WO2017018698A1 - Composition for preventing or treating tissue fibrosis using milk fat globule-egf factor (mfg-e8) - Google Patents

Composition for preventing or treating tissue fibrosis using milk fat globule-egf factor (mfg-e8) Download PDF

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WO2017018698A1
WO2017018698A1 PCT/KR2016/007611 KR2016007611W WO2017018698A1 WO 2017018698 A1 WO2017018698 A1 WO 2017018698A1 KR 2016007611 W KR2016007611 W KR 2016007611W WO 2017018698 A1 WO2017018698 A1 WO 2017018698A1
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mfg
fibrosis
tissue fibrosis
stem cells
mesenchymal stem
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PCT/KR2016/007611
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French (fr)
Korean (ko)
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김종훈
안수연
장유진
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고려대학교 산학협력단
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Priority to CN201680002518.6A priority Critical patent/CN106794223B/en
Publication of WO2017018698A1 publication Critical patent/WO2017018698A1/en

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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
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • 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
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility

Definitions

  • the present invention relates to the use of MFG-E8 used in the prevention or treatment of tissue fibrosis through the fibrosis reduction effect of Milk fat globule-EGF factor 8 (MFG-E8), a protein secreted from mesenchymal stem cells.
  • MFG-E8 Milk fat globule-EGF factor 8
  • mesenchymal mesenchymal stem cells may cross-differentiate into endoderm hepatocytes beyond the germological limits of germ cells.
  • this is not a result of cross-differentiation, but rather by activating factors secreted by mesenchymal stem cells after transplantation, the death of hepatocytes in host tissues is suppressed and the regeneration of damaged host liver tissues itself is promoted. It is suggested that it can be effects.
  • hepatitis C causes viral hepatitis, including hepatitis C, fatty liver, alcoholic liver disease, liver cancer, acute and chronic liver cirrhosis, and congenital metabolic abnormalities (inherited). This includes a variety of diseases, including metabolic diseases and bile duct disease or genetic liver damage.
  • Liver fibrosis is a disease caused by abnormal accumulation of ECM that can lead to cirrhosis or liver cancer.
  • chemokines secreted from damaged hepatocytes or vascular cells accumulate macrophages, and hepatic stellate cells present in hepatic tissue due to secreted TGF ⁇ secrete myofibroblast- like cells to produce ECM, but little is known about how to prevent and treat them.
  • renal fibrosis refers to a condition in which the tissues and / or blood vessels of the kidney are hardened
  • pulmonary fibrosis or pulmonary fibrosis is a disease mainly characterized by dry cough or dyspnea during labor, which is characterized by diffuse fibrosis on the alveolar wall.
  • the MFG-E8 protein is a protein found in mammals, and contains an arginine-glycine-aspartic acid motif as well as a phosphatidylserine binding domain, which can bind to intergreen. MFG-E8 binds to phosphatidylserine exposed on the surface of apoptotic cells and mediates the apoptosis of dead cells through opsonin action of apoptotic cells and binding to intergreens on the surface of phagocytes. It is known to contribute to the removal of collagen. In addition, it has been studied to inhibit the death of intestinal epithelial cells, reduce damage, and participate in neovascularization.
  • the purpose of the present invention is to investigate the effect on the fibrosis of secretory protein secreted from mesenchymal stem cells induced in mesenchymal stem cells, and based on the fibrosis inhibitory effect of MFG-E8, one of the secretoproteins, It provides a pharmaceutical use for the prophylaxis or treatment.
  • Another object of the present invention is to provide a use as a cell therapy for the treatment of tissue fibrosis of hepatocytes induced by MFG-E8 and mesenchymal stem cells.
  • Still another object of the present invention is to provide a method for screening a medicament for preventing or treating tissue fibrosis by measuring the expression or secretion level of the MFG-E8.
  • Another object of the present invention is to provide a method for selecting mesenchymal stem cells having improved tissue fibrosis therapeutic ability by measuring or determining the level of increased expression or secretion of MFG-E8 in mesenchymal stem cells.
  • the present invention provides a composition for preventing or treating tissue fibrosis comprising Milk fat globule-EGF factor 8 (MFG-E8).
  • MFG-E8 Milk fat globule-EGF factor 8
  • the invention also provides the use of MFG-E8 for the preparation of a pharmaceutical composition for the prevention or treatment of tissue fibrosis.
  • the invention also provides a method of treating tissue fibrosis comprising administering to a subject a pharmaceutically effective amount of MFG-E8.
  • the present invention also provides a composition for the prevention or treatment of tissue fibrosis comprising a secretome of hepatocytes induced and differentiated from mesenchymal stem cells.
  • the present invention also provides the use of a secretory of induced differentiated hepatocytes in mesenchymal stem cells for the preparation of a pharmaceutical composition for the prevention or treatment of tissue fibrosis.
  • the present invention also provides a method for the treatment of tissue fibrosis comprising administering to a subject a secretory of differentiated hepatocytes induced in mesenchymal stem cells.
  • the invention also relates to MFG-E8;
  • tissue fibrosis including hepatocytes.
  • the present invention also comprises the steps of contacting the candidate drug with a secretory or MFG-E8 of induced differentiated hepatocytes in mesenchymal stem cells; And determining whether the candidate drug increases the expression or secretion level of MFG-E8 to determine the candidate drug as a drug for the prevention or treatment of tissue fibrosis. to provide.
  • the present invention also provides a method for screening mesenchymal stem cells with improved tissue fibrosis therapeutic ability, comprising measuring or determining whether the level of MFG-E8 expression or secretion of any mesenchymal stem cells is increased compared to normal mesenchymal stem cells. To provide.
  • the present invention reduces the expression of collagen induced through TGF ⁇ / Smad signaling pathway, and in particular, serves to improve liver fibrosis by inhibiting the activity of hepatic stellate cells, and to reduce the degree of liver fibrosis in liver fibrosis disease model.
  • MFG-E8 protein that inhibits activation when treated in vitro cultured hepatic stellate cells, it can be used for the prevention or treatment of tissue fibrosis such as liver, lung, kidney, brain, heart or diaphragm.
  • hepatocytes induced and differentiated from mesenchymal stem cells can be used as a cell therapy for treating tissue fibrosis with the MFG-E8.
  • Figure 1a is a liver model of chronic liver disease, hpUCMSC secreto, hpUCMSC secreto treated with MFG-E8 antibody treated with the creatine and MFG-E8 synthetic protein, respectively, liver and H & E, serial red and MT staining
  • Figure 1b is a graph comparing the structure of the tissue and the degree of fibrosis
  • Figure 1b is a graph showing the results confirmed in Figure 1a
  • Figure 1c is a serious red liver collagen by treatment with MFG-E8 protein concentration in a mouse model of chronic liver disease
  • FIG. 1D shows the same experiment as that of FIG. 1C in the hepatic fibrosis mouse model
  • FIG. 1E shows a photograph of the results of FIG. 1D observed with an optical microscope.
  • Figure 2a is a photograph showing the expression of ⁇ -SMA by treatment of three synthetic proteins of decorin, PEDF and MFG-E8 after activating hTert-HSCs, a hepatic stellate cell line with TGF ⁇ 1
  • Figure 2b is the result of Figure 2a
  • Figure 2c shows a Western blot
  • Figure 2c shows the activity of hepatic stellate cells (HSCs) after reducing the activity of the protein by treating the antibody of the three proteins to hpUCMSC secreto
  • Figure 2d is the primary culture of human As a result of confirming the expression of ⁇ -SMA by treating three proteins in the HSCs (Human primary HSCs), FIG.
  • FIG. 2E is a result of treating MFG-E8 with concentrations in hTert-HSCs.
  • Figure 2g confirms the expression of MFG-E8 protein in various mesenchymal stem cell secretes
  • Figure 2h shows the result confirmed by the ELISA results of Figure 2g.
  • the present invention relates to a composition for preventing or treating tissue fibrosis comprising milk fat globule-EGF factor 8 (MFG-E8).
  • MFG-E8 milk fat globule-EGF factor 8
  • the present invention also provides the use of MFG-E8 for the preparation of a pharmaceutical composition for the prevention or treatment of tissue fibrosis.
  • the tissue fibrosis is caused by accumulation of extracellular matrix (ECM) through TGF ⁇ / Smad signaling pathway in lung, kidney, liver, brain, heart or diaphragm tissue, and MFG-E8 through TGF ⁇ / Smad signaling pathway. It is characterized by reducing the expression of collagen or inhibiting the activation of hepatic stellate cells to reduce hepatic fibrosis.
  • ECM extracellular matrix
  • ⁇ -SMA Smooth Muscle Actin
  • a marker of liver tissue Expression decreased and fibrosis decreased.
  • the antibody against the MFG-E8 protein was treated with the secreto and administered to a mouse model of chronic liver disease, and the effect of reducing ⁇ -SMA expression did not occur.
  • MFG-E8 synthetic protein was administered to a mouse model of chronic liver disease, and it was confirmed that ⁇ -SMA expression similar to the secretory hepatic cell was reduced.
  • MFG-E8 protein administration reduces the accumulation of collagen, and this reducing effect is manifested in a concentration dependent manner of MFG-E8.
  • MFG-E8 protein also showed a reduction in fibrosis in the liver fibrosis mouse model.
  • MFG-E8 is a variety of mesenchymal stem cells, such as Umbilical Cord Mesenchymal Stem Cells (UCMSC), stem cells from Human Exfoliated Deciduous teeth (SHED), bone marrow-derived stem cells Bone Marrow Stem Cell (BMSC) and the like are expressed in the secretory or secretory of hepatocytes derived from these stem cells, but not in human embryonic stem cells.
  • UMSC Umbilical Cord Mesenchymal Stem Cells
  • SHED Human Exfoliated Deciduous teeth
  • BMSC bone marrow-derived stem cells
  • MFG-E8 can be used for the prevention or treatment of tissue fibrosis through a fibrosis inhibitory function.
  • the MFG-E8 comprises a natural or recombinant MFG-E8, or a protein having a physiological activity substantially equivalent to these. Proteins having substantially equivalent physiological activity include native / recombinant MFG-E8 and its functional equivalents and functional derivatives.
  • the “functional equivalent” refers to an amino acid sequence variant in which some or all of the natural protein amino acids are substituted or a part of the amino acids are deleted or added, and have substantially the same physiological activity as the native MFG-E8.
  • мно derivative is meant a protein that has been modified to increase or decrease the physicochemical properties of the MFG-E8 protein and has substantially the same physiological activity as the native MFG-E8.
  • the MFG-E8 may be a protein derived from mammals such as humans, mice, and rats.
  • the MFG-E8 can be prepared by a genetic engineering method known to those skilled in the art from a known sequence, such as the sequence of human MFG-E8 published in GenBank NM_005928.
  • Recombinant MFG-E8 can be separated by a conventional column chromatography method, and the degree of purification of the protein can be confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). have.
  • the present invention also relates to a composition for the prevention or treatment of tissue fibrosis, including the secretory hepatocellular induced induced differentiation of mesenchymal stem cells.
  • the present invention also provides the use of a secretory of induced hepatocytes derived from mesenchymal stem cells for the preparation of a pharmaceutical composition for the prevention or treatment of tissue fibrosis.
  • the secretory of hepatocytes induced and differentiated from mesenchymal stem cells contains the MFG-E8, and the secretory of hepatocytes induced from mesenchymal stem cells can be used for the prevention or treatment of tissue fibrosis. have.
  • the “differentiation” refers to the process of changing the structure and shape from stem cells to specific cells, and refers to the process of changing the structure and shape suitable for performing each function.
  • the differentiation includes spontaneous differentiation and induced differentiation. Induced differentiation from the stem cells to specific cells can be performed using various methods known in the art or by applying the same.
  • the secreto generally refers to a mixture of organic and inorganic elements secreted from cells, tissues, organs, organisms, and more specifically, refers to the secreted protein, induced in mesenchymal stem cells of the present invention
  • the secretory of differentiated hepatocytes contains MFG-E8.
