficiently display bioregulatory functions in addition to nutrient supply, and can be prepared as tablets, capsules, pills, granules, powders, liquids, flakes, pastes, syrups, gels, jellies, bars, or film formulations.
  • “functionality” means obtaining useful effects for health purposes, such as adjusting nutrients for the structure and function of the human body or physiological functions.
  • foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. It includes all foods designed to be sufficiently expressed in vivo.
  • food additives relate to substances used in food additives, mixing, infiltration, and other methods in manufacturing, processing, or preserving food, and should be harmless to the human body when taken for a long period of time, such as health functional foods.
  • the food additive may be added to the composition of the present invention as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.
  • the mixing amount of active ingredients may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).
  • the composition of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material.
  • the amount may be less than 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.
  • the composition may include various food supplements acceptable in food science, and may further include appropriate carriers, excipients, and diluents commonly used in food production.
  • the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like.
  • the composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not very important, but is generally selected from the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto, and is optimal according to the type and function of the product used.
  • composition according to the present invention may be added to health drinks, and may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional drinks.
  • natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • sweetener natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used.
  • the ratio of the natural carbohydrate may be generally about 0.01 to 0.20 g, or about 0.04 to 0.10 g per 100 mL of the composition of the present invention, but is not limited thereto, and may be a general amount added in the art, or may include the maximum range to improve the efficacy of the composition of the present invention, and may include an optimal, arbitrary amount in consideration of synergistic effects with other substances added together.
  • the peptide of the present invention or a composition containing it as an active ingredient When used as a food additive, it can be added as it is or used together with other foods or food ingredients, and can be appropriately used according to conventional methods.
  • the mixing amount of active ingredients may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).
  • the freshwater extract or the compound of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material.
  • the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.
  • Adipose-derived stem cell culture medium-derived peptides transformed with the mRNA of the present invention were prepared in the following manner.
  • adipose-derived stem cells were cultured in DMEM low glucose medium supplemented with 10% inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) at 37°C and 5% CO 2 conditions.
  • FBS inactivated fetal bovine serum
  • P/S penicillin-streptomycin
  • miR150 was transfected into adipose-derived stem cells using a transfection reagent (lipofectamine, invitrogen) according to the manufacturer's instructions, and then the adipose-derived stem cells were cultured for 24 hours.
  • a transfection reagent lipofectamine, invitrogen
  • the culture medium of miR150-transformed adipose-derived stem cells was exchanged with FBS-Free DMEM low glucose, and the culture medium of adipose-derived stem cells was secured 24 hours after the medium exchange.
  • the obtained culture medium was analyzed by performing LC/MS. Among the peptides analyzed by LC/MS, PTX3 with increased expression was identified.
  • PTX3 is a single unglycosylated polypeptide chain produced in (Recombinant Human) E. coli, containing 381 amino acids and having a molecular mass of 44.4 kDa.
  • PTX3 has a 36 amino acid His Tag fused to the N-terminus and has been purified by chromatography.
  • one having a purity of 90% or more by SDS-PAGE analysis was used, and the PTX3 protein (1 mg/ml) contained 20 mM Tris-HCl buffer pH 8.0, 1 mM DTT and 10% glycerol, and had the amino acid sequence of SEQ ID NO: 1.
  • PTX3 in the present invention was purchased from Mybiosource and used in the experiment.
  • Example 2 Confirmation of the effect of increasing hepatocyte proliferation by the culture medium-derived peptide according to the present invention in a liver injury model
  • TAA Thioacetic acid
  • a liver injury model was implemented by treating normal liver cell line AML12 with TAA, and it was confirmed that cell proliferation of hepatocytes, which was reduced when treated with PTX3, increased (FIG. 1).
  • Example 3 Confirmation of hepatic stellate cell proliferation reduction effect of the culture medium-derived peptide according to the present invention in hepatic stellate cell activation model
  • hepatic stellate cell lines were treated with 5 mM TAA to implement a hepatic stellate cell activation model.
  • Hepatic stellate cell line LX-2 was treated with TAA to implement a liver injury model, and it was confirmed that the cell proliferation of hepatic stellate cells, which was increased when treated with PTX3, was reduced (FIG. 2).
  • Example 4 Analysis of liver regeneration factor expression of the culture medium-derived peptide composition according to the present invention
  • the expression levels of HGF and VEGF which are liver regeneration factors, were analyzed at the RNA level and the expression levels were compared.
  • HGF and VEGF which are liver regeneration factors
  • a liver injury model was constructed by TAA treatment and then treated with PTX3 alone
  • the expression of liver regeneration factors HGF and VEGF, which were reduced when treated with TAA alone increased, and it was confirmed that the peptide composition derived from the culture medium of the present invention had remarkably excellent liver regeneration ability (FIG. 3).
  • Example 5 Analysis of factors related to fibrosis of the culture medium-derived peptide composition according to the present invention using an animal model
  • TAA was administered to BALB/C Nude mouse (in vivo) mice at 200 mg/kg 3 times a week for 6 weeks, and PTX3 was administered 3 times a week for 2 weeks, then the mouse was sacrificed to extract liver tissue, and the level of fibrosis change in liver tissue was confirmed through Western blot.
  • the experimental group was divided into a control group (Ct), a PTX3 treatment group (PTX3), a TAA treatment group (TAA), and a TAA+PTX3 treatment group (TAA+PTX3).
  • the expression of TIMP1 was also found to be reduced, but such an effect of reducing TIMP1 expression is considered to be due to the following reasons.
  • the expression of TIMP1 is increased to suppress MMP, and as a result of confirming at the mRNA level, it was confirmed that the expression of TIMP1 is increased.
  • the reason for the decrease in the expression of TIMP1 in animal tissues is that the expression of TIMP1, which is increased at the mRNA level, is judged to be reduced at the protein level because the expression of MMP has already been reduced at the time of mouse sacrifice.
  • the level of fibrosis change in the liver tissue was analyzed through the expression of fibrosis-related factors.
  • the experimental group was divided into a control group (Ct), PTX3 treatment group (rPTX3), TAA treatment group (TAA), and TAA + PTX3 treatment group (TAA + rPTX3), and the experiment was conducted.
  • the expression levels of Collagen I, MMP, and TIMP1, which are factors related to fibrosis, were analyzed through RealtimePCR.
  • TAA-treated hepatic stellate cells are the main producers of extracellular matrix, and when hepatic stellate cells are activated, they change into a myofibroblast-like form, thereby increasing the production of CollagenI, an extracellular matrix material.
  • the rPTX3 group exhibited almost the same level of CollagenI and MMP expression as the control group.
  • the expression level of the factor in the TAA group increased about 6-fold, whereas the expression of CollagenI and MMP in the TAA+rPTX3 group was significantly reduced to about 50% compared to the TAA group.
  • the expression of TIMP1 was higher than that of the TAA alone group, and according to the results, it was confirmed that this was because the degree of fibrosis during PTX3 treatment was lower than that of the TAA alone group (FIG. 5).
  • Example 6 Analysis of histological characteristics of the culture medium-derived peptide composition according to the present invention using an animal model
  • mice After administering TAA to BALB/C Nude mouse (in vivo) mice at 200 mg/kg 3 times a week for 6 weeks and then administering PTX3 3 times a week for 2 weeks, the mice were sacrificed to extract liver tissue, and hematoxylin & eosin (H&E) staining was performed to observe histological changes (FIG. 10).
  • H&E hematoxylin & eosin
  • the extracted tissue was fixed with 10% formalin containing 0.1M phosphate buffer (pH 7.2, Sigma). After embedding each tissue in paraffin, sections were prepared. The prepared paraffin sections (4 ⁇ m thick) were dewaxed, hydrated, treated with 0.01% protease XXIV (Sigma) in phosphate-buffered saline at 37° C. for 20 minutes, and stained with hematoxylin and eosin.
  • the tissue pattern of the TAA+rPTX group was almost similar to that of the non-treated group (control), and it was confirmed that tissue damage was reduced in the TAA+rPTX-treated group compared to the TAA-only treated group (FIG. 6).
  • the present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis containing a peptide derived from a stem cell culture medium of stem cells transformed with miR150 or a food composition for improving liver fibrosis containing a peptide derived from a stem cell culture medium of stem cells transformed with miR150.

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Abstract

The present invention relates to an antifibrotic inhibitory composition, and to: a pharmaceutical composition for preventing or treating liver fibrosis, comprising, as an active ingredient, a peptide derived from a stem cell culture solution of stem cells transformed with miR150; or a food composition for alleviating liver fibrosis, comprising a peptide derived from a stem cell culture solution of stem cells transformed with miR150. It has been identified that a composition of the present invention exhibits an excellent liver fibrosis inhibitory effect in in vitro and in vivo liver injury models, and thus is expected to be used in the field of liver fibrosis treatment, especially in clinical application technology.

Description

항섬유화 억제용 조성물Composition for inhibiting antifibrosis
본 발명은 miRNA로 형질전환된 줄기세포의 줄기세포 배양액 유래 펩타이드를 포함하는 조성물 및 이의 용도에 관한 것이다.The present invention relates to a composition comprising a peptide derived from a stem cell culture medium of stem cells transformed with miRNA and a use thereof.
본 출원은 2022년 1월 20일에 출원된 한국특허출원 제10-2022-0008369호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다. This application claims priority based on Korean Patent Application No. 10-2022-0008369 filed on January 20, 2022, and all contents disclosed in the specification and drawings of the application are incorporated into this application.
간 섬유화는 간경변증의 전구병변으로 만성 간질환을 일으키는 심한 간 손상의 결과로 여러 가지 사이토카인과 성장인자의 작용에 의해 시작된다.Liver fibrosis is a precursor to liver cirrhosis and is initiated by the action of various cytokines and growth factors as a result of severe liver damage that causes chronic liver disease.
일반적으로 간 섬유증은 가역적이고 얇은 섬유(fibril)로 구성되며 결절 형성이 없고 간 손상의 원인이 한시적인 경우에는 세포사멸(apoptosis) 과정과 매트릭스 메탈로프로테이나아제(matrix metalloproteinases, MMP)에 의해서 증가된 세포외기질(extracellular matrix, ECM)이 분해되어 정상 회복이 가능하나, 간 섬유증 과정이 반복적으로 지속되면 두터운 섬유(thick fibril)를 형성하고 결절이 있는 간경변으로 진행하게 된다. 또한, 다양한 염증 유발요인으로 인해 간세포가 손상되어 콜라겐을 포함한 비정상적인 세포외기질 단백질이 축적되는 간 섬유증의 과정을 통하여 간경변증이 유발되기 때문에 간경변증의 발현 조절을 위해서는 세포외기질의 축적을 조절하는 것이 중요하다. 한편, 간경화는 조직학적으로는 콜라겐을 생성하고 분해하는 대사과정의 균형이 깨져 간 섬유화(liver fibrosis)가 일어나 간 조직 내에 결합조직이 과도하게 축적되어 발생하며, 괴사나 염증이 동반된다. 간 섬유화 및 간경화는 초기 단계에서는 통증이나 자각증세가 나타나지 않고, 말기에서야 발견되기 때문에 그 치료가 쉽지 않으며, 사망률이 높아 사회적인 문제를 야기하고 있다.In general, liver fibrosis is reversible and consists of thin fibrils, and when there is no nodule formation and the cause of liver damage is temporary, normal recovery is possible due to the breakdown of extracellular matrix (ECM) increased by apoptosis and matrix metalloproteinases (MMP). will proceed to In addition, since liver cirrhosis is induced through the process of liver fibrosis in which abnormal extracellular matrix proteins, including collagen, are accumulated due to liver cell damage due to various inflammatory factors, it is important to control the accumulation of extracellular matrix to control the expression of liver cirrhosis. On the other hand, liver cirrhosis is histologically caused by excessive accumulation of connective tissue in the liver tissue due to an imbalance in the metabolic process of collagen production and decomposition, resulting in liver fibrosis, accompanied by necrosis or inflammation. Since liver fibrosis and cirrhosis do not show pain or subjective symptoms in the early stages and are discovered only in the late stages, their treatment is not easy, and the mortality rate is high, causing social problems.
간 섬유화는 조직손상의 복구 과정에서 발생하는 생체적응 반응이지만 생체 내 물질의 대사 및 담즙분비 등의 고유기능을 전혀 수행할 수 없는 섬유조직으로 간이 대체된다는 점에서 간 기능의 저하가 필연적으로 나타난다. 간 섬유화 현상이 지속적으로 반복될 때에는 간경화로 발전되어 사망에까지 이르게 한다는 점에서 적절한 치료제의 개발은 의약품 개발의 중요한 과제로 수행되어 왔다. 그러나 현재까지는 간 섬유화의 기전 자체가 명확하게 밝혀져 있지 않으므로 적당한 치료 약물이 개발되지 않은 실정이다.Liver fibrosis is a bioadaptive reaction that occurs in the process of repairing tissue damage, but inevitably deteriorates liver function in that the liver is replaced with fibrous tissue that cannot perform its own functions such as metabolism and bile secretion. When liver fibrosis is continuously repeated, it develops into cirrhosis and leads to death. Therefore, the development of an appropriate therapeutic agent has been performed as an important task in drug development. However, until now, since the mechanism itself of liver fibrosis has not been clearly identified, appropriate therapeutic drugs have not been developed.
간 섬유화 및 간경화를 포함하는 다양한 간질환에서 miRNA의 중요성이 광범위하게 연구되어 보고되고 있는데, miRNA는 간 섬유화의 다양한 과정을 조절하는 것으로 알려져 있다. 또한, miRNA에 의해 간경화의 핵심세포인 간 성상세포(HSC)의 활성화가 조절된다. 이와 같이 miRNA가 간 질환 치료 과정에서 중요한 역할을 차지함에도 불구하고, miRNA를 이용한 간 질환 치료에 대한 연구는 미흡한 상황이다.The importance of miRNAs has been extensively studied and reported in various liver diseases including liver fibrosis and liver cirrhosis, and miRNAs are known to regulate various processes of liver fibrosis. In addition, the activation of hepatic stellate cells (HSCs), a key cell of liver cirrhosis, is regulated by miRNAs. Although miRNA plays an important role in liver disease treatment, studies on liver disease treatment using miRNA are insufficient.
한편, 줄기세포는 신체를 구성하는 다양한 종류의 세포로 분화할 능력을 가진 세포로서, 최근에는 줄기세포를 간 질환에 대한 치료제로서 직접 사용하는 기술이 활발하게 연구되고 있는 중이다. 이러한 줄기세포는 크게 배아줄기세포 및 성체줄기세포로 분류는데, 배아줄기세포는 분화능력이 매우 뛰어나지만 윤리적인 문제 및 대량 획득이 어려운 단점이 있고, 성체줄기세포는 세포수를 많이 얻을 수 있으나 타인에게 이식할 때 감염의 위험이나 분화 능력이 상대적으로 부족한 단점이 있으며, 줄기세포의 채취 및 유지에도 많은 비용이 요구된다. 이러한 줄기세포 치료법의 단점을 극복하기 위해, 줄기세포의 치료 효과가 줄기세포의 파라크린(paracrine) 활성에 의한 것임에 착안하여 줄기세포 자체보다는 줄기세포가 분비하는 다양한 물질을 포함하는 배양액(secretome)의 치료 효능에 관한 연구가 활발하게 진행 중이다.On the other hand, stem cells are cells that have the ability to differentiate into various types of cells constituting the body, and recently, technologies for directly using stem cells as a treatment for liver diseases are being actively researched. These stem cells are largely classified into embryonic stem cells and adult stem cells. Embryonic stem cells have excellent differentiation ability, but have ethical problems and difficult to obtain in large quantities, and adult stem cells can obtain a large number of cells. However, there is a disadvantage in that the risk of infection or differentiation ability is relatively low when transplanted to others, and a lot of cost is required to collect and maintain stem cells. In order to overcome the disadvantages of stem cell therapy, studies on the therapeutic efficacy of a secretome containing various substances secreted by stem cells rather than stem cells themselves are being actively conducted, noting that the therapeutic effect of stem cells is due to the paracrine activity of stem cells.
이에, 본 발명자들은 상기와 같은 줄기세포 치료법의 단점을 극복하기 위해, 지방유래 줄기세포가 분비하는 물질을 포함하는 배양액(secretome)으로부터 항섬유화 기능을 지닌 펩타이드를 발굴하여 간 질환 치료 효과에 대하여 연구한 결과, 간 경변 치료에 관련된 miRNA를 형질전환시킨 지방줄기세포 배양액으로부터 유래한 펩타이드가 간 섬유화 억제 효과가 있음을 확인하여 본 발명을 완성하였다.Therefore, in order to overcome the above disadvantages of stem cell therapy, the present inventors discovered peptides having anti-fibrotic function from a secretome containing substances secreted by adipose-derived stem cells and studied the effect of treating liver diseases. As a result, it was confirmed that peptides derived from adipose stem cell cultures transformed with miRNAs related to liver cirrhosis treatment had an inhibitory effect on liver fibrosis, thereby completing the present invention.
본 발명의 목적은 PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물을 유효성분으로 포함하는 간 섬유증의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.An object of the present invention is to provide a pharmaceutical composition for preventing or treating liver fibrosis comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
본 발명의 다른 목적은 하기 단계를 포함하는 miR150으로 형질전환된 줄기세포 배양물 제조방법을 제공하는 것이다:Another object of the present invention is to provide a method for preparing a stem cell culture transformed with miR150, comprising the following steps:
(a) miR150을 줄기세포에 형질전환시키는 단계; 및(a) transforming miR150 into stem cells; and
(b) 상기 형질전환된 줄기세포를 무혈청 배지에서 배양하여 배양물을 획득하는 단계.(b) culturing the transformed stem cells in a serum-free medium to obtain a culture product.
본 발명의 또 다른 목적은 하기 단계를 포함하는 miR150으로 형질전환된 줄기세포의 배양물로부터 PTX3 단백질을 분리 정제하는 방법을 제공하는 것이다:Another object of the present invention is to provide a method for separating and purifying PTX3 protein from a culture of stem cells transformed with miR150, comprising the following steps:
miR150으로 줄기세포를 형질전환하는 단계; 및 transforming stem cells with miR150; and
형질전환된 줄기세포의 배양물에서 PTX3 단백질을 분리 정제하는 단계.Separating and purifying the PTX3 protein from the transformed stem cell culture.
본 발명의 또 다른 목적은 PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물을 유효성분으로 포함하는 간 섬유증 개선용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for improving liver fibrosis comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당해 기술분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
본 발명은 PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물을 유효성분으로 포함하는 간 섬유증의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating liver fibrosis, comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
본 발명의 일 실시예에 있어서, 상기 miR150으로 형질전환된 줄기세포 배양물은 PTX3 단백질을 포함할 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the stem cell culture transformed with miR150 may contain PTX3 protein, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 PTX3 단백질은 miR150으로 형질전환된 줄기세포 배양물에서 분리된 것일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the PTX3 protein may be isolated from a stem cell culture transformed with miR150, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 PTX3 단백질은 하기 중 어느 하나 이상의 특징을 포함할 수 있으나, 이에 제한되는 것은 아니다:In one embodiment of the present invention, the PTX3 protein may include any one or more of the following characteristics, but is not limited thereto:
서열번호 1로 이루어짐; consisting of SEQ ID NO: 1;
44.4kDA의 분자량; 및molecular weight of 44.4 kDA; and
N-말단에 36 아미노산 His Tag이 융합됨. A 36 amino acid His Tag is fused to the N-terminus.
본 발명의 일 실시예에 있어서, 상기 줄기세포는 지방유래 줄기세포 (Adipose-derived stem cell; ADSC)일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the stem cells may be adipose-derived stem cells (ADSC), but are not limited thereto.
본 발명의 일 실시예에 있어서, 상기 간 섬유증은 간 독소에 의해 유발될 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the liver fibrosis may be caused by liver toxin, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 간 독소는 티오아세트아마이드 (thioacetamide)일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the liver toxin may be thioacetamide, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 PTX3 단백질 또는 상기 배양물은 간세포의 증식을 증가시키고, 간 성상세포의 증식을 감소시킬 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the PTX3 protein or the culture may increase the proliferation of hepatocytes and decrease the proliferation of hepatic stellate cells, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 PTX3 단백질 또는 상기 배양물은In one embodiment of the present invention, the PTX3 protein or the culture
하기 (a)로 이루어진 군으로부터 선택되는 어느 하나 이상의 발현을 증가시키거나;To increase the expression of any one or more selected from the group consisting of (a);
하기 (b)로 이루어진 군으로부터 선택되는 어느 하나 이상의 발현을 감소시킬 수 있으나, 이에 제한되는 것은 아니다:The expression of one or more selected from the group consisting of the following (b) may be reduced, but is not limited thereto:
(a) HGF (Hepatocyte growth factor), VEGF (vascular endothelial growth factor), 및 TIMP1 (metallopeptidase inhibitor 1); 및(a) Hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and metallopeptidase inhibitor 1 (TIMP1); and
(b) α-SMA (alpha-smooth muscle actin), MMP-2 (matrix metalloproteinase-2), CollagenⅠ, 및 MMP.(b) α-SMA (alpha-smooth muscle actin), MMP-2 (matrix metalloproteinase-2), collagen I, and MMP.
본 발명은 하기 단계를 포함하는 miR150으로 형질전환된 줄기세포 배양물 제조방법을 제공한다:The present invention provides a method for preparing a stem cell culture transformed with miR150 comprising the following steps:
(a) miR150을 줄기세포에 형질전환시키는 단계; 및(a) transforming miR150 into stem cells; and
(b) 상기 형질전환된 줄기세포를 무혈청 배지에서 배양하여 배양물을 획득하는 단계.(b) culturing the transformed stem cells in a serum-free medium to obtain a culture product.
본 발명은 하기 단계를 포함하는 miR150으로 형질전환된 줄기세포의 배양물로부터 PTX3 단백질을 분리 정제하는 방법을 제공한다:The present invention provides a method for separating and purifying PTX3 protein from a culture of stem cells transformed with miR150, comprising the following steps:
miR150으로 줄기세포를 형질전환하는 단계; 및 transforming stem cells with miR150; and
형질전환된 줄기세포의 배양물에서 PTX3 단백질을 분리 정제하는 단계.Separating and purifying the PTX3 protein from the transformed stem cell culture.
본 발명의 일 실시예에 있어서, 상기 줄기세포는 지방유래 줄기세포 (Adipose-derived stem cell; ADSC)일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the stem cells may be adipose-derived stem cells (ADSC), but are not limited thereto.
본 발명은 PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물을 유효성분으로 포함하는 간 섬유증 개선용 식품 조성물을 제공한다.The present invention provides a food composition for improving liver fibrosis comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
본 발명의 일 실시예에 있어서, 상기 식품은 건강기능식품일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the food may be a health functional food, but is not limited thereto.
또한, 본 발명은 PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물, 또는 상기 단백질 또는 상기 배양물 중 어느 하나 이상을 유효성분으로 포함하는 조성물을 이를 필요로 하는 개체에게 투여하는 방법을 포함하는 간 섬유증의 예방, 개선 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing, improving or treating liver fibrosis, including a method of administering a stem cell culture transformed with PTX3 protein or miR150, or a composition containing at least one of the protein or the culture as an active ingredient to a subject in need thereof.
또한, 본 발명은 PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물, 또는 상기 단백질 또는 상기 배양물 중 어느 하나 이상을 유효성분으로 포함하는 조성물의 간 섬유증의 예방, 개선 또는 치료 용도를 제공한다.In addition, the present invention provides a stem cell culture transformed with PTX3 protein or miR150, or a composition comprising at least one of the protein or the culture as an active ingredient to prevent, improve, or treat liver fibrosis.
또한, 본 발명은 간 섬유증의 예방, 개선 또는 치료제를 제조하기 위한 PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물, 또는 상기 단백질 또는 상기 배양물 중 어느 하나 이상을 유효성분으로 포함하는 조성물의 용도를 제공한다.In addition, the present invention provides the use of a stem cell culture transformed with PTX3 protein or miR150, or a composition containing at least one of the protein or the culture as an active ingredient for preparing a preventive, ameliorative or therapeutic agent for liver fibrosis.
또한, 본 발명은 줄기세포 배양액 유래 펩타이드인 PTX3, 또는 줄기세포 배양액을 제조하기 위한 miR150 또는 지방유래 줄기세포의 용도를 제공한다.In addition, the present invention provides the use of miR150 or adipose-derived stem cells for preparing PTX3, a stem cell culture medium-derived peptide, or stem cell culture medium.
본 발명의 miR150로 형질전환된 줄기세포의 줄기세포 배양액 유래 펩타이드를 포함하는 간 섬유증의 예방 또는 치료용 약학적 조성물은 간 섬유증를 유발시키는 섬유화 인자의 발현을 억제하고 항섬유화 인자의 발현을 증가시킴으로써 간 섬유화를 효과적으로 억제할 수 있어 간 섬유증을 효과적으로 치료 또는 예방할 수 있다. 따라서, 상기 약학적 조성물은 간 섬유화 치료 분야, 특히 임상적 적용 기술로 응용될 수 있다. 또한, 본 발명의 miR150로 형질전환된 줄기세포의 줄기세포 배양액 유래 펩타이드를 포함하는 식품 조성물은 간 섬유증의 개선에 사용될 수 있다.The pharmaceutical composition for preventing or treating liver fibrosis comprising the peptide derived from stem cell culture medium of stem cells transformed with miR150 of the present invention suppresses the expression of fibrotic factors that induce liver fibrosis and increases the expression of anti-fibrotic factors, thereby effectively inhibiting liver fibrosis, thereby effectively treating or preventing liver fibrosis. Therefore, the pharmaceutical composition can be applied to the field of liver fibrosis treatment, particularly clinical application technology. In addition, a food composition containing peptides derived from stem cell culture medium of stem cells transformed with miR150 of the present invention can be used to improve liver fibrosis.
도 1은 본 발명에 따른 배양액 유래 펩타이드 조성물의 간손상 모델에서의 간세포 증식 증가 효과를 나타낸 것이다.1 shows the effect of the culture medium-derived peptide composition according to the present invention on increasing hepatocyte proliferation in a liver injury model.
도 2는 본 발명에 따른 배양액 유래 펩타이드 조성물의 간 성상세포 활성화 모델에서의 간 성상세포 증식 감소 효과를 나타낸 것이다.Figure 2 shows the effect of reducing hepatic stellate cell proliferation in a hepatic stellate cell activation model of the culture medium-derived peptide composition according to the present invention.
도 3은 본 발명에 따른 배양액 유래 펩타이드 조성물의 간 재생인자 발현 분석을 나타낸 것이다.Figure 3 shows the liver regeneration factor expression analysis of the culture medium-derived peptide composition according to the present invention.