  • the secreto may be obtained by differentiating mesenchymal stem cells in serum medium and concentrating the culture after a certain time of culturing, but not limited thereto.
  • TGF- ⁇ myofibroblast, myofibroblasts
  • myofibroblasts myofibroblasts
  • tissue fibrosis may be fibrosis occurring in any one of the tissues of the lung, kidney, liver, brain, heart or diaphragm.
  • composition for preventing or treating tissue fibrosis of the present invention may further include a pharmaceutically acceptable carrier.
  • Such pharmaceutically acceptable carriers include carriers and vehicles commonly used in the pharmaceutical arts, and in particular, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer materials (eg, Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g.
  • protamine sulfate disodium hydrogen phosphate, carbohydrogen phosphate, sodium chloride and zinc salts
  • gelatinous Silica magnesium trisilicate
  • polyvinylpyrrolidone polyvinylpyrrolidone
  • cellulosic substrates polyethylene glycols, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycols or wool, and the like.
  • composition of the present invention may further include a lubricant, a humectant, an emulsifier, a suspending agent, or a preservative in addition to the above components.
  • the composition according to the invention may be prepared in an aqueous solution for parenteral administration, preferably a buffered solution such as Hanks' solution, Ringer's solution or physically buffered saline. Can be used.
  • Aqueous injection suspensions can be added with a substrate that can increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.
  • compositions of the present invention may be administered systemically or topically and may be formulated in suitable formulations by known techniques for such administration.
  • oral administration it can be administered by mixing with an inert diluent or an edible carrier, sealed in hard or soft gelatin capsules, or pressed into tablets.
  • the active compounds can be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like.
  • MFG-E8 is well soluble in saline or buffer solution, store it in a freeze-dried state, and then administer an effective amount of MFG-E8 in saline or buffer in a form suitable for intravenous, subcutaneous, intramuscular, intraperitoneal, or transdermal administration. It may be administered by formulating a solution immediately before.
  • Suitable dosages of the compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of the patient, food, time of administration, route of administration, rate of excretion and response to reaction. have.
  • the dosage of the composition of the present invention may be administered to an adult in an amount of 0.1 to 1000 mg / kg, preferably in a dose of 10 to 100 mg / kg, once to several times daily.
  • the invention also provides a method of treating tissue fibrosis comprising administering to a subject a pharmaceutically effective amount of MFG-E8.
  • the present invention also provides a method for treating tissue fibrosis comprising administering to a subject a secreto of induced hepatocyte differentiated mesenchymal stem cells.
  • the secretory and administration method of MFG-E8 or mesenchymal stem cells derived from the mesenchymal stem cells used in the method for the treatment of tissue fibrosis follows the criteria and the administration method of the above-described pharmaceutical composition, and therefore, common content between the two. Is omitted in order to avoid excessive complexity of the present specification.
  • the subject may be a dog, a cat, a mouse, a human, or the like, but is not limited thereto.
  • the tissue fibrosis may be fibrosis occurring in any one of the tissues of the lung, kidney, liver, brain, heart or diaphragm.
  • the invention also relates to MFG-E8;
  • It relates to a cell therapy for the treatment of tissue fibrosis comprising hepatocytes.
  • the cell therapy can further increase the therapeutic effect when treating tissue fibrosis through cell transplantation of hepatocytes.
  • the hepatocytes may be hepatocytes induced and differentiated from mesenchymal stem cells, but are not particularly limited thereto.
  • Hepatocytes induced and differentiated from the mesenchymal stem cells may be used interchangeably with the term “hepatocyte-like cells” or "like hepatocytes.”
  • fibrosis which occurs in the brain, heart, and diaphragm, is also caused by TGF- ⁇ -activated astrocytic cells (myofibroblast, myofibroblasts), and thus the brain, heart, diaphragm, and the like through cell therapy using MFG-E8 and hepatocytes. It can also be used to treat fibrosis.
  • the present invention also comprises the steps of contacting the candidate drug with a secretory or MFG-E8 of induced differentiated hepatocytes in mesenchymal stem cells; And determining whether the candidate drug increases the expression or secretion level of MFG-E8, and thus, determining the candidate drug as a drug for preventing or treating tissue fibrosis. It is about.
  • the candidate drug has a potential as a drug that promotes or inhibits MFG-E8 gene sequencing, translation or translation into MFG-E8 gene, or a drug that enhances or inhibits the function or activity of MFG-E8 protein.
  • the expression of the MFG-E8 gene, the amount of protein or the activity of the protein can be measured in the cells treated with the candidate drug.
  • the candidate drug may be determined as a substance capable of treating or preventing tissue fibrosis.
  • the method of measuring the expression amount of the gene, the amount of the protein or the activity of the protein in the above may be carried out through a variety of methods known in the art, for example, but not limited to reverse transcriptase polymerase chain reaction (reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion (radioimmunodiffusion) And immunoprecipitation assays.
  • reverse transcriptase polymerase chain reaction reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion (radioimmunodiffusion)
  • immunoprecipitation assays for example, but not limited to reverse transcriptase polymerase
  • Such candidate candidates for treating tissue fibrosis may act as leading compounds in the development of tissue fibrosis therapeutics in the future, and may be effective in promoting the function of the MFG-E8 gene or a protein expressed therefrom.
  • new therapeutic agents for tissue fibrosis can be developed.
  • the present invention also provides a method for screening mesenchymal stem cells with improved tissue fibrosis treatment, comprising measuring or determining whether the level of MFG-E8 expression or secretion of any mesenchymal stem cells is increased compared to normal mesenchymal stem cells. It is about a method.
  • the present invention discloses for the first time that the active ingredient of mesenchymal stem cells for treating tissue fibrosis is secreted from MFG-E8, so that the mesenchymal stem cells with increased expression or secretion level of MFG-E8 are improved in the ability to treat tissue fibrosis. can see.
  • expression or secretion levels of MFG-E8 in mesenchymal stem cells may serve as a basis for improving the pharmacological therapeutic effect of mesenchymal stem cells used in the treatment of tissue fibrosis.
  • the mesenchymal stem cells used in this experiment were derived from umbilical cord blood and attached to 3 ⁇ 10 4 cells / cm 2 in a collagen-coated culture dish for differentiation into hepatocytes.
  • MesenPro RSTM medium (Gibco) was used as the basic culture medium. After 70-80% of the culture dish was filled, Epidermal growth factor (EGF; Peprotech EC Ltd, London, England, 20 ng /) was used in Iscove's modified Dulbecco's medium (IMDM; Invitrogen, Carlsbad, CA, USA). mL) and basic fibroblast growth factor (bFGF; Peprotech EC Ltd, 10 ng / mL) were added and incubated for 2 days.
  • EGF Epidermal growth factor
  • IMDM Iscove's modified Dulbecco's medium
  • bFGF basic fibroblast growth factor
  • HGF hepatocyte growth factor
  • bFGF ng / mL
  • nicotinamide Sigma, 0.61 g / L
  • insulin-transferrin- Incubated for 10 days with the addition of selenium (ITS) premix Invitrogen.
  • oncostatin M (Peprotech EC Ltd, 20 ng / mL), dexamethasone (Sigma, 1 ⁇ mol / L), 1% ITS was added to basal medium IMDM and incubated for 10 days.
  • FBS 0.05% FBS was added to basal medium IMDM, and cultured for 24 hours, followed by concentration of 25-fold with 3-kDa cutoff filter ultrafiltration units (Millipore).
  • mice C57BL / C were intraperitoneally injected with saline solution diluted Thioacetamide (TAA) to 200mg / kg body weight. Intraperitoneal injections were repeated three times a week for a total of eight weeks. Eight weeks later, the concentrated protein of creatre was quantified by Bradford assay and then intraperitoneally injected in an amount of 500 ⁇ g. After the secreto infusion, mice were analyzed for liver fibrosis 3 days and 3 days later.
  • TAA Thioacetamide
  • MFG-E8 protein (R & D systems) is administered based on 160 ⁇ g / kg body weight, and in order to confirm the effect of each concentration, 32 ⁇ g / kg body weight is low, 160 ⁇ g / kg body weight is medium and 800 ⁇ g / kg body weight was set to a high concentration was administered to the abdominal cavity. Three days after administration of MFG-E8 protein, the degree of hepatic fibrosis was analyzed.
  • liver tissue was fixed in 4% paraformaldehyde to confirm the degree of fibrosis of liver tissue.
  • the liver tissues were fixed in paraffin and obtained as tissue sections. H & E, Masson's trichrome and serial red staining were performed under an optical microscope.
  • tissue sections obtained from paraffin fixation were deparaffinized and re-fixed in Bouin's solution, followed by staining in Weigert hematoxylin / Biebrich scarlet-acid fuchsin-aniline blue solution or 2% light green. It was. Fibrous tissue appears in blue, cytoplasm, muscle, keratin in red, and nucleus in dark brown.
  • tissue sections obtained by paraffin fixation were deparaffinized, nucleus stained with hematoxylin, and then stained with a picro-sirius red dye for 1 hour.
  • the liver fibrosis-induced areas appear red.
  • tissue sections obtained by paraffin immobilization were blocked with 10% donkey serum after deparaffinization, followed by reaction with ⁇ -SMA and F4 / 80 antibody. After the reaction was added to the secondary antibody with a fluorescent label was observed by fluorescence microscope.
  • HPUCMSC secreto and the HPUCMSC secreto treated with MFG-E8 antibody were treated with the chronic liver disease mouse model made with TAA and MFG-E8 synthetic protein, respectively, and H & E, serial red,
  • fibrosis was significantly reduced in the hpUCMSC secreto and MFG-E8 protein groups.
  • the group treated with MFG-E8 antibody developed more fibrosis than sham.
  • the expression of ⁇ -SMA was also decreased in the group treated with hpUCMSC secreto and MFG-E8 proteins than sham (FIGS. 1A and 1B).
  • F4 / 80 a marker of macrophages, was identified as a control, but there was no difference between groups.
  • MFG-E8 protein was treated by concentration to confirm collagen accumulation in serial red, and fibrosis was reduced compared to sham (FIG. 1C).
  • hepatic stellate cell lines were cultured in culture medium containing 10% FBS, 100 units of penicillin, and 100 ⁇ g of streptomycin in basal medium DMEM.
  • culture medium added 0.2% FBS to the basal medium DMEM was incubated for 24 hours, and then treated with 10 ng / mL TGF ⁇ 1 protein in the same medium to activate the hepatic stellate cell line.
  • the MFG-E8 protein used in the experiment was basically treated with TGF ⁇ 1 protein to be 500ng / mL and incubated for 48 hours. If the treatment by concentration, incubated to 100ng / mL, 250ng / mL, 500ng / mL, 1 ⁇ g / mL, 5 ⁇ g / mL. In order to neutralize the protein in the secreto, the concentration of the antibody was 20 ⁇ g / mL and then mixed with the secreto and used for 1 hour at room temperature.
  • Figure 2a is a result of processing three synthetic proteins after activating hTert-HSCs with TGF ⁇ 1, the hepatic stellate cell line is inactivated in the serum-free state, the cells are observed to be thin and thin, but when treated with TGF ⁇ 1 is activated in a broad shape ⁇ -SMA expression is increased. At this time, the decorin, PEDF and MFG-E8 treatment all reduced the expression of ⁇ -SMA. Similar results were confirmed in Western blot results (FIG. 2B).
  • hepatic stellate cell lines proteins were extracted and Western blot was performed.
  • Cell proteins were extracted using RIPA buffer containing protease inhibitors.
  • 40 ⁇ g of protein of each group was separated using SDS-PAGE gel and transferred to polyvinylidene fluoride transfer membrane. Ponceau S solution was used to confirm that the protein was well transferred to the membrane.