도 4는 동물 모델에서 본 발명에 따른 배양액 유래 펩타이드 조성물의 섬유화와 관련된 인자의 Western blot 분석 결과를 나타낸 것이다.Figure 4 shows the results of Western blot analysis of factors related to fibrosis of the culture medium-derived peptide composition according to the present invention in an animal model.
도 5는 동물 모델에서 본 발명에 따른 배양액 유래 펩타이드 조성물의 섬유화와 관련된 인자의 RealtimePCR 분석 결과를 나타낸 것이다.Figure 5 shows the results of RealtimePCR analysis of factors related to fibrosis of the culture medium-derived peptide composition according to the present invention in an animal model.
도 6은 동물 모델에서 본 발명에 따른 배양액 유래 펩타이드 조성물의 조직학적 특징 분석 결과를 나타낸 것이다.6 shows the histological characteristics analysis results of the culture medium-derived peptide composition according to the present invention in an animal model.
본 발명에서의 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 출원에서 개시된 다양한 요소들의 모든 조합이 본 출원의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 출원의 범주가 제한된다고 볼 수 없다.Each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below.
본 발명은 PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물을 유효성분으로 포함하는 간 섬유증의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating liver fibrosis, comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
본 발명의 일 실시예에 있어서, 상기 miR150으로 형질전환된 줄기세포 배양물은 PTX3 단백질을 포함할 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the stem cell culture transformed with miR150 may contain PTX3 protein, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 PTX3 단백질은 miR150으로 형질전환된 줄기세포 배양물에서 분리될 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the PTX3 protein may be isolated from stem cell cultures transformed with miR150, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 PTX3 단백질은 하기 중 어느 하나 이상의 특징을 포함할 수 있으나, 이에 제한되는 것은 아니다:In one embodiment of the present invention, the PTX3 protein may include any one or more of the following characteristics, but is not limited thereto:
서열번호 1로 이루어짐; consisting of SEQ ID NO: 1;
44.4kDA의 분자량; 및molecular weight of 44.4 kDA; and
N-말단에 36 아미노산 His Tag이 융합됨. A 36 amino acid His Tag is fused to the N-terminus.
본 발명에서의 “miRNA (마이크로 RNA)”는 19 내지 25 뉴클레오타이드 (nucleotide) 길이의 단일 가닥 RNA (single strand RNA, ssRNA) 분자로서 내재적 (endogenous) 헤어핀-구조 전사체 (hairpin-shaped transcript)에 의해 생성된다. miRNA는 표적 mRNA의 3’ 비번역 영역 (UTRs)에 상보적으로 결합하여 전사 후 유전자 억압자 (post-transcriptional gene suppressor)로서 작용하며, 번역 억제와 메신저RNA (mRNA) 불안정화를 유도함으로써 표적 유전자를 억제한다. miRNA는 발달 (development), 분화, 증식, 세포사멸 및 신진대사 (metabolism)과 같은 다양한 과정에서 중요한 역할을 한다. 상기 miRNA는 화학합성에 의해 루프 및 스템으로 구성된 헤어핀 구조의 단일가닥 RNA (shRNA, short hairpin RNA) 또는 이중가닥 RNA (siRNA, small intefering RNA)로 합성될 수 있으며, 상기 miRNA를 코드하는 DNA 서열을 siRNA 발현용 바이러스 또는 플라스미드 벡터에 삽입하여 제조한 재조합 벡터로 플라스미드 벡터의 형태로 제공될 수 있다. 상기 발현벡터는 siRNA 발현에 효율적인 프로모터를 포함하는데, 상기 프로모터는 RNA polymerase II 또는 RNA polymerase III에 의해 인식되는 것이 바람직하며, 특히 RNA polymerase III에 의해 인식되는 것이 더욱 바람직하며, U6 프로모터 또는 H1 프로모터인 것이 가장 바람직하다. 상기와 같은 siRNA 발현벡터는 pSilencer (Ambion, Inc.), pSiEx (Novagen, Inc.), siXpress (Takara Bio, Inc.) 및 pBLOCK-iTTM (Invitrogen, Inc.), 및 SilenCircleTM (Allele) 등을 사용할 수 있으나, 이에 제한되는 것은 아니다. 특히, 본 발명에 따른 miR122, miR150 및 miR214는 간경화 억제 효과를 나타내는 마이크로 RNA로서 Samchully Pharmaceutical사로부터 구입하여 사용하였다.In the present invention, “miRNA (micro RNA)” is a single-stranded RNA (ssRNA) molecule with a length of 19 to 25 nucleotides and is generated by endogenous hairpin-shaped transcript. miRNAs complementarily bind to the 3' untranslated regions (UTRs) of target mRNAs, act as post-transcriptional gene suppressors, and suppress target genes by inducing translational repression and messengerRNA (mRNA) destabilization. miRNAs play important roles in various processes such as development, differentiation, proliferation, apoptosis and metabolism. The miRNA may be synthesized into single-stranded RNA (shRNA, small interfering RNA) or double-stranded RNA (siRNA, small interfering RNA) having a hairpin structure composed of a loop and a stem by chemical synthesis, and a DNA sequence encoding the miRNA A recombinant vector prepared by inserting into a virus or plasmid vector for expression may be provided in the form of a plasmid vector. The expression vector includes a promoter efficient for siRNA expression. The promoter is preferably recognized by RNA polymerase II or RNA polymerase III, more preferably recognized by RNA polymerase III, and most preferably a U6 promoter or H1 promoter. Such siRNA expression vectors include pSilencer (Ambion, Inc.), pSiEx (Novagen, Inc.), siXpress (Takara Bio, Inc.), pBLOCK-iT TM (Invitrogen, Inc.), and SilenCircle TM (Allele), but are not limited thereto. In particular, miR122, miR150, and miR214 according to the present invention were purchased from Samchully Pharmaceuticals and used as microRNAs exhibiting liver cirrhosis inhibitory effects.
본 발명의 일 실시예에 있어서, 상기 줄기세포는 지방유래 줄기세포 (Adipose-derived stem cell; ADSC)일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the stem cells may be adipose-derived stem cells (ADSC), but are not limited thereto.
본 발명에서의 “줄기세포 (stem cells)”는 미분화 상태에서 적절한 조건을 맞춰주면 다양한 조직 세포로 분화될 수 있는 만능세포로서 다양한 유래의 세포를 의미한다. 바람직하게, 상기 “줄기세포”는 골수유래줄기세포, 제대혈유래줄기세포 또는 지방유래줄기세포일 수 있다. 또한, 상기 골수유래줄기세포, 제대혈유래줄기세포 또는 지방유래줄기세포는 인체 또는 동물유래 줄기세포인 것일 수 있다. In the present invention, “stem cells” refer to cells of various origins as pluripotent cells capable of differentiating into various tissue cells when appropriate conditions are met in an undifferentiated state. Preferably, the “stem cells” may be bone marrow-derived stem cells, cord blood-derived stem cells, or adipose-derived stem cells. In addition, the bone marrow-derived stem cells, cord blood-derived stem cells, or adipose-derived stem cells may be human or animal-derived stem cells.
본 발명에서의 “지방유래 줄기세포”는 지방세포, 골모세포, 연골모세포, 근섬유모세포 등 대부분의 중간엽 세포로 분화할 수 있는 지방조직으로부터 분리된 줄기세포로서, 지방전구세포, 기질세포, 다분화능 지방유래 세포 (multipotent adipose-derived cells) 또는 지방유래 성체줄기세포 (adipose derived adult stem cells) 등으로 불려온 세포를 의미한다. 상기 지방유래 줄기세포는 특별히 이에 제한되지는 않으나, 인간에게 이식할 수 있는 돼지, 소, 영장류 및 인간 등을 포함하는 포유류 유래일 수 있다. 한편, 중간엽 줄기세포는 성체 줄기세포의 전형을 나타내므로, 본 발명에서의 지방유래 줄기세포는 통상적으로 지방-유래 중간엽 줄기세포 (Adipose-derived Mesenchymal Stem Cell)로 사용된다. 중간엽 줄기세포 (MSC)는 골수 (Bone Marrow, BM) 흡인물로부터 통상적으로 분리되고, 다능성 세포군으로 검증되어 지방, 골 및 연골 마커를 발현하도록 유도될 수 있다."Adipose-derived stem cells" in the present invention are stem cells isolated from adipose tissue capable of differentiating into most mesenchymal cells, such as adipocytes, osteoblasts, chondroblasts, and myofibroblasts. The adipose-derived stem cells may be derived from mammals including, but not limited to, pigs, cattle, primates, and humans that can be transplanted into humans. Meanwhile, since mesenchymal stem cells represent the typical adult stem cells, the adipose-derived stem cells in the present invention are generally used as adipose-derived mesenchymal stem cells. Mesenchymal stem cells (MSCs) are routinely isolated from bone marrow (BM) aspirates and validated as pluripotent cell populations that can be induced to express fat, bone and cartilage markers.
본 발명에서의 “줄기세포 마커”는 분화가 진행되기 전의 줄기세포와 미분화된 줄기세포를 구분하여 판별할 수 있는 물질로서, 분화가 개시, 진행 또는 완료된 줄기세포에서 증가 또는 감소 양상을 보이는 단백질 또는 핵산, 지질, 당지질, 당단백질 등과 같은 유기 생체 분자 등을 포함한다.The "stem cell marker" in the present invention is a substance that can discriminate between stem cells before differentiation proceeds and undifferentiated stem cells, and includes proteins or nucleic acids showing an increased or decreased pattern in stem cells that have initiated, progressed, or completed differentiation, organic biomolecules such as lipids, glycolipids, and glycoproteins.
본 발명에서의 “CD90” 및 “CD105”는 중간엽 줄기세포의 표면 마커로서 분화가 진행되면 이의 발현이 감소되는 특징을 나타낸다. CD90 및 CD105의 발현 감소는 지방유래 줄기세포 (ADSC), 및 중간엽 줄기세포 (MSC)의 분화에 대한 일반적인 특징이 될 수 있다.In the present invention, "CD90" and "CD105" are surface markers of mesenchymal stem cells, and their expression is reduced when differentiation proceeds. Decreased expression of CD90 and CD105 may be a common feature for the differentiation of adipose-derived stem cells (ADSC) and mesenchymal stem cells (MSC).
본 발명에서의 “배양액”이란 줄기세포를 배양하여 얻은 배지에 포함된 구성 성분을 포함하는 물질을 의미한다. 또한, 본 발명에서의 줄기세포 배양액 유래 펩타이드는 miR150이 형질전환된 지방유래 줄기세포의 배양배지에 대하여 LC/MS를 수행하여 확보한 펩타이드 중에서 발현이 증가한 펩타이드를 의미한다. 바람직하게, 상기 배양액 유래 펩타이드는 (Recombinant) PTX3 일 수 있다.The term "culture medium" in the present invention refers to a substance containing components contained in a medium obtained by culturing stem cells. In addition, in the present invention, the stem cell culture medium-derived peptide refers to a peptide with increased expression among peptides obtained by performing LC/MS on the culture medium of miR150-transformed adipose-derived stem cells. Preferably, the culture medium-derived peptide may be (Recombinant) PTX3.
본 발명에서의 “조정 배지 (conditioned medium, CM)”는 세포를 세포분열 최성기인 대수성장기에 도달했을 때 무혈청 배지로 교환한 후 재배양하여 배양액만 수거한 배지를 의미하는 것으로, 분열 중인 세포로부터 배지 내에서 추출되어 나오는 성장 인자, 사이토카인 등을 포함하는 배지를 의미한다.In the present invention, "conditioned medium (CM)" refers to a medium in which only the culture medium is collected by replacing the cells with a serum-free medium when the cells reach the logarithmic growth phase, which is the peak of cell division, and then re-culturing. It means a medium containing growth factors, cytokines, etc.
본 발명에서의 “세크리톰 (secretome)”은 일반적으로 줄기세포에서 분비되거나 표면에 존재하는 분자의 집합으로써, 줄기세포 분비물의 총칭을 의미한다. 세크리톰은 사이토카인, 케모카인, 성장인자 등과 같은 생물활성 펩타이드 (bioactive peptide)를 포함할 수 있으며, 배양액에 포함되어 자가분비 (autocrine), 측분비(paracrine), 내분비 (endocrine) 등의 기능을 통해 세포에 영향을 줄 수 있다.In the present invention, "secretome" is generally a collection of molecules secreted from or present on the surface of stem cells, and refers to a generic term for stem cell secretions. The secretome may contain bioactive peptides such as cytokines, chemokines, and growth factors, and may affect cells through autocrine, paracrine, and endocrine functions when included in the culture medium.
본 발명에서, “형질전환 (transformation)”이란 주입된 핵산, 즉, DNA 또는 RNA에 의하여 생물의 유전적인 성질이 변하는 것을 총칭하며, “형질전환체 (transgenic organism)”란 분자유전학적 방법으로 외부의 유전자를 주입하여 제조된 생명체 또는 물질을 의미할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, "transformation" is a generic term for changes in the genetic properties of an organism by an injected nucleic acid, that is, DNA or RNA, and "transgenic organism" is a molecular genetic method. It may mean a living organism or material produced by injecting an external gene, but is not limited thereto.