  • the membrane was allowed to stand overnight in a solution containing each antibody of ⁇ -SMA, MFG-E8 and GAPDH. After reacting the secondary antibody for each antibody for 1 hour at room temperature, the expression of the protein was confirmed using a chemiluminescence kit (chemiluminescence kit).
  • chemiluminescence kit chemiluminescence kit
  • MFG-E8 protein is induced and differentiated from various stem cells (umbilical cord blood-derived mesenchymal stem cells (UCMSC), degenerating stem cells (SHED), bone marrow-derived stem cells (BMSC)) and these stem cells Hepatocytes (hpUCMSCs, hpSHEDs, hpBMSCs) are expressed in the secretory, but not in embryonic stem cells.
  • UMSC umbilical cord blood-derived mesenchymal stem cells
  • SHED degenerating stem cells
  • BMSC bone marrow-derived stem cells
  • the present invention can be used in the field of preventing or treating tissue fibrosis.

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Abstract

The present invention relates to a composition for preventing or treating tissue fibrosis using milk fat globule-EGF factor 8 (MFG-E8). More specifically, MFG-E8 has a role in suppressing the expression of collagen induced via TGFβ/Smad signal transmission and in suppressing the activity of hepatic stellate cells and so improving liver fibrosis, and reduces the extent of fibrosis in a mouse model having liver fibrosis disease, and suppresses activation when used to treat hepatic stellate cells cultured in vitro, so having the advantage of being able to be used as an agent for preventing or treating tissue fibrosis.

Description

Ｍｉｌｋ ｆａｔ ｇｌｏｂｕｌｅ－ＥＧＦ ｆａｃｔｏｒ（ＭＦＧ－Ｅ８）을 이용한 조직섬유화 예방 또는 치료용 조성물Composition for the prevention or treatment of tissue fibrosis using Ｍｉｌｋ ｔａｔ gｌｏｂｕｌｅ-ＥＧＦ
본 발명은 중간엽 줄기세포에서 분비된 단백질인 Milk fat globule-EGF factor 8 (MFG-E8)의 섬유화 감소 효과를 통해 조직섬유화의 예방 또는 치료에 사용하는 MFG-E8의 용도에 관한 것이다.The present invention relates to the use of MFG-E8 used in the prevention or treatment of tissue fibrosis through the fibrosis reduction effect of Milk fat globule-EGF factor 8 (MFG-E8), a protein secreted from mesenchymal stem cells.
중간엽 줄기세포(mesenchymal stem cell)에서 간세포로의 분화에 대한 연구의 경우, 초반에는 중배엽성 중간엽 줄기세포가 배엽의 발생학적 한계성을 넘어 내배엽성 간세포로 교차분화(transdifferentiation) 할 가능성이 있다고 보고되었으나, 최근 축적되는 연구결과에 의하면 이는 교차분화의 결과가 아니라 중간엽 줄기세포가 이식된 후 분비하는 활성인자에 의해 호스트 조직 내의 간세포의 사멸이 억제되고 손상된 호스트 간조직 자체의 재생이 촉진(paracrine effects) 될 수 있다는 가능성이 제시되고 있다. Studies on the differentiation of mesenchymal stem cells into hepatocytes initially reported that mesenchymal mesenchymal stem cells may cross-differentiate into endoderm hepatocytes beyond the germological limits of germ cells. However, according to recent accumulating results, this is not a result of cross-differentiation, but rather by activating factors secreted by mesenchymal stem cells after transplantation, the death of hepatocytes in host tissues is suppressed and the regeneration of damaged host liver tissues itself is promoted. It is suggested that it can be effects.
줄기세포를 이용한 세포치료의 적응증 중 간 기능의 저해를 초래하는 원인으로는 C형 간염(hepatitis C)을 포함한 바이러스성 간염, 지방간, 알코올성 간 질환, 간암, 급성·만성 간경화, 선천성 대사 이상(inherited metabolic diseases) 및 담관질환(bile duct disease) 또는 유전적 간 기능 손상 등의 다양한 질병이 이에 해당된다.Among the indications for stem cell therapy using stem cells, hepatitis C causes viral hepatitis, including hepatitis C, fatty liver, alcoholic liver disease, liver cancer, acute and chronic liver cirrhosis, and congenital metabolic abnormalities (inherited). This includes a variety of diseases, including metabolic diseases and bile duct disease or genetic liver damage.
최근 줄기세포에서 분비되는 단백질이 각종 조직의 재생 및 면역조절에 관여한다는 증거가 축적되고 있다. 현재 단백질 의약품 시장은 생명공학 분야에서 대표적인 유망사업으로 급부상하고 있으며 소화기를 통한 흡수로 온몸에 영향을 주는 화학합성 의약품보다 치료효과가 높고 부작용이 적으며 기존 화학합성 의약품에 비해 개발기간이 짧으면서 가격은 높게 유지되는 시장가치적 강점이 있다. 따라서 세포치료제와 더불어 줄기세포 및 분화된 세포로부터 분비되어 조직재생 및 재건에 중요한 역할을 하는 분비단백질의 신규기능을 발굴할 경우 기능성 및 안정성 강화를 위한 단백질 공학적 공정을 거쳐 고부가가치의 단백질의약품으로 개발될 가능성이 있다.Recently, evidence is accumulating that proteins secreted from stem cells are involved in the regeneration and immune regulation of various tissues. Currently, the protein pharmaceutical market is emerging as a promising business in the biotechnology field, and it has a higher therapeutic effect and fewer side effects than the chemical synthetic drugs that affect the whole body by absorption through the digestive organs, and the development period is shorter than the conventional chemical synthetic drugs. Has market value strengths that remain high. Therefore, when discovering new functions of secretory proteins that are secreted from stem cells and differentiated cells as well as cell therapy products and play an important role in tissue regeneration and reconstruction, they are developed as high value-added protein drugs through protein engineering process to enhance functionality and stability. There is a possibility.
다양한 조직에서 중간엽 줄기세포의 파라크린(paracrine) 효과에 대해 보고가 이루어지고 있다. 그러나 분비된 물질에 대한 자세한 메커니즘이 알려져 있지 않고, 중간엽 줄기세포에서 분화된 세포의 특성에 대해 논란이 여전히 존재하기에 중간엽 줄기세포나 분화된 세포를 직접적으로 치료제로 사용하기는 제약이 따른다. 따라서 직접적인 세포를 이용한 치료제보다는 그로부터 분비된 물질을 이용한 치료제 발굴이 각광받고 있는 추세이다.There have been reports of paracrine effects of mesenchymal stem cells in various tissues. However, the detailed mechanisms of secreted substances are not known, and controversy still exists regarding the properties of differentiated cells in mesenchymal stem cells, which makes it difficult to use mesenchymal stem cells or differentiated cells directly as therapeutic agents. . Therefore, the discovery of therapeutic agents using materials secreted therefrom rather than direct therapeutic agents is in the spotlight.
간섬유화는 ECM이 비정상적으로 축적되어 생기는 질병으로 간경변이나 간암으로 발전할 수 있다. 간섬유화가 일어나면 손상된 간세포나 혈관세포에서 분비된 케모카인(chemokine)이 대식세포를 모이게 하고, 더불어 분비된 TGFβ로 인해 간조직내 존재하는 간성상세포(hepatic stellate cell)가 근섬유모세포 유사 세포(myofibroblast-like cell)이 되어 ECM 생산을 하게 되나, 이를 예방 및 치료하는 물질이나 방법에 대해서는 많이 알려진 바가 없다.Liver fibrosis is a disease caused by abnormal accumulation of ECM that can lead to cirrhosis or liver cancer. When hepatic fibrosis occurs, chemokines secreted from damaged hepatocytes or vascular cells accumulate macrophages, and hepatic stellate cells present in hepatic tissue due to secreted TGFβ secrete myofibroblast- like cells to produce ECM, but little is known about how to prevent and treat them.
또한, 신장섬유화는 신장의 조직 및/또는 혈관이 단단하게 굳어지는 증상을 의미하며, 폐섬유화 또는 폐섬유증은 폐포벽에 미만성 섬유 증식을 특징으로 하는 건성 기침이나 노동 시 호흡 곤란을 주 증상으로 하는 질환으로 알려져 있다.In addition, renal fibrosis refers to a condition in which the tissues and / or blood vessels of the kidney are hardened, and pulmonary fibrosis or pulmonary fibrosis is a disease mainly characterized by dry cough or dyspnea during labor, which is characterized by diffuse fibrosis on the alveolar wall. Known as
줄기세포 혹은 줄기세포에서 분화된 세포들이 이식된 후, 세포대체효과(repopulation by donor cells)와 더불어 손상된 조직주변을 다양한 분비성 인자들로서 자극함으로써 숙주조직 자체의 재생 및 복구(regeneration of host tissue)에 기여할 가능성을 시사하고 있으나 이에 관련된 연구는 현재까지 매우 미미한 실정이다. 나아가 현재까지 근본적으로 질병 자체를 치료할 수 있는 섬유화에 대한 알려진 치료제가 없다.After transplantation of stem cells or differentiated cells from stem cells, the regeneration of host tissues is regenerated and stimulated by various secretory factors, along with repopulation by donor cells. It suggests the possibility of contributing, but the research related to this is very small to date. Furthermore, to date there is no known cure for fibrosis that can cure the disease itself.
MFG-E8 단백질은 포유동물에서 발견되는 단백질로, 아르기닌-글리신-아스파르트산 모티프뿐만 아니라 포스파티딜세린 결합 도메인을 포함하고 있어 인터그린과 결합할 수 있다. MFG-E8은 세포사멸 세포의 표면에 노출된 포스파티딜세린과 결합하여 세포사멸 세포의 옵소닌 작용과 식세포의 표면에 있는 인터그린과의 결합을 통해 죽은 세포의 포식을 매개하는 기능이 있고, 축적된 콜라겐을 제거하는 데 기여한다고 알려져 있다. 또한, 장내 상피세포의 사멸을 억제하고, 손상을 줄여줌과 더불어 신생혈관 형성에 관여한다는 것에 대해 연구된 바 있다. 또한, 인간 배아줄기세포에서 유도 분화된 간세포에서 높은 발현율을 높이며, 간세포 증식 및 혈관 재생을 촉진하여 간재생 및 간질환 개선에 사용할 수 있다고 보고되어 있다(특허문헌 1 참조). 그러나 간조직 내 간섬유화 또는 이외의 다른 조직 내 섬유화 단계에서 어떤 영향을 끼치는지 보고된 바가 없는 실정이다.The MFG-E8 protein is a protein found in mammals, and contains an arginine-glycine-aspartic acid motif as well as a phosphatidylserine binding domain, which can bind to intergreen. MFG-E8 binds to phosphatidylserine exposed on the surface of apoptotic cells and mediates the apoptosis of dead cells through opsonin action of apoptotic cells and binding to intergreens on the surface of phagocytes. It is known to contribute to the removal of collagen. In addition, it has been studied to inhibit the death of intestinal epithelial cells, reduce damage, and participate in neovascularization. In addition, it has been reported that it can be used to improve liver regeneration and liver disease by increasing high expression rate in induced hepatocytes induced from human embryonic stem cells and promoting hepatocyte proliferation and blood vessel regeneration (see Patent Document 1). However, there has been no report on the effect of hepatic fibrosis in liver tissue or in other tissue fibrosis stages.
본 발명의 목적은 중간엽 줄기세포에서 유도 분화된 간세포에서 분비된 세크레톰 단백질의 섬유화에 미치는 효과를 규명하여 상기 세크레톰 단백질 중 하나인 MFG-E8의 섬유화 억제 효과를 기반으로 조직섬유화의 예방 또는 치료에 사용하는 약제학적 용도를 제공하는 것이다.The purpose of the present invention is to investigate the effect on the fibrosis of secretory protein secreted from mesenchymal stem cells induced in mesenchymal stem cells, and based on the fibrosis inhibitory effect of MFG-E8, one of the secretoproteins, It provides a pharmaceutical use for the prophylaxis or treatment.