특정 경우에 따라 상기 생명체는 미생물, 진핵세포, 곤충, 동물, 식물 등 생명이 있는 생물이라면 제한이 없이 포함될 수도 있고, 바람직하게는 대장균, 살모넬라, 바실러스, 효모, 동물 세포, 마우스, 래트, 개, 원숭이, 돼지, 말, 소, 아그로박테리움 튜머패시언스, 식물 등이나, 이에 제한되지 않는다. 또한, 물질로는 상기 핵산이 주입될 수 있는 모든 세포를 의미하고, 본 발명에서는 지방유래줄기세포일 수 있으나, 이에 제한되는 것은 아니다.According to a specific case, the living organism may be included without limitation as long as it is a living organism such as a microorganism, eukaryotic cell, insect, animal, plant, etc., preferably E. coli, salmonella, bacillus, yeast, animal cells, mice, rats, dogs, monkeys, pigs, horses, cows, Agrobacterium tumefaciens, plants, etc., but is not limited thereto. In addition, the material means any cell into which the nucleic acid can be injected, and in the present invention, it may be an adipose-derived stem cell, but is not limited thereto.
상기 형질전환체는 형질전환 (transformation), 형질감염 (transfection), 아그로박테리움(Agrobacterium)-매개 형질전환 방법, 입자 총 충격법 (particle gun bombardment), 초음파 처리법 (sonication), 미세주입법 (microinjection), 전기천공법 (electroporation), PEG (Polyethylen glycol)-매개 형질전환 방법, 바이러스 감염법 (viral infection), 직접근육주입법 (direct muscle injection), 인슐레이터 (insulator) 및 트랜스포존 (trnasposon) 등의 방법으로 제조될 수 있으나, 이에 제한되지 않는다. 또한, 세포 내로 도입하는 방법으로, G-fectin, Mirus TrasIT-TKO 지질친화성 시약, 리포펙틴, 리포펙타민, 셀펙틴 (cellfectin), 양이온성 인지질 나노입자, 양이온성 고분자, 양이온성 미셀, 양이온성 에멀젼 또는 리포좀을 포함하는 전달시약과 함께 세포 내로 도입되거나, 폴리에틸렌글리콜과 같은 생체적합성 고분자를 접합하여 세포 내 흡수를 증가시킬 수 있으나, 이에 제한되지 않는다.The transformants are transformation, transfection, Agrobacterium-mediated transformation method, particle gun bombardment, sonication, microinjection, electroporation, PEG (Polyethylen glycol)-mediated transformation method, viral infection method, direct muscle injection method, insulin It may be prepared by methods such as insulator and transposon, but is not limited thereto. In addition, as a method of introducing into cells, G-fectin, Mirus TrasIT-TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticles, cationic polymers, cationic micelles, cationic emulsions, or liposomes may be introduced into cells together with delivery reagents, or biocompatible polymers such as polyethylene glycol may be conjugated to increase uptake into cells, but is not limited thereto.
진핵 세포에 형질 전환시키는 경우에는 숙주세포로서, 효모 (Saccharomyce cerevisiae), 곤충세포, 사람세포 (예컨대, CHO 세포주 (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN 및 MDCK 세포주) 및 식물세포 등이 이용 될 수 있으나, 이에 제한되는 것은 아니다.In the case of transformation into eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells may be used, but are not limited thereto.
숙주세포 내로 운반하는 방법은, 숙주 세포가 원핵 세포인 경우, CaCl2 방법, 하나한 방법 (Hanahan, D, J Mol Biol, 166:557-580 (1983)) 및 전기천공 방법 등에 의해 실시될 수 있다. 또한, 숙주세포가 진핵세포인 경우에는, 미세주입법, 칼슘포스페이트 침전법, 전기천공법, 리포좀-매개 형질감염법, DEAE-덱스트란 처리법, 및 유전자 밤바드먼트 등에 의해 벡터를 숙주세포 내로 주입할 수 있으나, 이에 제한되는 것은 아니다.The transfer method into the host cell, when the host cell is a prokaryotic cell, may be carried out by the CaCl 2 method, the Hanhan method (Hanahan, D, J Mol Biol, 166:557-580 (1983)), and the electroporation method. In addition, when the host cell is a eukaryotic cell, the vector may be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment, but is not limited thereto.
본 발명에서의 “프라이머 (primer)”는 짧은 자유 3 말단 수산화기를 갖는 핵산서열로 상보적인 주형 (template)과 염기쌍 (base pair)을 형성할 수 있고 주형 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응을 위한 시약 (DNA 중합효소 또는 역전사 효소) 및 상이한 4가지 dNTP (deoxynucleoside triphosphate)의 존재 하에서 DNA 합성을 개시할 수 있다.In the present invention, a “primer” is a nucleic acid sequence having a short free 3-terminal hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying a template strand. It means a short nucleic acid sequence. The primer may initiate DNA synthesis in the presence of a reagent (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphosphates (dNTPs) for polymerization at an appropriate buffer solution and temperature.
본 발명에서의 “중합효소연쇄반응 (PCR, Polymerase Chain Reaction)”은 아주 적은 양의 DNA 만으로도 특정 부위의 DNA 염기서열을 기하급수적으로 증폭시킬 수 있는 간단하고 편리한 방법으로, 증폭하고자 하는 DNA에 특이적으로 결합하여 고온에서도 안정한 Taq DNA 중합효소를 사용하여 변성 (denaturation), 어닐링 (annealing), 연장 (extension)의 과정을 반복적으로 실행함으로써 특정 DNA를 증폭시키게 된다. PCR을 이용한 목적 유전자의 검출은 유전자를 인식하는 프라이머를 이용하여 이를 증폭 및 확인하는 것이다."Polymerase Chain Reaction (PCR)" in the present invention is a simple and convenient method that can exponentially amplify the DNA sequence of a specific site with only a very small amount of DNA. Specific DNA is amplified by repeatedly executing the processes of denaturation, annealing, and extension using Taq DNA polymerase, which is stable at high temperatures by specifically binding to the DNA to be amplified. Detection of a gene of interest using PCR is to amplify and confirm the gene using primers that recognize the gene.
본 발명에서의 “역전사 중합효소 반응 (RT-PCR)”은 역전사효소를 이용하여 RNA를 상보적인 DNA (cDNA)로 역전사 시킨 후에 만들어진 cDNA를 주형 (template)으로 하여 타겟 프라이머와 표지를 포함하는 타겟 프로브를 이용해 타겟을 증폭하여 이를 검출해 내는 분자생물학적 방법을 의미한다.In the present invention, "reverse transcription polymerase reaction (RT-PCR)" refers to a molecular biological method of amplifying and detecting a target using a target probe containing a target primer and a label using cDNA as a template after reverse transcription of RNA into complementary DNA (cDNA) using reverse transcriptase.
본 발명에서의 “발현 수준 측정”은 세포에서 증가 또는 감소되는 폴리펩타이드 (또는 단백질) 또는 핵산 (또는 mRNA) 등과 같은 생체 분자의 발현 수준을 측정하는 것을 의미한다. 본 발명에서는, 교모세포종 내 BIS (또는 BAG3), SOX2 및 SOX4의 DNA, mRNA, 또는 단백질 발현 수준을 측정하는 것을 포함하나, 이에 제한되지는 않는다.In the present invention, "measurement of expression level" means measuring the expression level of biological molecules such as polypeptide (or protein) or nucleic acid (or mRNA) that is increased or decreased in cells. In the present invention, it includes, but is not limited to, measuring the DNA, mRNA, or protein expression levels of BIS (or BAG3), SOX2, and SOX4 in glioblastoma.
상기의 “mRNA 발현 수준 측정”은 목적하는 유전자의 mRNA 존재 여부와 발현 정도를 확인하는 과정으로서, 이를 위한 분석 방법으로는 역전사 중합효소 반응 (RT-PCR), 경쟁적 역전사 중합효소 반응 (Competitive RT-PCR), 실시간 역전사 중합효소 반응 (Real-time RT-PCR), RNase 보호 분석법 (RPA: RNase Protection Assay), 노던 블랏팅 (Northern Blotting) 또는 DNA 칩 등이 있으나, 이에 제한되지는 않는다.The “mRNA expression level measurement” is a process of identifying the presence and expression of the mRNA of the desired genes. For this purpose, the analysis method is the reverse transcriptional polymerase reaction (RT-PCR), the competitive reverse transcriptional polymerase reaction (Competitive RT-PCR), the real-time polymerase RT-PC R), RNase protection assay (RPA), Northern Blotting or DNA chip, but are not limited thereto.
상기의 “단백질 발현 수준 측정”은 생물학적 시료에서 목적하는 유전자로부터 발현된 단백질의 존재 여부와 발현 정도를 확인하는 과정으로, 바람직하게는 상기 목적하는 유전자의 단백질에 대하여 특이적으로 결합하는 항체를 이용하여 단백질의 양을 확인할 수 있다. 이를 위한 분석 방법으로는 웨스턴 블랏팅 (Western Blotting), ELISA (Enzyme Linked Immunosorbent Assay), 방사선면역분석 (RIA: Rodioimmunoassay), 방사 면역 확산법 (Radioimmunodiffusion), 오우크테로니 (Ouchterolony) 면역 확산법, 로케트 (rochet) 면역전기영동, 조직면역 염색, 면역 침전 분석법 (Immunoprecipitation Assay), 보체 고정 분석법 (Complement Fixation Assay), 유세포분석 (Fluorescence Activated Cell Sorter, FACS), 단백질 칩 (Protein Chip) 등이 있으나, 이에 제한되지는 않는다.The above “measurement of protein expression level” is a process of confirming the presence and expression level of a protein expressed from a gene of interest in a biological sample, preferably using an antibody that binds specifically to the protein of the gene of interest. Analysis methods for this include Western Blotting, ELISA (Enzyme Linked Immunosorbent Assay), Radioimmunoassay (RIA: Rodioimmunoassay), Radioimmunodiffusion, Ouchterolony immunodiffusion method, Rochet immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay (Complement Fixation Assay), flow cytometry (Fluorescence Activated Cell Sorter, FACS), protein chip (Protein Chip), etc., but are not limited thereto.
본 발명의 일 실시예에서, 상기 간 섬유증은 간 독소에 의해 유발될 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the liver fibrosis may be caused by liver toxins, but is not limited thereto.
본 발명에서의 “간 섬유증 (Liver fibrosis)”은 간질환이 거듭되어 간조직의 손상과 염증세포의 침윤에 의해 간 성상세포 (Hepatic stellate cell; 이하 HSC)가 활성화되어 HSC에 의해 섬유화의 주요 물질인 콜라젠 (collagen)을 분비하여 간에 두꺼운 섬유성 격벽 (fibrous septa)이 형성되는 현상을 의미한다.In the present invention, "Liver fibrosis" refers to a phenomenon in which liver tissue damage and infiltration of inflammatory cells as a result of repeated liver disease activates hepatic stellate cells (HSCs), which secrete collagen, a major material for fibrosis, to form a thick fibrous septa in the liver.
본 발명에서의 “간 성상세포 (Hepatic stellate cell; 이하 HSC)”는 간 내 휴지기 (quiescent)에 간세포 (hepatocyte)와 혈관내피세포 (sinusoidal endothelial cell) 사이의 Disse라는 공간에 상주하면서 신체에 필요한 레티놀 (retinol; vitamin A)의 70%를 저장하고 필요에 따라 이를 공급하는 기능을 하고 있다. 그러나 만성 간 손상시 휴지기의 간 성상세포들은 활성화 (activation)되고 이 과정 중에 저장된 레티놀은 사라지게 되며 결국 근섬유아세포 (myofibroblast like cells)와 비슷한 세포로 변환 (transformation)되어 간 내 콜라겐 (collagen) 생산 및 분비를 증가시킴으로써 간 섬유화 (liver fibrosis)를 유도할 수 있다.In the present invention, “Hepatic stellate cells (hereinafter referred to as HSCs)” reside in the space called Disse between hepatocytes and sinusoidal endothelial cells during the quiescent period in the liver, and function to store 70% of retinol (vitamin A) required by the body and supply it as needed. However, during chronic liver damage, hepatic stellate cells in the resting phase are activated, and retinol stored during this process is lost, and eventually transformed into cells similar to myofibroblast-like cells. By increasing collagen production and secretion in the liver, liver fibrosis can be induced.
본 발명에서의 “간세포 (hepatocyte)”는 간에서 기능을 하는 실질 (parenchyma) 세포로, 간세포의 손상과 사멸은 간 섬유화를 촉진하는 세포인 간 성상세포를 활성화시키고 섬유소를 비정상적으로 축적시킬 수 있으나, 이에 제한되는 것은 아니다."Hepatocyte" in the present invention is a parenchyma cell that functions in the liver, and damage and death of hepatocytes may activate hepatic stellate cells, which are cells that promote liver fibrosis, and abnormally accumulate fibrin, but is not limited thereto.
본 발명의 일 실시예에서, 상기 펩타이드 또는 상기 조성물은 간 손상 모델에서 간세포의 증식을 증가시키고, 간 섬유화를 유도하는 간 성상세포의 증식을 감소시킬 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the peptide or the composition may increase the proliferation of hepatocytes in a liver injury model and decrease the proliferation of hepatic stellate cells that induce liver fibrosis, but is not limited thereto.
본 발명에서의 “간 경변 (liver cirrhosis)”은 간 섬유화가 더욱 진행되어 간기능이 감소되는 현상으로, 간 경변증의 원인은 HBV, HCV, 알코올성 간질환, 독성간질환 등이 있으며 간 경변증이 더욱 심해지면 간암으로 발전하게 된다. 또한, 간 경변증은 복수 (ADSCites), 간염, 간성혼수, 정맥류 출혈 등의 합병증 발생의 원인이 된다.In the present invention, "liver cirrhosis" is a phenomenon in which liver fibrosis further progresses and liver function decreases. The causes of liver cirrhosis include HBV, HCV, alcoholic liver disease, toxic liver disease, and the like, and when liver cirrhosis becomes more severe, liver cancer develops. In addition, liver cirrhosis causes complications such as ascites (ADSCites), hepatitis, hepatic coma, and variceal bleeding.