본 발명의 다른 목적은 상기 MFG-E8과 중간엽 줄기세포에서 유도 분화된 간세포의 조직섬유화 치료용 세포치료제로서의 용도를 제공하는 것이다.Another object of the present invention is to provide a use as a cell therapy for the treatment of tissue fibrosis of hepatocytes induced by MFG-E8 and mesenchymal stem cells.
본 발명의 또 다른 목적은 상기 MFG-E8의 발현 또는 분비 수준의 측정을 통해 조직섬유화의 예방 또는 치료용 의약의 스크리닝 방법을 제공하는 것이다.Still another object of the present invention is to provide a method for screening a medicament for preventing or treating tissue fibrosis by measuring the expression or secretion level of the MFG-E8.
본 발명의 또 다른 목적은 중간엽 줄기세포에서 MFG-E8의 증가된 발현 또는 분비 수준의 측정 또는 결정을 통해 조직섬유화 치료능이 개선된 중간엽 줄기세포의 선별방법을 제공하는 것이다.Another object of the present invention is to provide a method for selecting mesenchymal stem cells having improved tissue fibrosis therapeutic ability by measuring or determining the level of increased expression or secretion of MFG-E8 in mesenchymal stem cells.
상기 목적을 달성하기 위하여, 본 발명은 Milk fat globule-EGF factor 8 (MFG-E8)을 포함하는 조직섬유화의 예방 또는 치료용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for preventing or treating tissue fibrosis comprising Milk fat globule-EGF factor 8 (MFG-E8).
본 발명은 또한 조직섬유화의 예방 또는 치료를 위한 약제학적 조성물의 제조를 위한 MFG-E8의 용도를 제공한다.The invention also provides the use of MFG-E8 for the preparation of a pharmaceutical composition for the prevention or treatment of tissue fibrosis.
본 발명은 또한 약학적 유효량의 MFG-E8을 개체에 투여하는 단계를 포함하는 조직섬유화의 치료방법을 제공한다.The invention also provides a method of treating tissue fibrosis comprising administering to a subject a pharmaceutically effective amount of MFG-E8.
본 발명은 또한 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰(secretome)을 포함하는 조직섬유화의 예방 또는 치료용 조성물을 제공한다.The present invention also provides a composition for the prevention or treatment of tissue fibrosis comprising a secretome of hepatocytes induced and differentiated from mesenchymal stem cells.
본 발명은 또한 조직섬유화의 예방 또는 치료를 위한 약제학적 조성물의 제조를 위한 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰의 용도를 제공한다. The present invention also provides the use of a secretory of induced differentiated hepatocytes in mesenchymal stem cells for the preparation of a pharmaceutical composition for the prevention or treatment of tissue fibrosis.
본 발명의 또한 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰을 개체에 투여하는 단계를 포함하는 조직섬유화의 치료방법을 제공한다.The present invention also provides a method for the treatment of tissue fibrosis comprising administering to a subject a secretory of differentiated hepatocytes induced in mesenchymal stem cells.
본 발명은 또한 MFG-E8; 및The invention also relates to MFG-E8; And
간세포를 포함하는 조직섬유화 치료용 세포치료제를 제공한다.Provided is a cell therapy for treating tissue fibrosis, including hepatocytes.
본 발명은 또한 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰 또는 MFG-E8을 후보약물과 접촉시키는 단계; 및 상기 후보약물이 MFG-E8의 발현 또는 분비 수준을 증가시키는지 측정하여 상기 후보약물을 조직섬유화의 예방 또는 치료용 의약으로 판정하는 단계를 포함하는 조직섬유화의 예방 또는 치료용 의약의 스크리닝 방법을 제공한다.The present invention also comprises the steps of contacting the candidate drug with a secretory or MFG-E8 of induced differentiated hepatocytes in mesenchymal stem cells; And determining whether the candidate drug increases the expression or secretion level of MFG-E8 to determine the candidate drug as a drug for the prevention or treatment of tissue fibrosis. to provide.
본 발명은 또한 정상 중간엽 줄기세포 대비 임의의 중간엽 줄기세포의 MFG-E8의 발현 또는 분비 수준의 증가 여부를 측정 또는 결정하는 단계를 포함하는 조직섬유화 치료능이 개선된 중간엽 줄기세포의 선별방법을 제공한다.The present invention also provides a method for screening mesenchymal stem cells with improved tissue fibrosis therapeutic ability, comprising measuring or determining whether the level of MFG-E8 expression or secretion of any mesenchymal stem cells is increased compared to normal mesenchymal stem cells. To provide.
본 발명은 TGFβ/Smad 신호전달경로를 통해 유도된 콜라겐의 발현을 감소하고, 특히, 간성상세포의 활성을 억제하여 간섬유화를 호전시키는 역할을 하며, 간섬유화 질환 모델에서 간섬유화 정도를 감소시키고, 체외 배양된 간성상세포에 처리하면 활성화를 억제시키는 MFG-E8 단백질의 특성을 기반으로 하여 간, 폐, 신장, 뇌, 심장 또는 횡경막 등의 조직섬유화의 예방 또는 치료에 사용할 수 있다.The present invention reduces the expression of collagen induced through TGFβ / Smad signaling pathway, and in particular, serves to improve liver fibrosis by inhibiting the activity of hepatic stellate cells, and to reduce the degree of liver fibrosis in liver fibrosis disease model. Based on the properties of MFG-E8 protein that inhibits activation when treated in vitro cultured hepatic stellate cells, it can be used for the prevention or treatment of tissue fibrosis such as liver, lung, kidney, brain, heart or diaphragm.
또한, 중간엽 줄기세포에서 유도 분화된 간세포는 상기 MFG-E8과 함께 조직섬유화 치료용 세포치료제로 사용할 수 있다.In addition, hepatocytes induced and differentiated from mesenchymal stem cells can be used as a cell therapy for treating tissue fibrosis with the MFG-E8.
도 1a는 만성 간질환 마우스 모델에 hpUCMSC 세크레톰, hpUCMSC 세크레톰에 MFG-E8 항체를 처리한 세크레톰 및 MFG-E8 합성 단백질을 각각 처리하여 H&E, 씨리어스 레드 및 MT 염색을 통해 간조직의 구조 및 섬유화 정도를 비교한 사진도, 도 1b는 상기 도 1a에서 확인한 결과를 그래프로 나타낸 도면, 도 1c는 만성 간질환 마우스 모델에서 MFG-E8 단백질을 농도별로 처리하여 씨리어스 레드로 콜라겐 축적을 확인한 결과, 도 1d는 간섬유화 마우스 모델에서 상기 도 1c와 같은 실험을 수행한 결과, 도 1e는 상기 도 1d의 결과를 광학현미경으로 관찰한 사진도를 나타낸 것이다.Figure 1a is a liver model of chronic liver disease, hpUCMSC secreto, hpUCMSC secreto treated with MFG-E8 antibody treated with the creatine and MFG-E8 synthetic protein, respectively, liver and H & E, serial red and MT staining Figure 1b is a graph comparing the structure of the tissue and the degree of fibrosis, Figure 1b is a graph showing the results confirmed in Figure 1a, Figure 1c is a serious red liver collagen by treatment with MFG-E8 protein concentration in a mouse model of chronic liver disease As a result of confirming the accumulation, FIG. 1D shows the same experiment as that of FIG. 1C in the hepatic fibrosis mouse model, and FIG. 1E shows a photograph of the results of FIG. 1D observed with an optical microscope.
도 2a는 간성상세포주인 hTert-HSCs를 TGFβ1으로 활성화시킨 후 데코린, PEDF 및 MFG-E8의 3가지 합성 단백질을 처리하여 α-SMA의 발현을 보여주는 사진도, 도 2b는 상기 도 2a의 결과를 웨스턴 블랏으로 보여주는 사진도, 도 2c는 hpUCMSC 세크레톰에 3가지 단백질의 항체를 처리하여 단백질의 활성을 감소시킨 후 간성상세포(HSCs)의 활성을 확인한 결과, 도 2d는 사람의 초대 배양된 HSCs(Human primary HSCs)에서 3가지 단백질을 처리하여 α-SMA의 발현을 확인한 결과, 도 2e는 hTert-HSCs에 MFG-E8을 농도별로 처리한 결과, 도 2f는 사람의 초대 배양된 HSCs에 MFG-E8을 농도별로 처리한 결과, 도 2g는 다양한 중간엽 줄기세포 세크레톰에서 MFG-E8 단백질의 발현을 확인한 결과, 도 2h는 상기 도 2g의 결과를 ELISA를 통해 확인한 결과를 나타낸 것이다.Figure 2a is a photograph showing the expression of α-SMA by treatment of three synthetic proteins of decorin, PEDF and MFG-E8 after activating hTert-HSCs, a hepatic stellate cell line with TGFβ1, Figure 2b is the result of Figure 2a Figure 2c shows a Western blot, Figure 2c shows the activity of hepatic stellate cells (HSCs) after reducing the activity of the protein by treating the antibody of the three proteins to hpUCMSC secreto, Figure 2d is the primary culture of human As a result of confirming the expression of α-SMA by treating three proteins in the HSCs (Human primary HSCs), FIG. 2E is a result of treating MFG-E8 with concentrations in hTert-HSCs. As a result of treatment of MFG-E8 by concentration, Figure 2g confirms the expression of MFG-E8 protein in various mesenchymal stem cell secretes, Figure 2h shows the result confirmed by the ELISA results of Figure 2g.
이하, 본 발명의 구성을 구체적으로 설명한다.EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.
본 발명은 Milk fat globule-EGF factor 8 (MFG-E8)을 포함하는 조직섬유화의 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating tissue fibrosis comprising milk fat globule-EGF factor 8 (MFG-E8).
또한, 본 발명은 조직섬유화의 예방 또는 치료를 위한 약제학적 조성물의 제조를 위한 MFG-E8의 용도를 제공한다.The present invention also provides the use of MFG-E8 for the preparation of a pharmaceutical composition for the prevention or treatment of tissue fibrosis.
상기 조직섬유화는 폐, 신장, 간, 뇌, 심장 또는 횡경막 조직 등에서 TGFβ/Smad 신호전달경로를 통한 세포외기질(ECM)의 축적으로 인해 생기는 것으로, MFG-E8이 TGFβ/Smad 신호전달경로를 통해 유도된 콜라겐의 발현을 억제하거나, 간성상세포의 활성화를 억제하여 간섬유화를 감소시키는 특징이 있다.The tissue fibrosis is caused by accumulation of extracellular matrix (ECM) through TGFβ / Smad signaling pathway in lung, kidney, liver, brain, heart or diaphragm tissue, and MFG-E8 through TGFβ / Smad signaling pathway. It is characterized by reducing the expression of collagen or inhibiting the activation of hepatic stellate cells to reduce hepatic fibrosis.
더 구체적으로, 본 발명의 일 구체예에 따르면, 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰을 만성 간질환 마우스 모델에 투여한 결과, 간조직의 마커인 α-SMA(Smooth Muscle Actin)의 발현이 감소되고 섬유화가 감소되었다. 상기 간세포의 세크레톰의 간섬유화 억제 효과를 확인하기 위해 MFG-E8 단백질에 대한 항체를 세크레톰에 처리하여 만성 간질환 마우스 모델에 투여한 결과, α-SMA 발현 감소 효과가 일어나지 않았다. 이를 기반으로 MFG-E8 합성 단백질을 만성 간질환 마우스 모델에 투여한 결과, 간세포의 세크레톰과 유사한 α-SMA 발현 감소 효과를 확인할 수 있었다. 또한, MFG-E8 단백질 투여는 콜라겐의 축적을 감소시키며, 이러한 감소 효과는 MFG-E8의 농도 의존적 방식으로 나타난다. 아울러, MFG-E8 단백질은 간섬유화 마우스 모델에서도 섬유화 감소 효과를 나타냈다. More specifically, according to one embodiment of the present invention, as a result of administering a secretory hepatocellular induced induced differentiation of mesenchymal stem cells to a mouse model of chronic liver disease, α-SMA (Smooth Muscle Actin), a marker of liver tissue Expression decreased and fibrosis decreased. In order to confirm the hepatic fibrosis inhibitory effect of the hepatocytes, the antibody against the MFG-E8 protein was treated with the secreto and administered to a mouse model of chronic liver disease, and the effect of reducing α-SMA expression did not occur. Based on this, the MFG-E8 synthetic protein was administered to a mouse model of chronic liver disease, and it was confirmed that α-SMA expression similar to the secretory hepatic cell was reduced. In addition, MFG-E8 protein administration reduces the accumulation of collagen, and this reducing effect is manifested in a concentration dependent manner of MFG-E8. In addition, MFG-E8 protein also showed a reduction in fibrosis in the liver fibrosis mouse model.