본 발명에서의 “간 독소”는 간 조직에 손상 또는 질환을 유발하는 물질로서 사염화탄소 (CCl4), tBHP (tert-butyl hydroperoxide), 아세트아미노펜 (acetaminophen), 티오아세트아마이드 (thioacetamide), 타크린 (tacrine), 루브라톡신 B (rubratoxin B) 및 과산화수소 (H2O2)로 구성된 군으로부터 선택된 어느 하나이지만, 이에 제한되지는 않는다. "Liver toxin" in the present invention is a substance that causes damage or disease to liver tissue, and is any one selected from the group consisting of carbon tetrachloride (CCl 4 ), tert-butyl hydroperoxide (tBHP), acetaminophen, thioacetamide, tacrine, rubratoxin B, and hydrogen peroxide (H 2 O 2 ), but is not limited thereto.
본 발명의 일 실시예에서, 상기 간 독소는 티오아세트아마이드 (thioacetamide)일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the liver toxin may be thioacetamide, but is not limited thereto.
본 발명에서, “섬유화와 관련된 인자”는 섬유화의 개선, 악화 등과 관련된 모든 인자를 포함할 수 있고, 이는 섬유화 인자, 및 항섬유화 인자로 분류될 수 있다. 본 발명에서 “섬유화 인자”란 섬유화 유발 인자를 포함하는 개념이고, 이 때 섬유화 유발 인자란 간 섬유화를 유발시키거나, 간 섬유화 유발 시 증가하는 물질을 모두 포함할 수 있으며, 섬유화 유발 이외에도 질환의 증장, 진행, 악화, 섬유화에 의한 합병증을 발생시키는 등과 관련된 인자를 모두 포함할 수 있다. 예를 들어, procollagen-1 alpha1, MMP-2, TGF-beta1, TGF-beta2의 mRNA과 담도세포 표지자(ITGb-6) 등이 있을 수 있으나, 이에 제한되는 것은 아니다. 또한, 본 발명에서 “항섬유화 인자”란 섬유화의 유발, 진행, 섬유화로 인한 증상 악화, 합병증의 발생 등을 치료, 개선, 완화의 마커가 되는 인자를 모두 의미할 수 있으며, 본원발명의 일 실시예에서는 상기 마커로 TIMP1, 간 재생인자를 확인하였으나, 이에 제한되는 것은 아니다.In the present invention, "factors related to fibrosis" may include all factors related to improvement or aggravation of fibrosis, which may be classified into fibrotic factors and anti-fibrotic factors. In the present invention, "fibrosis factor" is a concept including a fibrosis-inducing factor, and at this time, the fibrosis-inducing factor may include all substances that induce liver fibrosis or increase when liver fibrosis is induced, and may include all factors related to growth, progression, exacerbation, and complications caused by fibrosis in addition to causing fibrosis. For example, there may be procollagen-1 alpha1, MMP-2, TGF-beta1, TGF-beta2 mRNA and biliary cell marker (ITGb-6), etc., but are not limited thereto. In addition, in the present invention, "anti-fibrotic factor" may mean all factors that are markers of treatment, improvement, and alleviation of the induction and progression of fibrosis, aggravation of symptoms due to fibrosis, and occurrence of complications, and the like.
본 발명에서, α-SMA는 인간의 ACTA2 유전자에 의해 암호화되는 단백질이며 세포 수축 시스템의 핵심 구성 요소로, α-SMA의 발현은 간 성상세포 활성의 핵심 마커일 수 있고, 본 발명에서, MMP-2는 섬유화 촉진 역할을 할 수 있다.In the present invention, α-SMA is a protein encoded by the human ACTA2 gene and is a key component of the cell contraction system. Expression of α-SMA may be a key marker of hepatic stellate cell activity, and in the present invention, MMP-2 may play a role in promoting fibrosis.
본 발명의 펩타이드, 또는 이를 포함하는 조성물은 섬유화 인자의 발현을 감소시키고 항섬유화 인자의 발현을 증가시킬 수 있다.The peptide of the present invention, or a composition containing the same, can reduce the expression of fibrotic factors and increase the expression of anti-fibrotic factors.
본 발명의 일 실시예에서, 상기 PTX3 단백질 또는 상기 배양물은In one embodiment of the present invention, the PTX3 protein or the culture
하기 (a)로 이루어진 군으로부터 선택되는 어느 하나 이상의 발현을 증가시키거나;To increase the expression of any one or more selected from the group consisting of (a);
하기 (b)로 이루어진 군으로부터 선택되는 어느 하나 이상의 발현을 감소시킬 수 있으나, 이에 제한되는 것은 아니다:The expression of one or more selected from the group consisting of the following (b) may be reduced, but is not limited thereto:
(a) HGF (Hepatocyte growth factor), VEGF (vascular endothelial growth factor), 및 TIMP1 (metallopeptidase inhibitor 1); 및(a) Hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and metallopeptidase inhibitor 1 (TIMP1); and
(b) α-SMA (alpha-smooth muscle actin), MMP-2 (matrix metalloproteinase-2), CollagenⅠ, 및 MMP.(b) α-SMA (alpha-smooth muscle actin), MMP-2 (matrix metalloproteinase-2), collagen I, and MMP.
본 발명의 일 실시예에서 사용된 PTX3은 서열번호 1로 표시될 수 있으나, 이에 제한되는 것은 아니다. 예를 들어, 본 발명의 PTX3은 상기 아미노산 서열의 변이체를 포함할 수 있다. 예를 들어, 본 발명의 서열번호 1로 표시되는 PTX3의 아미노산 서열은 상기 아미노산 서열과 각각 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 아미노산 서열을 포함할 수 있다. 예를 들면, 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%의 서열 상동성을 가질 수 있다.PTX3 used in one embodiment of the present invention may be represented by SEQ ID NO: 1, but is not limited thereto. For example, PTX3 of the present invention may include a variant of the above amino acid sequence. For example, the amino acid sequence of PTX3 represented by SEQ ID NO: 1 of the present invention may include an amino acid sequence having 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more sequence homology with the amino acid sequence. 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.
아미노산 서열 및/또는 핵산 서열과 관련하여 본원에 사용된 용어 “퍼센트 동일성”, “서열 동일성”, “백분율 유사성”, “서열 유사성” 등은 정렬된 아미노산 잔기 또는 뉴클레오티드 사이의 유사성을 최대화하고 동일하거나 유사한 잔기 또는 뉴클레오티드의 수, 총 잔기 또는 뉴클레오티드의 수, 및 서열 정렬에서 갭의 존재 및 길이의 함수인 서열의 정렬에 기초한 2개의 서열의 유사성 정도의 척도를 지칭할 수 있으나, 이에 제한되는 것은 아니다.The terms "percent identity," "sequence identity," "percent similarity," "sequence similarity," and the like, as used herein with reference to amino acid sequences and/or nucleic acid sequences, can refer to, but are not limited to, a measure of the degree of similarity of two sequences based on an alignment of the sequences that maximizes similarity between aligned amino acid residues or nucleotides and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment.
본 발명에 따른 약학적 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 상기 부형제는 예를 들어, 희석제, 결합제, 붕해제, 활택제, 흡착제, 보습제, 필름-코팅 물질, 및 제어방출첨가제로 이루어진 군으로부터 선택된 하나 이상일 수 있다. The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.
본 발명에 따른 약학적 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 서방형 과립제, 장용과립제, 액제, 점안제, 엘실릭제, 유제, 현탁액제, 주정제, 트로키제, 방향수제, 리모나아데제, 정제, 서방형정제, 장용정제, 설하정, 경질캅셀제, 연질캅셀제, 서방캅셀제, 장용캅셀제, 환제, 틴크제, 연조엑스제, 건조엑스제, 유동엑스제, 주사제, 캡슐제, 관류액, 경고제, 로션제, 파스타제, 분무제, 흡입제, 패취제, 멸균주사용액, 또는에어로졸 등의 외용제 등의 형태로 제형화하여 사용될 수 있으며, 상기 외용제는 크림, 젤, 패치, 분무제, 연고제, 경고제, 로션제, 리니멘트제, 파스타제 또는 카타플라스마제 등의 제형을 가질 수 있다. The pharmaceutical compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, solutions, eye drops, elsilic agents, emulsions, suspensions, spirits, troches, perfumes, limonadese, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, and soft drinks, respectively, according to conventional methods. It can be formulated and used in the form of external preparations such as extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, warning agents, lotions, pastes, sprays, inhalants, patches, sterilized injection solutions, or aerosols.
본 발명에 따른 약학적 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 올리고당, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, and propylhydroxy. hydroxybenzoate, talc, magnesium stearate and mineral oil.
제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
본 발명에 따른 정제, 산제, 과립제, 캡슐제, 환제, 트로키제의 첨가제로 옥수수전분, 감자전분, 밀전분, 유당, 백당, 포도당, 과당, 디-만니톨, 침강탄산칼슘, 합성규산알루미늄, 인산일수소칼슘, 황산칼슘, 염화나트륨, 탄산수소나트륨, 정제 라놀린, 미결정셀룰로오스, 덱스트린, 알긴산나트륨, 메칠셀룰로오스, 카르복시메칠셀룰로오스나트륨, 카올린, 요소, 콜로이드성실리카겔, 히드록시프로필스타치, 히드록시프로필메칠셀룰로오스 (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, 프로필렌글리콜, 카제인, 젖산칼슘, 프리모젤 등 부형제; 젤라틴, 아라비아고무, 에탄올, 한천가루, 초산프탈산셀룰로오스, 카르복시메칠셀룰로오스, 카르복시메칠셀룰로오스칼슘, 포도당, 정제수, 카제인나트륨, 글리세린, 스테아린산, 카르복시메칠셀룰로오스나트륨, 메칠셀룰로오스나트륨, 메칠셀룰로오스, 미결정셀룰로오스, 덱스트린, 히드록시셀룰로오스, 히드록시프로필스타치, 히드록시메칠셀룰로오스, 정제쉘락, 전분호, 히드록시프로필셀룰로오스, 히드록시프로필메칠셀룰로오스, 폴리비닐알코올, 폴리비닐피롤리돈 등의 결합제가 사용될 수 있으며, 히드록시프로필메칠셀룰로오스, 옥수수전분, 한천가루, 메칠셀룰로오스, 벤토나이트, 히드록시프로필스타치, 카르복시메칠셀룰로오스나트륨, 알긴산나트륨, 카르복시메칠셀룰로오스칼슘, 구연산칼슘, 라우릴황산나트륨, 무수규산, 1-히드록시프로필셀룰로오스, 덱스트란, 이온교환수지, 초산폴리비닐, 포름알데히드처리 카제인 및 젤라틴, 알긴산, 아밀로오스, 구아르 고무 (Guar gum), 중조, 폴리비닐피롤리돈, 인산칼슘, 겔화전분, 아라비아고무, 아밀로펙틴, 펙틴, 폴리인산나트륨, 에칠셀룰로오스, 백당, 규산마그네슘알루미늄, 디-소르비톨액, 경질무수규산 등 붕해제; 스테아린산칼슘, 스테아린산마그네슘, 스테아린산, 수소화식물유 (Hydrogenated vegetable oil), 탈크, 석송자, 카올린, 바셀린, 스테아린산나트륨, 카카오지, 살리실산나트륨, 살리실산마그네슘, 폴리에칠렌글리콜 4000, 6000, 유동파라핀, 수소첨가대두유 (Lubri wax), 스테아린산알루미늄, 스테아린산아연, 라우릴황산나트륨, 산화마그네슘, 마크로골 (Macrogol), 합성규산알루미늄, 무수규산, 고급지방산, 고급알코올, 실리콘유, 파라핀유, 폴리에칠렌글리콜지방산에테르, 전분, 염화나트륨, 초산나트륨, 올레인산나트륨, dl-로이신, 경질무수규산 등의 활택제가 사용될 수 있다.Corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium hydrogen carbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methyl cellulose, carboxymethyl cellulose sodium, kaol excipients such as lean, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch starch, hydroxypropylcellulose, hydroxypropylmethyl Binders such as cellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used, and 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-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde disintegrants such as hydrotreated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, di-sorbitol liquid, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopod, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, sodium stearate A lubricant such as lead, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid, etc. may be used.
본 발명에 따른 액제의 첨가제로는 물, 묽은 염산, 묽은 황산, 구연산나트륨, 모노스테아린산슈크로스류, 폴리옥시에칠렌소르비톨지방산에스텔류 (트윈에스텔), 폴리옥시에칠렌모노알킬에텔류, 라놀린에텔류, 라놀린에스텔류, 초산, 염산, 암모니아수, 탄산암모늄, 수산화칼륨, 수산화나트륨, 프롤아민, 폴리비닐피롤리돈, 에칠셀룰로오스, 카르복시메칠셀룰로오스나트륨 등이 사용될 수 있다.Additives of the liquid formulation 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, carr boxymethylcellulose sodium and the like can be used.
본 발명에 따른 시럽제에는 백당의 용액, 다른 당류 혹은 감미제 등이 사용될 수 있으며, 필요에 따라 방향제, 착색제, 보존제, 안정제, 현탁화제, 유화제, 점조제 등이 사용될 수 있다.In the syrup according to the present invention, a solution of white sugar, other sugars, or a sweetener may be used, and aromatics, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. may be used as necessary.
본 발명에 따른 유제에는 정제수가 사용될 수 있으며, 필요에 따라 유화제, 보존제, 안정제, 방향제 등이 사용될 수 있다.Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.
본 발명에 따른 현탁제에는 아카시아, 트라가칸타, 메칠셀룰로오스, 카르복시메칠셀룰로오스, 카르복시메칠셀룰로오스나트륨, 미결정셀룰로오스, 알긴산나트륨, 히드록시프로필메칠셀룰로오스, HPMC 1828, HPMC 2906, HPMC 2910 등 현탁화제가 사용될 수 있으며, 필요에 따라 계면활성제, 보존제, 안정제, 착색제, 방향제가 사용될 수 있다.Acacia, tragacantha, methylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, HPMC 2910, etc. may be used in the suspension agent according to the present invention, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as necessary.
본 발명에 따른 주사제에는 주사용 증류수, 0.9%염화나트륨주사액, 링겔주사액, 덱스트로스주사액, 덱스트로스+염화나트륨주사액, 피이지 (PEG), 락테이티드 링겔주사액, 에탄올, 프로필렌글리콜, 비휘발성유-참기름, 면실유, 낙화생유, 콩기름, 옥수수기름, 올레인산에칠, 미리스트산 이소프로필, 안식향산벤젠과 같은 용제; 안식향산나트륨, 살리실산나트륨, 초산나트륨, 요소, 우레탄, 모노에칠아세트아마이드, 부타졸리딘, 프로필렌글리콜, 트윈류, 니정틴산아미드, 헥사민, 디메칠아세트아마이드와 같은 용해보조제; 약산 및 그 염 (초산과 초산나트륨), 약염기 및 그 염 (암모니아 및 초산암모니움), 유기화합물, 단백질, 알부민, 펩톤, 검류와 같은 완충제; 염화나트륨과 같은 등장화제; 중아황산나트륨 (NaHSO3)이산화탄소가스, 메타중아황산나트륨 (Na2S2O5), 아황산나트륨 (Na2SO3),질소가스 (N2),에칠렌디아민테트라초산과 같은 안정제; 소디움비설파이드 0.1%, 소디움포름알데히드 설폭실레이트, 치오우레아, 에칠렌디아민테트라초산디나트륨, 아세톤소디움비설파이트와 같은 황산화제; 벤질알코올, 클로로부탄올, 염산프로카인, 포도당, 글루콘산칼슘과 같은 무통화제; 시엠시나트륨, 알긴산나트륨, 트윈 80, 모노스테아린산알루미늄과 같은 현탁화제를 포함할 수 있다.The injection according to the present invention includes injected distilled water, 0.9%sodium chloride liquid, ring gel injection, dextros+sodium chloride injection, fiji (PEG), lactated ring gel injection, ethanol, propylene glycol Sorghum oil, oleic acid, mirist isopropyl, and sabbatic hyangsan benzene; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twins, nijuntinamide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, and gums; tonicity 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 ), ethylenediaminetetraacetic acid; Sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, ethylenediamine disodium tetraacetate, acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; Suspending agents such as Siemesis sodium, sodium alginate, Tween 80, aluminum monostearate may be included.
본 발명에 따른 좌제에는 카카오지, 라놀린, 위텝솔, 폴리에틸렌글리콜, 글리세로젤라틴, 메칠셀룰로오스, 카르복시메칠셀룰로오스, 스테아린산과 올레인산의 혼합물, 수바날 (Subanal), 면실유, 낙화생유, 야자유, 카카오버터+콜레스테롤, 레시틴, 라네트왁스, 모노스테아린산글리세롤, 트윈 또는 스판, 임하우젠 (Imhausen), 모놀렌 (모노스테아린산프로필렌글리콜), 글리세린, 아뎁스솔리두스 (Adeps solidus), 부티룸 태고-G (Buytyrum Tego-G), 세베스파마 16 (Cebes Pharma 16), 헥사라이드베이스 95, 코토마 (Cotomar), 히드록코테 SP, S-70-XXA, S-70-XX75 (S-70-XX95), 히드록코테 (Hydrokote) 25, 히드록코테 711, 이드로포스탈 (Idropostal), 마사에스트라리움 (Massa estrarium, A, AS, B, C, D, E, I, T), 마사-MF, 마수폴, 마수폴-15, 네오수포스탈-엔, 파라마운드-B, 수포시로 (OSI, OSIX, A, B, C, D, H, L), 좌제기제 IV 타입 (AB, B, A, BC, BBG, E, BGF, C, D, 299), 수포스탈 (N, Es), 웨코비 (W, R, S, M ,Fs), 테제스터 트리글리세라이드 기제 (TG-95, MA, 57)와 같은 기제가 사용될 수 있다.The suppository according to the present invention includes cacao butter, lanolin, witapsol, 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, twin or span, Imhausen, monolen (monosteol) Propylene Glycol Phosphate), Glycerin, Adeps Solidus, Buytyrum Tego-G, Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrocote (Hydr okote) 25, Hydrocote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository type IV (AB, B , A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), Testester triglyceride bases (TG-95, MA, 57).
경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로스 (sucrose) 또는 락토오스 (lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the extract with at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
경구 투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
본 발명에 따른 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, “약학적으로 유효한 양”은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level may be determined according to the type of patient's disease, severity, drug activity, drug sensitivity, administration time, administration route and discharge rate, treatment period, factors including concurrently used drugs, and other factors well known in the medical field.
본 발명에 따른 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 본 발명이 속하는 기술분야에 통상의 기술자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention belongs.
본 발명의 약학적 조성물은 개체에게 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구 복용, 피하 주사, 복강 투여, 정맥 주사, 근육 주사, 척수 주위 공간 (경막내) 주사, 설하 투여, 볼점막 투여, 직장 내 삽입, 질 내 삽입, 안구 투여, 귀 투여, 비강 투여, 흡입, 입 또는 코를 통한 분무, 피부 투여, 경피 투여 등에 따라 투여될 수 있다.The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.
본 발명의 약학적 조성물은 치료할 질환, 투여 경로, 환자의 연령, 성별, 체중 및 질환의 중등도 등의 여러 관련 인자와 함께 활성성분인 약물의 종류에 따라 결정된다.The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient together with various related factors such as the disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient.
본 발명에서 “개체”란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는 인간 또는 비-인간인 영장류, 생쥐 (mouse), 쥐 (rat), 개, 고양이, 말, 및 소 등의 포유류를 의미한다.In the present invention, "individual" means a subject in need of treatment of a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow. It means.
본 발명에서 “투여”란 임의의 적절한 방법으로 개체에게 소정의 본 발명의 조성물을 제공하는 것을 의미한다. In the present invention, "administration" means providing a given composition of the present invention to a subject by any suitable method.
본 발명에서 “예방”이란 목적하는 질환의 발병을 억제하거나 지연시키는 모든 행위를 의미하고, “치료”란 본 발명에 따른 약학적 조성물의 투여에 의해 목적하는 질환과 그에 따른 대사 이상 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미하며, “개선”이란 본 발명에 따른 조성물의 투여에 의해 목적하는 질환과 관련된 파라미터, 예를 들면 증상의 정도를 감소시키는 모든 행위를 의미한다.In the present invention, "prevention" means any action that inhibits or delays the onset of a desired disease, and "treatment" means any action that improves or beneficially changes a desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention, and "improvement" means any action that reduces a parameter related to a desired disease, for example, the severity of symptoms, by administration of the composition according to the present invention.
또한, 본 발명은 하기 단계를 포함하는 miR150으로 형질전환된 줄기세포 배양물 제조방법을 제공한다:In addition, the present invention provides a method for preparing a stem cell culture transformed with miR150 comprising the following steps:
(a) miR150을 줄기세포에 형질전환시키는 단계; 및(a) transforming miR150 into stem cells; and
(b) 상기 형질전환된 줄기세포를 무혈청 배지에서 배양하여 배양물을 획득하는 단계.(b) culturing the transformed stem cells in a serum-free medium to obtain a culture product.
또한, 본 발명은 하기 단계를 포함하는 miR150으로 형질전환된 줄기세포의 배양물로부터 PTX3 단백질을 분리 정제하는 방법을 제공한다:In addition, the present invention provides a method for separating and purifying the PTX3 protein from a culture of stem cells transformed with miR150, comprising the following steps:
miR150으로 줄기세포를 형질전환하는 단계; 및 transforming stem cells with miR150; and
형질전환된 줄기세포의 배양물에서 PTX3 단백질을 분리 정제하는 단계.Separating and purifying the PTX3 protein from the transformed stem cell culture.
또한, 본 발명은 PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물을 유효성분으로 포함하는 간 섬유증 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for improving liver fibrosis comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
본 발명에 있어서, “식품”이란 영양소를 한 가지 또는 그 이상 함유하고 있는 천연물 또는 가공품을 의미하며, 바람직하게는 어느 정도의 가공 공정을 거쳐 직접 먹을 수 있는 상태가 된 것을 의미하며, 통상적인 의미로서, 건강기능성 식품, 음료, 식품 첨가제 및 음료 첨가제 등을 모두 포함하는 의미이다. In the present invention, "food" means a natural product or processed product containing one or more nutrients, preferably means a state that can be directly eaten through a certain degree of processing, and as a general meaning, health functional food, It means to include all beverages, food additives and beverage additives.
본 발명에서 용어, “식품학적으로 허용 가능한 염”이란 식품학적으로 허용되는 유기산, 무기산, 또는 염기로부터 유도된 염을 포함한다.In the present invention, the term "acceptable salt in food science" includes salts derived from organic acids, inorganic acids, or bases acceptable in food science.
본 발명의 일 실시예에 있어서, 상기 식품은 건강기능식품일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the food may be a health functional food, but is not limited thereto.
본 발명에 있어서, 상기 “건강기능성 식품(functional food)”이란, 특정보건용 식품(food for special health use, FoSHU)과 동일한 용어로, 영양 공급 외에도 생체조절기능이 효율적으로 나타나도록 가공된 의학, 의료효과가 높은 식품을 의미하며, 정제, 캡슐제, 환제, 과립제, 분말, 액제, 편상(flake), 페이스트, 시럽제, 겔, 젤리, 바(bar), 또는 필름 제형으로 제조될 수 있다. 여기서 “기능성”이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다.In the present invention, the "functional food" is the same term as food for special health use (FoSHU), and means a food with high medical and medical effects processed to efficiently display bioregulatory functions in addition to nutrient supply, and can be prepared as tablets, capsules, pills, granules, powders, liquids, flakes, pastes, syrups, gels, jellies, bars, or film formulations. Here, “functionality” means obtaining useful effects for health purposes, such as adjusting nutrients for the structure and function of the human body or physiological functions.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 특히 통상적인 의미에서의 해당 식품의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체방어리듬조절, 질병 방지와 회복 등에 관한 체내조절기능을 생체에 대하여 충분히 발현하도록 설계된 식품을 모두 포함한다.There is no particular limitation on the type of food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. It includes all foods designed to be sufficiently expressed in vivo.
본 발명에 있어서, “식품 첨가물”은 식품을 제조, 가공 또는 보존을 함에 있어 식품에 첨가, 혼합, 침윤, 기타의 방법으로 사용되는 물질에 관한 것으로, 건강기능식품과 같이 장기적으로 복용하였을 때 인체에 무해하여야한다.In the present invention, "food additives" relate to substances used in food additives, mixing, infiltration, and other methods in manufacturing, processing, or preserving food, and should be harmless to the human body when taken for a long period of time, such as health functional foods.
본 발명의 조성물을 식품 첨가물로 사용할 경우, 상기 식품첨가물은 본 발명의 조성물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. When the composition of the present invention is used as a food additive, the food additive may be added to the composition of the present invention as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.
유효성분의 혼합양은 사용 목적 (예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시 본 발명의 조성물은 원료에 대하여 15 중량% 이하, 또는 10 중량% 이하의 양으로 첨가될 수 있다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의양으로도 사용될 수 있다.The mixing amount of active ingredients may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the composition of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than 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.
본 발명에 있어서, 상기 조성물에는 식품학적으로 허용 가능한 다양한 식품 보조 첨가제를 포함할 수 있으며, 식품의 제조에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더욱 포함할 수 있다.In the present invention, the composition may include various food supplements acceptable in food science, and may further include appropriate carriers, excipients, and diluents commonly used in food production.
또한, 상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 조성물은 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.01-0.20 중량부의 범위에서 선택되는 것이 일반적이나, 이에 제한되는 것은 아니고, 활용되는 제품의 종류, 기능 등에 따라 최적, 임의의 용량일 수 있다.In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not very important, but is generally selected from the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto, and is optimal according to the type and function of the product used.
또한, 본 발명에 따른 조성물은 건강음료에도 첨가될 수 있으며, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당 및 과당과 같은 모노사카라이드, 말토오스 및 수크로오스와 같은 디사카라이드, 덱스트린 및 시클로덱스트린과 같은 폴리사카라이드, 및 자일리톨, 소르비톨 및 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 mL당 일반적으로 약 0.01-0.20g, 또는 약 0.04-0.10g 일 수 있으나, 이에 제한되는 것은 아니며, 당업계에서 첨가되는 일반적인 용량일 수 있거나, 본 발명의 조성물의 효능을 제고하기 위하여 최대 범위를 포함할 수 있으며, 함께 첨가되는 기타 물질과의 상승 효과를 고려한 최적의, 임의의 용량을 포함할 수 있다.In addition, the composition according to the present invention may be added to health drinks, and may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional drinks. The aforementioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The ratio of the natural carbohydrate may be generally about 0.01 to 0.20 g, or about 0.04 to 0.10 g per 100 mL of the composition of the present invention, but is not limited thereto, and may be a general amount added in the art, or may include the maximum range to improve the efficacy of the composition of the present invention, and may include an optimal, arbitrary amount in consideration of synergistic effects with other substances added together.