다음으로, 간성상세포주를 TGFβ1으로 활성화시킨 후 데코린, PEDF(Pigment Epithelium-Derived Factor) 및 MFG-E8 단백질을 처리한 결과 α-SMA의 발현이 감소되었고, 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰에 MFG-E8 단백질에 대한 항체를 처리하여 단백질의 활성을 감소시킨 후 간성상세포(HSC)에 처리하여 HSC의 활성화를 확인한 결과, α-SMA의 발현이 높아졌고, MFG-E8 단백질을 처리한 경우에는 HSC에서 α-SMA의 발현이 농도 의존적 방식으로 감소됨을 통해 MFG-E8 단백질은 간섬유화를 억제함으로 알 수 있다. Next, after activation of hepatic stellate cell line with TGFβ1 and treatment with decorin, PEDF (Pigment Epithelium-Derived Factor) and MFG-E8 protein, the expression of α-SMA was decreased and hepatic cells induced by mesenchymal stem cells were induced. Increasing the expression of α-SMA and MFG-E8 was confirmed by treatment of antibodies to the MFG-E8 protein in the secretory of the protein to reduce the activity of the protein and then in the treatment of hepatic stellate cells (HSC). In the case of protein treatment, the expression of α-SMA in HSC is reduced in a concentration-dependent manner, indicating that MFG-E8 protein inhibits liver fibrosis.
또한, MFG-E8은 다양한 중간엽 줄기세포, 예컨대, 제대혈 유래 중간엽 줄기세포(Umbilical Cord Mesenchymal Stem Cell; UCMSC), 탈락유치 줄기세포(Stem cells from Human Exfoliated Deciduous teeth; SHED), 골수 유래 줄기세포(Bone Marrow Stem Cell; BMSC) 등의 세크레톰 또는 이들 줄기세포에서 유도 분화된 간세포의 세크레톰에서 발현되나, 인간 배아줄기세포에서는 발현되지 않았다.In addition, MFG-E8 is a variety of mesenchymal stem cells, such as Umbilical Cord Mesenchymal Stem Cells (UCMSC), stem cells from Human Exfoliated Deciduous teeth (SHED), bone marrow-derived stem cells Bone Marrow Stem Cell (BMSC) and the like are expressed in the secretory or secretory of hepatocytes derived from these stem cells, but not in human embryonic stem cells.
따라서, MFG-E8은 섬유화 억제 기능을 통해 조직섬유화의 예방 또는 치료 용도로 사용할 수 있다.Therefore, MFG-E8 can be used for the prevention or treatment of tissue fibrosis through a fibrosis inhibitory function.
상기 MFG-E8은 천연형 또는 재조합 MFG-E8, 또는 이들과 실질적으로 동등한 생리 활성을 갖는 단백질을 포함한다. 실질적으로 동등한 생리 활성을 갖는 단백질에는 천연형/재조합 MFG-E8과 그 기능적 동등물(functional equivalent) 및 기능적 유도체(functional derivative)가 포함된다. The MFG-E8 comprises a natural or recombinant MFG-E8, or a protein having a physiological activity substantially equivalent to these. Proteins having substantially equivalent physiological activity include native / recombinant MFG-E8 and its functional equivalents and functional derivatives.
상기 "기능적 동등물"에는 천연형 단백질 아미노산 중 일부 또는 전부가 치환되거나, 아미노산의 일부가 결실 또는 부가된 아미노산 서열 변형체로서 천연형 MFG-E8과 실질적으로 동등한 생리활성을 갖는 것을 말한다. The "functional equivalent" refers to an amino acid sequence variant in which some or all of the natural protein amino acids are substituted or a part of the amino acids are deleted or added, and have substantially the same physiological activity as the native MFG-E8.
"기능적 유도체"는 상기 MFG-E8 단백질의 물리 화학적 성질을 증가 또는 감소시키기 위한 변형을 가한 단백질로서 천연형 MFG-E8과 실질적으로 동등한 생리 활성을 갖는 것을 의미한다.By "functional derivative" is meant a protein that has been modified to increase or decrease the physicochemical properties of the MFG-E8 protein and has substantially the same physiological activity as the native MFG-E8.
상기 MFG-E8는 인간, 생쥐, 랫트 등의 포유동물 유래의 단백질일 수 있다.The MFG-E8 may be a protein derived from mammals such as humans, mice, and rats.
또한, 상기 MFG-E8는 공지의 서열, 예컨대 GenBank NM_005928에 공개된 사람 MFG-E8의 서열로부터 당업자에 공지된 유전공학적 방법으로 제조할 수 있다.In addition, the MFG-E8 can be prepared by a genetic engineering method known to those skilled in the art from a known sequence, such as the sequence of human MFG-E8 published in GenBank NM_005928.
재조합 MFG-E8는 통상의 컬럼 크로마토그래피 방법 등을 통해 분리할 수 있고, 단백질의 정제 정도는 소듐 도데실 술페이트-폴리아크릴아마이드 젤 전기영동 (SDS-polyacrylamide gel electrophoresis (PAGE))등으로 확인할 수 있다.Recombinant MFG-E8 can be separated by a conventional column chromatography method, and the degree of purification of the protein can be confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). have.
본 발명은 또한 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰을 포함하는 조직섬유화의 예방 또는 치료용 조성물에 관한 것이다.The present invention also relates to a composition for the prevention or treatment of tissue fibrosis, including the secretory hepatocellular induced induced differentiation of mesenchymal stem cells.
또한, 본 발명은 조직섬유화의 예방 또는 치료를 위한 약제학적 조성물의 제조를 위한 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰의 용도를 제공한다.The present invention also provides the use of a secretory of induced hepatocytes derived from mesenchymal stem cells for the preparation of a pharmaceutical composition for the prevention or treatment of tissue fibrosis.
본 발명에 따르면, 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰은 상기 MFG-E8을 포함하고 있어 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰은 조직섬유화의 예방 또는 치료에 사용할 수 있다.According to the present invention, the secretory of hepatocytes induced and differentiated from mesenchymal stem cells contains the MFG-E8, and the secretory of hepatocytes induced from mesenchymal stem cells can be used for the prevention or treatment of tissue fibrosis. have.
상기 "분화"란 줄기세포로부터 특정 세포로 구조와 형태를 변화시키는 과정을 총칭하는데, 각각의 기능을 수행하기에 알맞은 구조와 형태를 변화시키는 과정을 말한다. 상기 분화는 자연발생적 분화 및 유도 분화를 포함한다. 상기 줄기세포로부터 특정 세포로의 유도 분화는 당업계에 공지된 다양한 방법을 사용 또는 이를 응용하여 수행될 수 있다.The "differentiation" refers to the process of changing the structure and shape from stem cells to specific cells, and refers to the process of changing the structure and shape suitable for performing each function. The differentiation includes spontaneous differentiation and induced differentiation. Induced differentiation from the stem cells to specific cells can be performed using various methods known in the art or by applying the same.
상기 "세크레톰"은 일반적으로 세포, 조직, 기관, 생물체에서 분비되는 유기 및 무기 요소들의 혼합을 의미하는 것으로, 보다 구체적으로, 분비된 단백질을 지칭하며, 본 발명의 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰은 MFG-E8를 포함하고 있다.The "secreto" generally refers to a mixture of organic and inorganic elements secreted from cells, tissues, organs, organisms, and more specifically, refers to the secreted protein, induced in mesenchymal stem cells of the present invention The secretory of differentiated hepatocytes contains MFG-E8.
상기 세크레톰은 중간엽 줄기세포를 혈청 배지에서 분화시키고 일정시간 배양 후 배양물을 농축시켜 얻을 수 있으나, 이에 제한하는 것은 아니다. The secreto may be obtained by differentiating mesenchymal stem cells in serum medium and concentrating the culture after a certain time of culturing, but not limited thereto.
상기 MFG-E8 단백질과 간세포를 이용한 간, 폐 또는 신장의 섬유화 치료용 세포치료제는 간, 폐 또는 신장의 섬유화 외에도, 뇌, 심장, 횡격막 등에서 발생하는 섬유화증 역시 TGF-β에 의해 활성화된 성상세포(myofibroblast, 근섬유아세포)가 원인이므로 상기 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰은 뇌, 심장, 횡격막 등의 섬유화 치료에도 사용할 수 있다.Cell therapy for the treatment of liver, lung or kidney fibrosis using the MFG-E8 protein and hepatocytes, in addition to fibrosis of the liver, lung or kidney, fibrosis occurring in the brain, heart, diaphragm, etc. is also activated by TGF-β (myofibroblast, myofibroblasts) is the cause of the induced mesenchymal stem cells derived from the mesenchymal stem cells can be used to treat the fibrosis of the brain, heart, diaphragm and the like.
따라서, 상기 조직섬유화는 폐, 신장, 간, 뇌, 심장 또는 횡경막 중 어느 하나의 조직에서 생기는 섬유화일 수 있다.Thus, the tissue fibrosis may be fibrosis occurring in any one of the tissues of the lung, kidney, liver, brain, heart or diaphragm.
본 발명의 조직섬유화 예방 또는 치료용 조성물은 약제학적으로 허용 가능한 담체를 더 포함할 수 있다.The composition for preventing or treating tissue fibrosis of the present invention may further include a pharmaceutically acceptable carrier.
상기 약제학적으로 허용 가능한 담체는 의약 분야에서 통상 사용되는 담체 및 비히클을 포함하며, 구체적으로 이온 교환 수지, 알루미나, 알루미늄 스테아레이트, 레시틴, 혈청 단백질(예, 사람 혈청 알부민), 완충 물질(예, 각종 인산염, 글리신, 소르브산, 칼륨 소르베이트, 포화 식물성 지방산의 부분적인 글리세라이드 혼합물), 물, 염 또는 전해질(예, 프로타민 설페이트, 인산수소이나트륨, 인산수소캄륨, 염화나트륨 및 아연 염), 교질성 실리카, 마그네슘 트리실리케이트, 폴리비닐피롤리돈, 셀룰로즈계 기질, 폴리에틸렌 글리콜, 나트륨 카르복시메틸셀룰로즈, 폴리아릴레이트, 왁스, 폴리에틸렌 글리콜 또는 양모지 등을 포함하나 이에 제한되지 않는다. Such pharmaceutically acceptable carriers include carriers and vehicles commonly used in the pharmaceutical arts, and in particular, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer materials (eg, Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, carbohydrogen phosphate, sodium chloride and zinc salts), gelatinous Silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substrates, polyethylene glycols, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycols or wool, and the like.
또한, 본 발명의 조성물은 상기 성분들 이외에 윤활제, 습윤제, 유화제, 현탁제, 또는 보존제 등을 추가로 포함할 수 있다.In addition, the composition of the present invention may further include a lubricant, a humectant, an emulsifier, a suspending agent, or a preservative in addition to the above components.
한 양태로서, 본 발명에 따른 조성물은 비경구 투여를 위한 수용성 용액으로 제조할 수 있으며, 바람직하게는 한스 용액(Hank's solution), 링거 용액(Ringer's solution) 또는 물리적으로 완충된 염수와 같은 완충 용액을 사용할 수 있다. 수용성 주입(injection) 현탁액은 소듐 카르복시메틸셀룰로즈, 솔비톨 또는 덱스트란과 같이 현탁액의 점도를 증가시킬 수 있는 기질을 첨가할 수 있다.In one embodiment, the composition according to the invention may be prepared in an aqueous solution for parenteral administration, preferably a buffered solution such as Hanks' solution, Ringer's solution or physically buffered saline. Can be used. Aqueous injection suspensions can be added with a substrate that can increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.
본 발명의 조성물은 전신계 또는 국소적으로 투여될 수 있으며, 이러한 투여를 위해 공지의 기술로 적합한 제형으로 제제화될 수 있다. 예를 들어, 경구 투여 시에는 불활성 희석제 또는 식용 담체와 혼합하거나, 경질 또는 연질 젤라틴 캡슐에 밀봉되거나 또는 정제로 압형하여 투여할 수 있다. 경구 투여용의 경우, 활성 화합물은 부형제와 혼합되어 섭취형 정제, 협측 정제, 트로키, 캡슐, 엘릭시르, 서스펜션, 시럽, 웨이퍼 등의 형태로 사용될 수 있다. The compositions of the present invention may be administered systemically or topically and may be formulated in suitable formulations by known techniques for such administration. For example, in oral administration, it can be administered by mixing with an inert diluent or an edible carrier, sealed in hard or soft gelatin capsules, or pressed into tablets. For oral administration, the active compounds can be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like.
주사용, 비경구 투여용 등의 각종 제형은 당해 기술 분야 공지된 기법 또는 통용되는 기법에 따라 제조할 수 있다. MFG-E8은 식염수 또는 완충용액에 잘 용해되므로 냉동 건조 상태로 보관한 후, 유효량의 MFG-E8을 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 투여 등에 적합한 형태로 식염수 또는 완충액에 투여 직전에 용액으로 제제화하여 투여할 수도 있다.Various formulations, such as for injection and parenteral administration, can be prepared according to techniques known in the art or commonly used techniques. Since MFG-E8 is well soluble in saline or buffer solution, store it in a freeze-dried state, and then administer an effective amount of MFG-E8 in saline or buffer in a form suitable for intravenous, subcutaneous, intramuscular, intraperitoneal, or transdermal administration. It may be administered by formulating a solution immediately before.
본 발명의 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 예컨대, 본 발명의 조성물의 투여량은 성인에게 1일에 0.1 내지 1000 mg/㎏의 양을, 바람직하게는 10 내지 100 mg/㎏의 용량을, 일일 1회 내지 수회 투여할 수 있다.Suitable dosages of the compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of the patient, food, time of administration, route of administration, rate of excretion and response to reaction. have. For example, the dosage of the composition of the present invention may be administered to an adult in an amount of 0.1 to 1000 mg / kg, preferably in a dose of 10 to 100 mg / kg, once to several times daily.
본 발명은 또한 약학적 유효량의 MFG-E8을 개체에 투여하는 단계를 포함하는 조직섬유화의 치료방법을 제공한다.The invention also provides a method of treating tissue fibrosis comprising administering to a subject a pharmaceutically effective amount of MFG-E8.
또한, 본 발명은 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰을 개체에 투여하는 단계를 포함하는 조직섬유화의 치료방법을 제공한다.The present invention also provides a method for treating tissue fibrosis comprising administering to a subject a secreto of induced hepatocyte differentiated mesenchymal stem cells.
상기 조직섬유화의 치료방법에 사용되는 MFG-E8 또는 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰 및 투여 방법은 상술한 약학적 조성물의 기준과 투여방법을 따르므로, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.The secretory and administration method of MFG-E8 or mesenchymal stem cells derived from the mesenchymal stem cells used in the method for the treatment of tissue fibrosis follows the criteria and the administration method of the above-described pharmaceutical composition, and therefore, common content between the two. Is omitted in order to avoid excessive complexity of the present specification.
상기 개체는 개, 고양이, 마우스, 인간 등일 수 있으나, 이에 제한하는 것은 아니다.The subject may be a dog, a cat, a mouse, a human, or the like, but is not limited thereto.
상기 조직섬유화는 폐, 신장, 간, 뇌, 심장 또는 횡경막 중 어느 하나의 조직에서 생기는 섬유화일 수 있다.The tissue fibrosis may be fibrosis occurring in any one of the tissues of the lung, kidney, liver, brain, heart or diaphragm.
본 발명은 또한 MFG-E8; 및The invention also relates to MFG-E8; And
간세포를 포함하는 조직섬유화 치료용 세포치료제에 관한 것이다.It relates to a cell therapy for the treatment of tissue fibrosis comprising hepatocytes.
상기 세포치료제는 간세포의 세포이식을 통한 조직섬유화 치료 시 치료적 효과를 더욱 높일 수 있다.The cell therapy can further increase the therapeutic effect when treating tissue fibrosis through cell transplantation of hepatocytes.
이러한 측면에서, 상기 간세포는 중간엽 줄기세포에서 유도 분화된 간세포일 수 있으나, 이에 특별히 제한하는 것은 아니다.In this aspect, the hepatocytes may be hepatocytes induced and differentiated from mesenchymal stem cells, but are not particularly limited thereto.
상기 중간엽 줄기세포에서 유도 분화된 간세포는 "간세포-유사 세포" 또는 "유사 간세포"라는 용어와 혼용되어 사용될 수 있다. Hepatocytes induced and differentiated from the mesenchymal stem cells may be used interchangeably with the term "hepatocyte-like cells" or "like hepatocytes."
따라서, MFG-E8과 간세포를 이용한 세포 치료 요법을 통한 간, 폐 또는 신장의 섬유화 치료용 세포치료제로 사용할 수 있다. 아울러, 뇌, 심장, 횡격막 등에서 발생하는 섬유화증 역시 TGF-β에 의해 활성화된 성상세포(myofibroblast, 근섬유아세포)가 원인이므로 상기 MFG-E8과 간세포를 이용한 세포 치료 요법을 통해 뇌, 심장, 횡격막 등의 섬유화 치료에도 사용할 수 있다.Therefore, it can be used as a cell therapy for the treatment of fibrosis of liver, lung or kidney through cell therapy using MFG-E8 and hepatocytes. In addition, fibrosis, which occurs in the brain, heart, and diaphragm, is also caused by TGF-β-activated astrocytic cells (myofibroblast, myofibroblasts), and thus the brain, heart, diaphragm, and the like through cell therapy using MFG-E8 and hepatocytes. It can also be used to treat fibrosis.
본 발명은 또한 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰 또는 MFG-E8을 후보약물과 접촉시키는 단계; 및 상기 후보약물이 MFG-E8의 발현 또는 분비 수준을 증가시키는지 측정하여 상기 후보약물을 조직섬유화의 예방 또는 치료용 의약으로 판정하는 단계를 포함하는 조직섬유화의 예방 또는 치료용 의약의 스크리닝 방법에 관한 것이다.The present invention also comprises the steps of contacting the candidate drug with a secretory or MFG-E8 of induced differentiated hepatocytes in mesenchymal stem cells; And determining whether the candidate drug increases the expression or secretion level of MFG-E8, and thus, determining the candidate drug as a drug for preventing or treating tissue fibrosis. It is about.
상기 후보약물은 통상적인 선정방식에 따라 MFG-E8 유전자 염기서열에서 mRNA, 단백질로의 전사, 번역을 촉진하거나 억제하는 물질 또는 MFG-E8 단백질의 기능 또는 활성을 증진하거나 억제하는 의약으로서의 가능성을 지닌 것으로 추정되거나 또는 무작위적으로 선정된 개별적인 핵산, 단백질, 펩타이드, 기타 추출물 또는 천연물, 화합물 등이 될 수 있다.The candidate drug has a potential as a drug that promotes or inhibits MFG-E8 gene sequencing, translation or translation into MFG-E8 gene, or a drug that enhances or inhibits the function or activity of MFG-E8 protein. Individual nucleic acids, proteins, peptides, other extracts or natural products, compounds, or allegedly or randomly selected.
이후, 후보약물이 처리된 세포에서 MFG-E8 유전자의 발현양, 단백질의 양 또는 단백질의 활성을 측정할 수 있으며, 측정 결과, 상기 MFG-E8 유전자의 발현양, 단백질의 양 또는 단백질의 활성이 증가되는 것이 측정되면 상기 후보약물은 조직섬유화를 치료 또는 예방할 수 있는 물질로 판단할 수 있다.Thereafter, the expression of the MFG-E8 gene, the amount of protein or the activity of the protein can be measured in the cells treated with the candidate drug. When the increase is measured, the candidate drug may be determined as a substance capable of treating or preventing tissue fibrosis.
상기에서 유전자의 발현양, 단백질의 양 또는 단백질의 활성을 측정하는 방법은 당업계에 공지된 다양한 방법을 통해 수행될 수 있는데, 예를 들면, 이에 제한되지는 않으나, 역전사 중합효소 연쇄반응(reverse transcriptase-polymerase chain reaction), 실시간 중합효소 연쇄반응(real time-polymerase chain reaction), 웨스턴 블럿, 노던 블럿, ELISA(enzyme linked immunosorbent assay), 방사선면역분석(RIA: radioimmunoassay), 방사 면역 확산법(radioimmunodiffusion) 및 면역침전분석법(immunoprecipitation assay) 등을 이용하여 수행할 수 있다.The method of measuring the expression amount of the gene, the amount of the protein or the activity of the protein in the above may be carried out through a variety of methods known in the art, for example, but not limited to reverse transcriptase polymerase chain reaction (reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion (radioimmunodiffusion) And immunoprecipitation assays.
이와 같은 조직섬유화 치료제 후보물질은 이후의 조직섬유화 치료제 개발과정에서 선도물질(leading compound)로서 작용하게 되며, 선도물질이 MFG-E8 유전자 또는 그로부터 발현되는 단백질의 기능을 촉진하는 효과를 나타낼 수 있도록 그 구조를 변형시키고 최적화함으로써, 새로운 조직섬유화 치료제를 개발할 수 있다.Such candidate candidates for treating tissue fibrosis may act as leading compounds in the development of tissue fibrosis therapeutics in the future, and may be effective in promoting the function of the MFG-E8 gene or a protein expressed therefrom. By modifying and optimizing the structure, new therapeutic agents for tissue fibrosis can be developed.
본 발명에서 유전공학적 기술과 관련된 사항은 샘브룩 등의 문헌(Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N. Y. (2001)) 및 프레드릭 등의 문헌(Frederick M. Ausubel et al., Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994))에 개시되어 있는 내용에 의해 보다 명확하게 된다.Matters related to genetic engineering in the present invention are described in Sambrook et al. (Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (2001)) and Frederick et al. M. Ausubel et al., Current protocols in molecular biology volumes 1, 2, 3, John Wiley & Sons, Inc. (1994)).
본 발명은 또한 정상 중간엽 줄기세포 대비 임의의 중간엽 줄기세포의 MFG-E8의 발현 또는 분비 수준의 증가 여부를 측정 또는 결정하는 단계를 포함하는, 조직섬유화 치료능이 개선된 중간엽 줄기세포의 선별방법에 관한 것이다.The present invention also provides a method for screening mesenchymal stem cells with improved tissue fibrosis treatment, comprising measuring or determining whether the level of MFG-E8 expression or secretion of any mesenchymal stem cells is increased compared to normal mesenchymal stem cells. It is about a method.
본 발명은 조직섬유화를 치료하는 중간엽 줄기세포의 유효성분이 이로부터 분비되는 MFG-E8임을 최초로 개시하고 있어 MFG-E8의 발현 또는 분비 수준이 증가된 중간엽 줄기세포는 조직섬유화 치료능이 개선된 것으로 볼 수 있다.The present invention discloses for the first time that the active ingredient of mesenchymal stem cells for treating tissue fibrosis is secreted from MFG-E8, so that the mesenchymal stem cells with increased expression or secretion level of MFG-E8 are improved in the ability to treat tissue fibrosis. can see.