본 발명의 펩타이드, 또는 이를 유효성분으로 포함하는 조성물을 식품 첨가물로 사용할 경우, 이를 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효성분의 혼합양은 사용 목적 (예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시 본 발명의 담팔수 추출물 또는 상기 화합물은 원료에 대하여 15 중량% 이하, 또는 10 중량% 이하의 양으로 첨가될 수 있다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.When the peptide of the present invention or a composition containing it as an active ingredient is used as a food additive, it can be added as it is or used together with other foods or food ingredients, and can be appropriately used according to conventional methods. The mixing amount of active ingredients may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the freshwater extract or the compound of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.
이하, 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 지방유래줄기세포 배양액 유래 펩타이드 제작 및 발현 확인Example 1. Adipose-derived stem cell culture medium-derived peptide production and expression confirmation
본 발명의 mRNA로 형질전환된 지방유래줄기세포 배양액 유래 펩타이드를 아래와 같은 방식으로 제작하였다.Adipose-derived stem cell culture medium-derived peptides transformed with the mRNA of the present invention were prepared in the following manner.
먼저, 지방유래줄기세포를 10% FBS (inactivated fetal bovine serum) 및 1% P/S (penicillin-streptomycin)을 첨가한 DMEM low glucose 배지에서 37℃, 5% CO2조건으로 배양하였다.First, adipose-derived stem cells were cultured in DMEM low glucose medium supplemented with 10% inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) at 37°C and 5% CO 2 conditions.
이 후, miR150를 제조사의 지침에 따라 형질감염시약 (lipofectamine, invitrogen)을 사용하여 지방유래줄기세포에 형질전환 (Transfection)시킨 후, 지방유래줄기세포를 24시간 동안 배양하였다.Thereafter, miR150 was transfected into adipose-derived stem cells using a transfection reagent (lipofectamine, invitrogen) according to the manufacturer's instructions, and then the adipose-derived stem cells were cultured for 24 hours.
miR150이 형질전환된 지방유래 줄기세포의 배양배지를 FBS-Free DMEM low glucose로 배지를 교환하였으며, 배지 교환 후 24시간 경과 후 지방유래줄기세포의 배양배지를 확보하였다. 확보한 배양배지에 대하여 LC/MS를 수행하여 분석하였다. LC/MS로 분석한 펩타이드 중에서 발현이 증가한 PTX3를 확인하였다.The culture medium of miR150-transformed adipose-derived stem cells was exchanged with FBS-Free DMEM low glucose, and the culture medium of adipose-derived stem cells was secured 24 hours after the medium exchange. The obtained culture medium was analyzed by performing LC/MS. Among the peptides analyzed by LC/MS, PTX3 with increased expression was identified.
PTX3는 (Recombinant Human) E.Coli에서 생산되고 381개의 아미노산을 포함하고 있으며, 44.4 kDa의 분자 질량을 갖는 단일 비글리코화 폴리펩티드 사슬이다. 또한, PTX3는 N-terminus에 36 amino acid His Tag이 융합되어 있으며 크로마토그래피에 의하여 정제된 바 있다. 또한, SDS-PAGE 분석으로 90% 이상의 순도를 지니는 것을 사용하였고 PTX3 단백질 (1 mg/ml)에는 20mM Tris-HCl 버퍼 pH 8.0, 1 mM DTT 및 10% 글리세롤이 포함되어 있으며, 서열번호 1의 아미노산 서열을 갖는다. 한편, 본 발명에서의 PTX3는 Mybiosource사로부터 구매하여 실험에 사용하였다.PTX3 is a single unglycosylated polypeptide chain produced in (Recombinant Human) E. coli, containing 381 amino acids and having a molecular mass of 44.4 kDa. In addition, PTX3 has a 36 amino acid His Tag fused to the N-terminus and has been purified by chromatography. In addition, one having a purity of 90% or more by SDS-PAGE analysis was used, and the PTX3 protein (1 mg/ml) contained 20 mM Tris-HCl buffer pH 8.0, 1 mM DTT and 10% glycerol, and had the amino acid sequence of SEQ ID NO: 1. On the other hand, PTX3 in the present invention was purchased from Mybiosource and used in the experiment.
이 후, PTX3 단독의 in vitro, in vivo 간손상 모델에서 섬유화 억제능을 확인하였다.After that, the ability of PTX3 alone to inhibit fibrosis was confirmed in in vitro and in vivo liver injury models.
구분division 서열(N→C)Sequence (N→C)
서열번호 1SEQ ID NO: 1 MHLLAILFCALWSAVLAENSDDYDLMYVNLDNEIDNGLHPTEDPTPCACGQEHSEWDKLFIMLENSQMRERMLLQATDDVLRGELQRLREELGRLAESLARPCAPGAPAEARLTSALDELLQATRDAGRRLARMEGAEAQRPEEAGRALAAVLEELRQTRADLHAVQGWAARSWLPAGCETAILFPMRSKKIFGSVHPVRPMRLESFSACIWVKATDVLNKTILFSYGTKRNPYEIQLYLSYQSIVFVVGGEENKLVAEAMVSLGRWTHLCGTWNSEEGLTSLWVNGELAATTVEMATGHIVPEGGILQIGQEKNGCCVGGGFDETLAFSGRLTGFNIWDSVLSNEEIRETGGAESCHIRGNIVGWGVTEIQPHGGAQYVSMHLLAILFCALWSAVLAENSDDYDLMYVNLDNEIDNGLHPTEDPTPCACGQEHSEWDKLFIMLENSQMRERMLLQATDDVLRGELQRLREELGRLAESLARPCAPGAPAEARLTSALDELLQATRDAGRRLARMEGAEAQRPEEAGRALAAVLEELRQTRADLHAVQGWAARSWLPAGCETAILFPMRSKKIFGSVHPVRPMRLESFSACIWVKAT DVLNKTILFSYGTKRNPYEIQLYLSYQSIVFVVGGEENKLVAEAMVSLGRWTHLCGTWNSEEGLTSLWVNGELAATTVEMATGHIVPEGGILQIGQEKNGCCVGGGFDETLAFSGRLTGFNIWDSVLSNEEIRETGGAESCHIRGNIVGWGVTEIQPHGGAQYVS
실시예 2. 간 손상 모델에서의 본 발명에 따른 배양액 유래 펩타이드에 의한 간세포 증식 증가 효과 확인Example 2. Confirmation of the effect of increasing hepatocyte proliferation by the culture medium-derived peptide according to the present invention in a liver injury model
본 발명의 miR150이 형질전환된 지방유래줄기세포 배양액 유래 펩타이드 조성물에 의한 간세포 증식 증가 효과를 분석하기 위해, 정상 간세포주에 Thioacetic acid (TAA)를 처리하여 간 손상 모델을 구현하였다. 티오아세트아미드 (Thioacetamide, 이하 TAA)는 간 독성을 지닌 대표적인 약물로서 농도, 반응시간을 다르게 적용하면 간 성상세포를 활성화시켜서 간 섬유화를 유도하는 것으로 알려져 있는 물질이다. In order to analyze the effect of increasing hepatocyte proliferation by the miR150-transformed adipose-derived stem cell culture medium-derived peptide composition of the present invention, a normal hepatocyte cell line was treated with Thioacetic acid (TAA) to implement a liver injury model. Thioacetamide (hereinafter referred to as TAA) is a representative drug with liver toxicity and is known to activate hepatic stellate cells and induce liver fibrosis when different concentrations and reaction times are applied.
정상 간 세포주 AML12에 TAA를 처리하여 간 손상 모델을 구현하고 PTX3를 처리하였을 때 감소되었던 간세포의 세포 증식 (cell proliferation)이 증가하는 것으로 확인되었다 (도 1). A liver injury model was implemented by treating normal liver cell line AML12 with TAA, and it was confirmed that cell proliferation of hepatocytes, which was reduced when treated with PTX3, increased (FIG. 1).
실시예 3. 간 성상세포 활성화 모델에서의 본 발명에 따른 배양액 유래 펩타이드의 간 성상세포 증식 감소 효과 확인Example 3. Confirmation of hepatic stellate cell proliferation reduction effect of the culture medium-derived peptide according to the present invention in hepatic stellate cell activation model
본 발명의 miR150이 형질전환된 지방유래줄기세포 배양액 유래 펩타이드 조성물에 의한 간 성상세포의 분석하기 위해, 간 성상세포주에 5mM TAA를 처리하여 간 성상세포 활성화 모델을 구현하였다.In order to analyze hepatic stellate cells using the miR150-transformed adipose-derived stem cell culture medium-derived peptide composition of the present invention, hepatic stellate cell lines were treated with 5 mM TAA to implement a hepatic stellate cell activation model.
간 성상세포주 LX-2에 TAA를 처리하여 간 손상 모델을 구현하고 PTX3를 처리하였을 때 증가되었던 간 성상세포의 세포 증식이 감소하는 것으로 확인되었다 (도 2). Hepatic stellate cell line LX-2 was treated with TAA to implement a liver injury model, and it was confirmed that the cell proliferation of hepatic stellate cells, which was increased when treated with PTX3, was reduced (FIG. 2).
실시예 4. 본 발명에 따른 배양액 유래 펩타이드 조성물의 간 재생인자 발현 분석Example 4. Analysis of liver regeneration factor expression of the culture medium-derived peptide composition according to the present invention
본 발명의 miR150이 형질전환된 지방유래줄기세포 배양액 유래 펩타이드 조성물의 간 재생능을 확인하기 위하여 간 재생인자인 HGF, 및 VEGF의 발현을 RNA 수준에서 분석해 발현 수준을 비교하였다.In order to confirm the liver regeneration ability of the miR150-transformed adipose-derived stem cell culture medium-derived peptide composition of the present invention, the expression levels of HGF and VEGF, which are liver regeneration factors, were analyzed at the RNA level and the expression levels were compared.
그 결과, 본 발명의 miR150이 형질전환된 지방유래줄기세포 배양액 유래 펩타이드 조성물에 의하여 간 재생인자인 HGF 및 VEGF의 발현이 증가하는 것으로 확인되었다. 구체적으로, TAA 처리하여 간 손상 모델을 구축한 후 PTX3 단독으로 처리하면 TAA 단독 처리시에 감소되었던 간 재생인자인 HGF 및 VEGF의 발현이 증가하여, 본 발명의 배양액 유래 펩타이드 조성물의 간 재생능이 현저히 우수한 것으로 확인되었다 (도 3).As a result, it was confirmed that the expression of HGF and VEGF, which are liver regeneration factors, was increased by the adipose-derived stem cell culture medium-derived peptide composition transformed with miR150 of the present invention. Specifically, when a liver injury model was constructed by TAA treatment and then treated with PTX3 alone, the expression of liver regeneration factors HGF and VEGF, which were reduced when treated with TAA alone, increased, and it was confirmed that the peptide composition derived from the culture medium of the present invention had remarkably excellent liver regeneration ability (FIG. 3).
실시예 5. 동물 모델을 이용한 본 발명에 따른 배양액 유래 펩타이드 조성물의 섬유화와 관련된 인자 분석Example 5. Analysis of factors related to fibrosis of the culture medium-derived peptide composition according to the present invention using an animal model
간 섬유화와 관련된 인자에 대한 본 발명의 일 실시예에 따른 PTX3 단백질, 및 배양 조성물에 대한 활성을 확인하고자 BALB/C Nude mouse (in vivo)마우스에 TAA를 200㎎/㎏으로 주에 3번씩 6주간 투여하고 PTX3를 주 당 (per week) 3번씩 2주간 투여 후 mouse를 희생하여 간 조직을 추출하였고, 간 조직 내의 섬유화 변화 수준을 Western blot을 통하여 확인하였다. 실험군은 Control군 (Ct), PTX3 처리군 (PTX3), TAA 처리군 (TAA), 및 TAA+PTX3 처리군 (TAA+PTX3)으로 나누어 실험을 진행하였다. In order to confirm the activity of the PTX3 protein and the culture composition according to an embodiment of the present invention on factors related to liver fibrosis, TAA was administered to BALB/C Nude mouse (in vivo) mice at 200 mg/kg 3 times a week for 6 weeks, and PTX3 was administered 3 times a week for 2 weeks, then the mouse was sacrificed to extract liver tissue, and the level of fibrosis change in liver tissue was confirmed through Western blot. The experimental group was divided into a control group (Ct), a PTX3 treatment group (PTX3), a TAA treatment group (TAA), and a TAA+PTX3 treatment group (TAA+PTX3).
그 결과, TAA 처리군에서 증가되었던 섬유화 인자, 즉, HSC의 섬유화 마커인 α-SMA, 및 MMP-2의 발현이 PTX3 처리 시 현저히 감소되는 것으로 확인되었다 (도 4). As a result, it was confirmed that the expression of fibrosis factors, that is, HSC fibrosis markers α-SMA and MMP-2, which had been increased in the TAA-treated group, was significantly reduced upon PTX3 treatment (FIG. 4).