따라서, 중간엽 줄기세포 내 MFG-E8의 발현 또는 분비 수준은 조직섬유화 치료에 사용되는 중간엽 줄기세포의 약리적 치료 효과의 개선을 위한 기준으로 작용할 수 있다.Thus, expression or secretion levels of MFG-E8 in mesenchymal stem cells may serve as a basis for improving the pharmacological therapeutic effect of mesenchymal stem cells used in the treatment of tissue fibrosis.
이하, 본 발명에 따르는 실시예 통하여 본 발명을 보다 상세히 설명하나, 본 발명의 범위가 하기 제시된 실시예에 의해 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited to the following examples.
<실시예 1> 중간엽 줄기세포에서 분화된 간세포의 배양액 제조Example 1 Preparation of Culture Solution of Hepatocytes Differentiated from Mesenchymal Stem Cells
본 실험에서 사용된 중간엽 줄기세포는 제대혈에서 유래된 것으로 간세포로의 분화를 위해 콜라겐이 코팅된 배양접시에 3×104 cell/cm2로 부착하였다. The mesenchymal stem cells used in this experiment were derived from umbilical cord blood and attached to 3 × 10 4 cells / cm 2 in a collagen-coated culture dish for differentiation into hepatocytes.
기본 배양배지로는 MesenPro RSTM medium(Gibco)를 사용하였다. 배양접시에 70-80%이 채워지면 첫 번째 분화배지인 기초 배지 Iscove's modified Dulbecco's medium(IMDM; Invitrogen, Carlsbad, CA, USA)에 Epidermal growth factor(EGF; Peprotech EC Ltd, London, England, 20 ng/mL)와 basic fibroblast growth factor(bFGF; Peprotech EC Ltd, 10 ng/mL)를 넣고 2일간 배양하였다. MesenPro RSTM medium (Gibco) was used as the basic culture medium. After 70-80% of the culture dish was filled, Epidermal growth factor (EGF; Peprotech EC Ltd, London, England, 20 ng /) was used in Iscove's modified Dulbecco's medium (IMDM; Invitrogen, Carlsbad, CA, USA). mL) and basic fibroblast growth factor (bFGF; Peprotech EC Ltd, 10 ng / mL) were added and incubated for 2 days.
이후 두 번째 분화배지인 기초 배지 IMDM에 hepatocyte growth factor(HGF; Peprotech EC Ltd, 20 ng/mL), bFGF(10 ng/mL), nicotinamide(Sigma, 0.61 g/L), 1% insulin-transferrin-selenium(ITS) premix(Invitrogen)를 첨가하여 10일 동안 배양하였다. Subsequently, hepatocyte growth factor (HGF; Peprotech EC Ltd, 20 ng / mL), bFGF (10 ng / mL), nicotinamide (Sigma, 0.61 g / L), 1% insulin-transferrin- Incubated for 10 days with the addition of selenium (ITS) premix (Invitrogen).
마지막으로 기초 배지 IMDM에 oncostatin M(Peprotech EC Ltd, 20 ng/mL), dexamethasone(Sigma, 1 μmol/L), 1% ITS를 넣고 다시 10일 동안 배양하였다. Finally, oncostatin M (Peprotech EC Ltd, 20 ng / mL), dexamethasone (Sigma, 1 μmol / L), 1% ITS was added to basal medium IMDM and incubated for 10 days.
분화된 중간엽 줄기세포의 세크레톰을 얻기 위해 기초 배지 IMDM에 0.05%의 FBS를 첨가하여 24시간 동안 배양 후 3-kDa cutoff filter ultrafiltration units(Millipore)으로 25배 농축하였다.To obtain a secretory of differentiated mesenchymal stem cells, 0.05% FBS was added to basal medium IMDM, and cultured for 24 hours, followed by concentration of 25-fold with 3-kDa cutoff filter ultrafiltration units (Millipore).
<실시예 2> 만성 간질환 모델 및 간섬유화 모델에서 MFG-E8의 효과 실험Example 2 Effect of MFG-E8 in Chronic Liver Disease Model and Hepatic Fibrosis Model
만성 간질환 유발을 위해 5~6주령 마우스(C57BL/C)에 Thioacetamide(TAA)를 200mg/kg body weight이 되도록 식염수에 희석하여 복강 주사하였다. 일주일에 3번 총 8주간 복강 주사를 반복하였다. 8주가 지난 후 농축시킨 세크레톰의 단백질을 브래포드 분석(Bradford assay)로 정량한 뒤 500㎍이 되는 양을 복강 주사하였다. 세크레톰 주입 이후, 3일 또한 7일이 지난 후 마우스의 간섬유화 정도를 분석하였다. MFG-E8 단백질(R&D systems)은 160㎍/kg body weight을 기본으로 투여하고, 농도별 효과를 확인하기 위해서는 32㎍/kg body weight을 낮은 농도, 160㎍/kg body weight을 중간 농도, 800㎍/kg body weight을 높은 농도로 설정하여 복강에 투여하였다. MFG-E8 단백질 투여 후 3일 뒤 간섬유화 정도를 분석하였다.To induce chronic liver disease, 5-6 weeks old mice (C57BL / C) were intraperitoneally injected with saline solution diluted Thioacetamide (TAA) to 200mg / kg body weight. Intraperitoneal injections were repeated three times a week for a total of eight weeks. Eight weeks later, the concentrated protein of creatre was quantified by Bradford assay and then intraperitoneally injected in an amount of 500 μg. After the secreto infusion, mice were analyzed for liver fibrosis 3 days and 3 days later. MFG-E8 protein (R & D systems) is administered based on 160μg / kg body weight, and in order to confirm the effect of each concentration, 32μg / kg body weight is low, 160μg / kg body weight is medium and 800μg / kg body weight was set to a high concentration was administered to the abdominal cavity. Three days after administration of MFG-E8 protein, the degree of hepatic fibrosis was analyzed.
다음으로, 간섬유화 유발을 위해 5~6주령 마우스(BALB/c)에 사염화탄소(CCl4)를 올리브오일에 10%로 희석한 뒤 100㎕/20g body weight으로 복강 주사하였다. 일주일에 2번 총 6주간 복강 주사를 반복한다. 이후 세크레톰 주입과 MFG-E8 단백질 투여는 TAA를 통해 확립한 만성 간질환 모델과 같은 방법으로 시행하였다.Next, carbon tetrachloride (CCl 4 ) was diluted to 10% in olive oil in 5-6 week old mice (BALB / c) to induce hepatic fibrosis and then intraperitoneally injected at 100 μl / 20 g body weight. The abdominal injection is repeated twice a week for a total of six weeks. Subsequently, secreto infusion and MFG-E8 protein administration were performed in the same way as the chronic liver disease model established by TAA.
또한, 간조직의 섬유화 정도를 확인하기 위해 간조직을 4% 파라포름알데히드에 고정시켰다. 이 간조직을 파라핀에 고정하여 조직절편으로 얻은 후 H&E, Masson's trichrome, 씨리어스 레드 염색을 시행하여 광학현미경 하에서 관찰하였다. In addition, liver tissue was fixed in 4% paraformaldehyde to confirm the degree of fibrosis of liver tissue. The liver tissues were fixed in paraffin and obtained as tissue sections. H & E, Masson's trichrome and serial red staining were performed under an optical microscope.
Masson's trichrome 염색을 위해, 파라핀 고정으로 얻은 조직절편을 탈파라핀 과정을 거쳐 Bouin 액에 다시 고정 후, Weigert hematoxylin/Biebrich scarlet-acid fuchsin-aniline blue 액 또는 2% 라이트 그린(light green)에 염색 후 관찰하였다. 섬유화된 조직은 청색으로 나타나고, 세포질, 근육, 케라틴은 적색으로 나타나며, 세포핵은 흑갈색으로 나타난다.For Masson's trichrome staining, tissue sections obtained from paraffin fixation were deparaffinized and re-fixed in Bouin's solution, followed by staining in Weigert hematoxylin / Biebrich scarlet-acid fuchsin-aniline blue solution or 2% light green. It was. Fibrous tissue appears in blue, cytoplasm, muscle, keratin in red, and nucleus in dark brown.
씨리어스 레드 염색을 위해서는, 파라핀 고정으로 얻은 조직절편을 탈파라핀 과정을 거쳐 헤마톡실린(hematoxylin)으로 핵을 염색한 뒤 피크로-씨리어스 picro-sirius red 염색약으로 1시간 염색하였다. 간섬유화가 유발된 부분은 붉은색으로 나타난다.For serial red staining, tissue sections obtained by paraffin fixation were deparaffinized, nucleus stained with hematoxylin, and then stained with a picro-sirius red dye for 1 hour. The liver fibrosis-induced areas appear red.
면역형광염색을 위해서는, 파라핀 고정으로 얻은 조직절편을 탈파라핀 과정을 거쳐 10% donkey serum으로 블로킹을 진행한 후 α-SMA와 F4/80 항체를 넣어 반응시켰다. 이후 형광 표지가 붙어있는 2차 항체를 넣어 반응 후 형광현미경으로 관찰하였다.For immunofluorescence staining, tissue sections obtained by paraffin immobilization were blocked with 10% donkey serum after deparaffinization, followed by reaction with α-SMA and F4 / 80 antibody. After the reaction was added to the secondary antibody with a fluorescent label was observed by fluorescence microscope.
염색 결과, 콜라겐 축적으로 인한 섬유화를 조직에서 확인할 수 있었다.As a result of staining, fibrosis due to collagen accumulation could be confirmed in the tissue.
또한, TAA를 이용하여 만든 만성 간질환 마우스 모델에 hpUCMSC 세크레톰, hpUCMSC 세크레톰에 MFG-E8 항체를 처리한 세크레톰 및 MFG-E8 합성단백질을 각각 처리하고, H&E, 씨리어스 레드, MT로 염색하여 간조직의 구조 및 섬유화 정도를 비교한 결과, hpUCMSC 세크레톰과 MFG-E8 단백질을 처리한 그룹에서 섬유화가 확연히 감소하였다. MFG-E8 항체를 처리한 그룹은 sham보다 더 심한 섬유화가 진행되었다. α-SMA의 발현 또한 sham보다 hpUCMSC 세크레톰과 MFG-E8 단백질을 처리한 그룹에서 감소되었다(도 1a 및 1b). 이때 대조군으로 대식세포의 마커인 F4/80을 확인하였으나, 그룹별 차이가 나타나지 않았다. In addition, the HPUCMSC secreto and the HPUCMSC secreto treated with MFG-E8 antibody were treated with the chronic liver disease mouse model made with TAA and MFG-E8 synthetic protein, respectively, and H & E, serial red, As a result of staining with MT and comparing the structure and degree of fibrosis of liver tissues, fibrosis was significantly reduced in the hpUCMSC secreto and MFG-E8 protein groups. The group treated with MFG-E8 antibody developed more fibrosis than sham. The expression of α-SMA was also decreased in the group treated with hpUCMSC secreto and MFG-E8 proteins than sham (FIGS. 1A and 1B). At this time, F4 / 80, a marker of macrophages, was identified as a control, but there was no difference between groups.
상기 만성 간질환 마우스 모델에 MFG-E8 단백질을 농도별로 처리하여 씨리어스 레드로 콜라겐 축적을 확인한 결과, sham에 비해 섬유화가 감소하였다(도 1c).In the chronic liver disease mouse model, MFG-E8 protein was treated by concentration to confirm collagen accumulation in serial red, and fibrosis was reduced compared to sham (FIG. 1C).
그리고, 간섬유화 마우스 모델에서 세크레톰과 MFG-E8 단백질 투여 실험 결과, 도 1d 및 1e에 나타난 바와 같이, sham에 비해 섬유화가 감소하였다.As a result of the administration of the secreto and MFG-E8 proteins in the liver fibrosis mouse model, as shown in FIGS. 1D and 1E, fibrosis was reduced compared to sham.