한편, TIMP1의 발현 역시 감소되는 것으로 나타났으나, 이와 같은 TIMP1 발현 감소 효과는 다음과 같은 이유에 기인하는 것으로 사료된다. 먼저, TIMP1의 발현은 MMP를 억제하기 위해서 증가하게 되고, mRNA 수준에서 확인한 결과 TIMP1의 발현이 증가하는 것으로 확인되었다. 그러나 동물실험 조직에서의 TIMP1의 발현이 감소하는 이유는 마우스 희생시점에 이미 MMP의 발현이 감소되었기 때문에 mRNA 수준에서 증가해 있는 TIMP1이 단백질 수준에서 발현이 감소된 것으로 판단된다.On the other hand, the expression of TIMP1 was also found to be reduced, but such an effect of reducing TIMP1 expression is considered to be due to the following reasons. First, the expression of TIMP1 is increased to suppress MMP, and as a result of confirming at the mRNA level, it was confirmed that the expression of TIMP1 is increased. However, the reason for the decrease in the expression of TIMP1 in animal tissues is that the expression of TIMP1, which is increased at the mRNA level, is judged to be reduced at the protein level because the expression of MMP has already been reduced at the time of mouse sacrifice.
추가적으로, 상기 방법으로 mouse를 희생하여 간 조직을 추출한 후 간 조직 내의 섬유화 변화 수준을 섬유화 관련 인자의 발현을 통해 분석하였다. 구체적으로, 실험군은 Control 군 (Ct), PTX3 처리군 (rPTX3), TAA 처리군 (TAA), TAA+PTX3 처리군 (TAA+rPTX3)으로 나누어 실험을 진행하였고, 섬유화 변화 수준을 확인하기 위한 섬유화 관련 인자인, CollagenⅠ, MMP, 및 TIMP1의 발현 수준을 RealtimePCR을 통하여 분석하였다. 이 때, TAA 처리된 간 성상세포는 세포외기질을 생산하는 주 생산원으로서, 간 성상세포가 활성화되면 근섬유모세포 유사형태로 바뀌게 되고 이에 따라 세포외기질 물질인 CollagenI의 생성이 증가하게 된다. Additionally, after the liver tissue was extracted by sacrificing the mouse by the above method, the level of fibrosis change in the liver tissue was analyzed through the expression of fibrosis-related factors. Specifically, the experimental group was divided into a control group (Ct), PTX3 treatment group (rPTX3), TAA treatment group (TAA), and TAA + PTX3 treatment group (TAA + rPTX3), and the experiment was conducted. The expression levels of Collagen I, MMP, and TIMP1, which are factors related to fibrosis, were analyzed through RealtimePCR. At this time, TAA-treated hepatic stellate cells are the main producers of extracellular matrix, and when hepatic stellate cells are activated, they change into a myofibroblast-like form, thereby increasing the production of CollagenI, an extracellular matrix material.
그 결과, rPTX3군은 대조군과 거의 동일한 수준의 CollagenI 및 MMP 발현을 나타내는 것으로 확인되었다. 또한, TAA군에서의 상기 인자의 발현 수준은 약 6배로 증가한 반면에, TAA+rPTX3군에서의 CollagenI 및 MMP 발현은 TAA군 대비 약 50% 수준으로 현저히 감소되는 것으로 나타났다. 뿐만 아니라, 도 4에서 나타난 TIMP1 발현과 관련된 실험 결과와는 달리 TIMP1의 발현이 TAA 단독군보다 높게 발현되는 것으로 조사되었으며, 상기 결과에 따라 PTX3 처리 시 섬유화 정도가 TAA 단독군에 비해 낮기 때문인 것으로 확인되었다 (도 5).As a result, it was confirmed that the rPTX3 group exhibited almost the same level of CollagenI and MMP expression as the control group. In addition, the expression level of the factor in the TAA group increased about 6-fold, whereas the expression of CollagenI and MMP in the TAA+rPTX3 group was significantly reduced to about 50% compared to the TAA group. In addition, unlike the experimental results related to TIMP1 expression shown in FIG. 4, it was investigated that the expression of TIMP1 was higher than that of the TAA alone group, and according to the results, it was confirmed that this was because the degree of fibrosis during PTX3 treatment was lower than that of the TAA alone group (FIG. 5).
실시예 6. 동물 모델을 이용한 본 발명에 따른 배양액 유래 펩타이드 조성물의 조직학적 특징 분석Example 6. Analysis of histological characteristics of the culture medium-derived peptide composition according to the present invention using an animal model
BALB/C Nude mouse (in vivo)마우스에 TAA를 200㎎/㎏으로 주에 3번씩 6주간 투여 후 PTX3를 주당 3번씩 2주간 투여 후 mouse를 희생하여 간 조직을 추출하였고, Hematoxylin & eosin (H&E) 염색을 실시하여 조직학적 변화를 관찰하였다 (도 10). After administering TAA to BALB/C Nude mouse (in vivo) mice at 200 mg/kg 3 times a week for 6 weeks and then administering PTX3 3 times a week for 2 weeks, the mice were sacrificed to extract liver tissue, and hematoxylin & eosin (H&E) staining was performed to observe histological changes (FIG. 10).
구체적으로, 출된 조직은 0.1M 인산 완충액 (pH 7.2, Sigma)을 포함하는 10% 포르말린으로 고정하였다. 각 조직을 파라핀에 포매시킨 후, 절편을 제조하였다. 제조된 파라핀 절편 (4㎛ 두께)을 탈왁스, 수화처리하고 인산-완충 염분에 들어있는 0.01% 프로테아제 XXIV (Sigma)로 20분간 37℃에서 처리하였으며, 헤마톡실린 (hematoxylin) 및 에오신 (eosin)으로 염색하였다.Specifically, the extracted tissue was fixed with 10% formalin containing 0.1M phosphate buffer (pH 7.2, Sigma). After embedding each tissue in paraffin, sections were prepared. The prepared paraffin sections (4 μm thick) were dewaxed, hydrated, treated with 0.01% protease XXIV (Sigma) in phosphate-buffered saline at 37° C. for 20 minutes, and stained with hematoxylin and eosin.
그 결과, TAA+rPTX군에 의한 조직의 양상은 무처리군 (대조군, control)과 거의 유사하게 나타났고, TAA 단독 처리군에 비해 TAA+rPTX 처리군에서 경우 조직의 손상이 감소되는 것으로 확인되었다 (도 6).As a result, the tissue pattern of the TAA+rPTX group was almost similar to that of the non-treated group (control), and it was confirmed that tissue damage was reduced in the TAA+rPTX-treated group compared to the TAA-only treated group (FIG. 6).
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The above description of the present invention is for illustrative purposes, and those skilled in the art may understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.
본 발명은 miR150로 형질전환된 줄기세포의 줄기세포 배양액 유래 펩타이드를 포함하는 간 섬유증의 예방 또는 치료용 약학적 조성물 또는 miR150로 형질전환된 줄기세포의 줄기세포 배양액 유래 펩타이드를 포함하는 간 섬유증 개선용 식품 조성물에 대한 것으로, 본 발명의 약학적 조성물은 간 섬유화 치료 분야, 특히 임상적 적용 기술로 응용될 수 있으며, 본 발명의 식품 조성물은 간 섬유증의 개선에 사용될 수 있는 이점이 있다.The present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis containing a peptide derived from a stem cell culture medium of stem cells transformed with miR150 or a food composition for improving liver fibrosis containing a peptide derived from a stem cell culture medium of stem cells transformed with miR150.

Claims (17)

  1. PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물을 유효성분으로 포함하는 간 섬유증의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating liver fibrosis comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
  2. 제1항에 있어서,According to claim 1,
    상기 miR150으로 형질전환된 줄기세포 배양물은 PTX3 단백질을 포함하는 것인, 조성물.Wherein the stem cell culture transformed with the miR150 contains the PTX3 protein.
  3. 제1항에 있어서,According to claim 1,
    상기 PTX3 단백질은 miR150으로 형질전환된 줄기세포 배양물에서 분리된 것인, 조성물.Wherein the PTX3 protein is isolated from a stem cell culture transformed with miR150.
  4. 제1항에 있어서,According to claim 1,
    상기 PTX3 단백질은 하기 중 어느 하나 이상의 특징을 포함하는 것인, 조성물:The composition, wherein the PTX3 protein comprises any one or more of the following characteristics:
    서열번호 1로 이루어짐; consisting of SEQ ID NO: 1;
    44.4kDA의 분자량; 및molecular weight of 44.4 kDA; and
    N-말단에 36 아미노산 His Tag이 융합됨. A 36 amino acid His Tag is fused to the N-terminus.
  5. 제1항에 있어서,According to claim 1,
    상기 줄기세포는 지방유래 줄기세포 (Adipose-derived stem cell; ADSC)인 것인, 조성물.Wherein the stem cells are adipose-derived stem cells (ADSC).
  6. 제1항에 있어서,According to claim 1,
    상기 간 섬유증은 간 독소에 의해 유발되는 것인, 조성물.Wherein the liver fibrosis is caused by liver toxins.
  7. 제6항에 있어서,According to claim 6,
    상기 간 독소는 티오아세트아마이드 (thioacetamide)인 것인, 조성물.Wherein the liver toxin is thioacetamide.
  8. 제1항에 있어서,According to claim 1,
    상기 PTX3 단백질 또는 상기 배양물은 간세포의 증식을 증가시키고, 간 성상세포의 증식을 감소시키는 것인, 조성물.Wherein the PTX3 protein or the culture increases the proliferation of hepatocytes and reduces the proliferation of hepatic stellate cells.
  9. 제1항에 있어서,According to claim 1,
    상기 PTX3 단백질 또는 상기 배양물은The PTX3 protein or the culture
    하기 (a)로 이루어진 군으로부터 선택되는 어느 하나 이상의 발현을 증가시키거나;To increase the expression of any one or more selected from the group consisting of (a);
    하기 (b)로 이루어진 군으로부터 선택되는 어느 하나 이상의 발현을 감소시키는 것인, 조성물:To reduce the expression of any one or more selected from the group consisting of the following (b), the composition:
    (a) HGF (Hepatocyte growth factor), VEGF (vascular endothelial growth factor), 및 TIMP1 (metallopeptidase inhibitor 1); 및(a) Hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and metallopeptidase inhibitor 1 (TIMP1); and
    (b) α-SMA (alpha-smooth muscle actin), MMP-2 (matrix metalloproteinase-2), CollagenⅠ, 및 MMP.(b) α-SMA (alpha-smooth muscle actin), MMP-2 (matrix metalloproteinase-2), collagen I, and MMP.
  10. 하기 단계를 포함하는 miR150으로 형질전환된 줄기세포 배양물 제조방법:Method for producing a stem cell culture transformed with miR150 comprising the following steps:
    (a) miR150을 줄기세포에 형질전환시키는 단계; 및(a) transforming miR150 into stem cells; and
    (b) 상기 형질전환된 줄기세포를 무혈청 배지에서 배양하여 배양물을 획득하는 단계.(b) culturing the transformed stem cells in a serum-free medium to obtain a culture product.
  11. 하기 단계를 포함하는 miR150으로 형질전환된 줄기세포의 배양물로부터 PTX3 단백질을 분리 정제하는 방법:A method for separating and purifying PTX3 protein from a culture of stem cells transformed with miR150 comprising the following steps:
    miR150으로 줄기세포를 형질전환하는 단계; 및 transforming stem cells with miR150; and
    형질전환된 줄기세포의 배양물에서 PTX3 단백질을 분리 정제하는 단계.Separating and purifying the PTX3 protein from the transformed stem cell culture.
  12. 제10항 또는 제11항에 있어서,According to claim 10 or 11,
    상기 줄기세포는 지방유래 줄기세포 (Adipose-derived stem cell; ADSC)인 것인, 줄기세포 배양액의 제조방법.Wherein the stem cells are adipose-derived stem cells (ADSC), a method for producing a stem cell culture medium.
  13. PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물을 유효성분으로 포함하는 간 섬유증 개선용 식품 조성물.A food composition for improving liver fibrosis comprising a stem cell culture transformed with PTX3 protein or miR150 as an active ingredient.
  14. 제13항에 있어서,According to claim 13,
    상기 식품은 건강기능식품인 것인, 간 섬유증 개선용 식품 조성물.The food is a health functional food, a food composition for improving liver fibrosis.
  15. PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물, 또는 상기 단백질 또는 상기 배양물 중 어느 하나 이상을 유효성분으로 포함하는 조성물을 이를 필요로 하는 개체에게 투여하는 방법을 포함하는 간 섬유증의 예방, 개선 또는 치료 방법.Preventing, improving or treating liver fibrosis, including a method of administering a stem cell culture transformed with PTX3 protein or miR150, or a composition comprising at least one of the protein or the culture as an active ingredient to a subject in need thereof.
  16. PTX3 단백질 또는 miR150으로 형질전환된 줄기세포 배양물, 또는 상기 단백질 또는 상기 배양물 중 어느 하나 이상을 유효성분으로 포함하는 조성물의 간 섬유증의 예방, 개선 또는 치료 용도.Use of a stem cell culture transformed with PTX3 protein or miR150, or a composition comprising at least one of the protein or the culture as an active ingredient for preventing, improving or treating liver fibrosis.
  17. 간 섬유증의 예방, 개선 또는 치료제를 제조하기 위한 PTX3 단백질, 또는 miR150으로 형질전환된 줄기세포 배양물 또는 상기 단백질 또는 상기 배양물 중 어느 하나 이상을 유효성분으로 포함하는 조성물의 용도.Use of a stem cell culture transformed with PTX3 protein or miR150, or a composition containing at least one of the protein or the culture as an active ingredient for producing a preventive, ameliorative, or therapeutic agent for liver fibrosis.
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