<실시예 3> 간성상세포주(hTert-HSCs) 및 초대 배양된 간성상세포(primary HSCs)에서 MFG-E8의 효과 실험Example 3 Effect Test of MFG-E8 in Hepatic Stem Cell Lines (hTert-HSCs) and Primary Cultured Primary HSCs
간성상세포주 및 초대 배양된 간성상세포를 이용하여 세크레톰의 인 비트로 효과를 실험하였다. 이를 위해, 간성상세포주는 기초 배지 DMEM에 10% FBS와 100unit의 페니실린, 100㎍의 스트렙토마이신을 첨가한 배양액에 배양하였다. 실험 진행 시 비활성화된 간성상세포주를 만들기 위해 기초 배지 DMEM에 0.2% FBS를 첨가한 배양액에서 24시간 동안 배양 후 동일한 배지에 TGFβ1 단백질을 10ng/mL로 처리하여 간성상세포주를 활성화시켰다. 실험에 사용한 MFG-E8 단백질은 기본적으로 500ng/mL 이 되도록 TGFβ1 단백질과 함께 처리하여 48시간 동안 배양하였다. 농도별로 처리할 경우, 100ng/mL, 250ng/mL, 500ng/mL, 1㎍/mL, 5㎍/mL이 되도록 배양하였다. 세크레톰 내 단백질을 중화시키기 위해 항체의 농도는 20㎍/mL이 되도록 세크레톰과 섞은 후 상온에서 1시간 방치 후 사용하였다.The in vitro effect of the secreto was tested using hepatic stellate cell lines and primary cultured hepatic stellate cells. To this end, hepatic stellate cell lines were cultured in culture medium containing 10% FBS, 100 units of penicillin, and 100 μg of streptomycin in basal medium DMEM. In order to make the inactivated hepatic stromal cell line, the culture medium added 0.2% FBS to the basal medium DMEM was incubated for 24 hours, and then treated with 10 ng / mL TGFβ1 protein in the same medium to activate the hepatic stellate cell line. The MFG-E8 protein used in the experiment was basically treated with TGFβ1 protein to be 500ng / mL and incubated for 48 hours. If the treatment by concentration, incubated to 100ng / mL, 250ng / mL, 500ng / mL, 1㎍ / mL, 5㎍ / mL. In order to neutralize the protein in the secreto, the concentration of the antibody was 20 µg / mL and then mixed with the secreto and used for 1 hour at room temperature.
초대 배양된 간성상세포의 경우 SteCM(ScienCell) 배양액에서 기본 배양을 진행하고, 이후 실험 진행은 간성상세포주와 동일한 조건에서 배양하였다.In the case of primary cultured hepatic stellate cells, the basic culture was carried out in SteCM (ScienCell) culture, and then the experiment was incubated under the same conditions as the hepatic stellate cell line.
도 2a는 hTert-HSCs를 TGFβ1으로 활성화시킨 후 3가지 합성 단백질을 처리한 결과로, 무혈청 상태에서는 간성상세포주가 비활성화되어 세포가 얇고 가는 모양으로 관찰되나, TGFβ1을 처리하면 넓적한 모양으로 활성화되어 α-SMA 발현이 높아진다. 이때, 데코린, PEDF 및 MFG-E8 처리 시 모두 α-SMA의 발현을 감소시켰다. 웨스턴 블랏 결과에서도 유사한 결과를 확인하였다(도 2b).Figure 2a is a result of processing three synthetic proteins after activating hTert-HSCs with TGFβ1, the hepatic stellate cell line is inactivated in the serum-free state, the cells are observed to be thin and thin, but when treated with TGFβ1 is activated in a broad shape α-SMA expression is increased. At this time, the decorin, PEDF and MFG-E8 treatment all reduced the expression of α-SMA. Similar results were confirmed in Western blot results (FIG. 2B).
또한, hpUCMSC 세크레톰에 각 단백질의 항체를 처리하여 단백질의 활성을 감소시킨 후 HSCs의 활성을 확인한 결과, 항체를 처리하지 않은 hpUCMSC 세크레톰에 비해 α-SMA의 발현이 높아졌다(도 2c). 사람의 초대 배양된 HSCs에서도 3가지 단백질이 같은 효과를 나타내는지 확인한 결과 MFG-E8을 처리한 그룹에서 α-SMA의 발현이 확연히 감소하였다(도 2d). In addition, by treating the hpUCMSC secreto to the antibody of each protein to reduce the activity of the protein and confirmed the activity of HSCs, the expression of α-SMA was higher than that of the hpUCMSC secreto not treated with the antibody (Fig. 2c). . As a result of confirming that the three proteins have the same effect even in human primary cultured HSCs, the expression of α-SMA was significantly decreased in the group treated with MFG-E8 (FIG. 2D).
hTert-HSCs에 MFG-E8을 농도별로 처리한 결과 농도 의존적인 감소가 일어났으며(도 2e), 사람의 초대 배양된 HSCs에 MFG-E8을 농도별로 처리한 결과 농도 의존적인 감소가 일어남을 확인하였다(도 2f).Concentration-dependent reduction of MFG-E8 by concentration in hTert-HSCs occurred (Fig. 2e), and concentration-dependent decrease in MFG-E8 concentration in human primary cultured HSCs. (FIG. 2f).
<실시예 4> 웨스턴 블랏 및 ELISA를 이용한 MFG-E8 단백질 발현 확인Example 4 Confirmation of MFG-E8 Protein Expression Using Western Blot and ELISA
간성상세포주의 활성화 정도를 확인하기 위해 단백질을 추출하여 웨스턴 블랏을 진행하였다. 단백질분해효소 억제제가 포함된 RIPA 버퍼를 이용하여 세포의 단백질을 추출하였다. SDS-PAGE 젤을 이용하여 각 그룹의 40㎍의 단백질을 분리한 뒤 폴리비닐리덴 플루오라이드 트랜스퍼 멤브레인(polyvinylidene fluoride transfer membrane)에 옮겼다. Ponceau S 용액을 이용하여 단백질이 멤브레인에 잘 옮겨졌는지 확인하였다. 이후 TBS-T 버퍼에 5% 스킴 밀크(skim milk)를 첨가한 용액에 상온에서 블로킹한 뒤 멤브레인을 α-SMA 및 MFG-E8, GAPDH의 각 항체가 있는 용액에서 하룻밤 동안 냉장에서 방치하였다. 이후 각 항체에 맞는 2차 항체를 상온에서 1시간 동안 반응시킨 후 화학발광 키트(chemiluminescence kit)를 이용하여 해당 단백질의 발현을 확인하였다. In order to confirm the activation level of hepatic stellate cell lines, proteins were extracted and Western blot was performed. Cell proteins were extracted using RIPA buffer containing protease inhibitors. 40 μg of protein of each group was separated using SDS-PAGE gel and transferred to polyvinylidene fluoride transfer membrane. Ponceau S solution was used to confirm that the protein was well transferred to the membrane. After blocking at room temperature to a solution in which 5% skim milk was added to the TBS-T buffer, the membrane was allowed to stand overnight in a solution containing each antibody of α-SMA, MFG-E8 and GAPDH. After reacting the secondary antibody for each antibody for 1 hour at room temperature, the expression of the protein was confirmed using a chemiluminescence kit (chemiluminescence kit).
MFG-E8 단백질의 ELISA 실험의 경우, Cusabio에서 판매하는 사람 MFG-E8 ELISA 키트를 사용하였다.For ELISA experiments with MFG-E8 protein, a human MFG-E8 ELISA kit sold by Cusabio was used.
도 2g에 나타난 바와 같이, MFG-E8 단백질은 다양한 줄기세포(제대혈 유래 중간엽 줄기세포(UCMSC), 탈락유치 줄기세포(SHED), 골수 유래 줄기세포(BMSC))와 이들 줄기세포에서 유도 분화된 간세포(hpUCMSCs, hpSHEDs, hpBMSCs)의 세크레톰에서 발현되고 있으나, 배아줄기세포에서는 발현되지 않았다. ELISA 분석 결과 역시 이와 동일한 결과를 나타내었다(도 2h).As shown in Figure 2g, MFG-E8 protein is induced and differentiated from various stem cells (umbilical cord blood-derived mesenchymal stem cells (UCMSC), degenerating stem cells (SHED), bone marrow-derived stem cells (BMSC)) and these stem cells Hepatocytes (hpUCMSCs, hpSHEDs, hpBMSCs) are expressed in the secretory, but not in embryonic stem cells. The result of ELISA analysis also showed this same result (FIG. 2H).
본 발명은 조직섬유화 예방 또는 치료 분야에서 사용할 수 있다.The present invention can be used in the field of preventing or treating tissue fibrosis.

Claims (13)

  1. Milk fat globule-EGF factor 8 (MFG-E8)을 포함하는 조직섬유화의 예방 또는 치료용 조성물.A composition for preventing or treating tissue fibrosis comprising milk fat globule-EGF factor 8 (MFG-E8).
  2. 제1항에 있어서,The method of claim 1,
    조직섬유화는 폐, 신장, 간, 뇌, 심장 또는 횡경막 중 어느 하나의 조직에서 생기는 섬유화인, 조직섬유화의 예방 또는 치료용 조성물.Tissue fibrosis is a fibrosis occurring in any one of the tissues of the lung, kidney, liver, brain, heart or diaphragm, the composition for preventing or treating tissue fibrosis.
  3. 조직섬유화의 예방 또는 치료를 위한 약제학적 조성물의 제조를 위한 Milk fat globule-EGF factor 8 (MFG-E8)의 용도.Use of Milk fat globule-EGF factor 8 (MFG-E8) for the preparation of a pharmaceutical composition for the prevention or treatment of tissue fibrosis.
  4. 약학적 유효량의 Milk fat globule-EGF factor 8 (MFG-E8)을 개체에 투여하는 단계를 포함하는 조직섬유화의 치료방법.A method of treating tissue fibrosis comprising administering to a subject a pharmaceutically effective amount of Milk fat globule-EGF factor 8 (MFG-E8).
  5. 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰(secretome)을 포함하는 조직섬유화의 예방 또는 치료용 조성물.A composition for preventing or treating tissue fibrosis comprising a secretome of hepatocytes induced and differentiated from mesenchymal stem cells.
  6. 제5항에 있어서,The method of claim 5,
    세크레톰은 Milk fat globule-EGF factor 8 (MFG-E8)을 포함하는 것인, 조직섬유화의 예방 또는 치료용 조성물.Secreto is Milk fat globule-EGF factor 8 (MFG-E8) that comprises, the composition for the prevention or treatment of tissue fibrosis.
  7. 제5항에 있어서,The method of claim 5,
    조직섬유화는 폐, 신장, 간, 뇌, 심장 또는 횡경막 중 어느 하나의 조직에서 생기는 섬유화인, 조직섬유화의 예방 또는 치료용 조성물.Tissue fibrosis is a fibrosis occurring in any one of the tissues of the lung, kidney, liver, brain, heart or diaphragm, the composition for preventing or treating tissue fibrosis.
  8. 조직섬유화의 예방 또는 치료를 위한 약제학적 조성물의 제조를 위한 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰(secretome)의 용도.Use of secretomes of induced differentiated hepatocytes in mesenchymal stem cells for the preparation of pharmaceutical compositions for the prevention or treatment of tissue fibrosis.
  9. 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰(secretome)을 개체에 투여하는 단계를 포함하는 조직섬유화의 치료방법.A method of treating tissue fibrosis comprising administering to a subject a secreto of secreted hepatocytes induced in mesenchymal stem cells.
  10. Milk fat globule-EGF factor 8 (MFG-E8); 및Milk fat globule-EGF factor 8 (MFG-E8); And
    간세포를 포함하는 조직섬유화 치료용 세포치료제.Cell therapy for the treatment of tissue fibrosis comprising hepatocytes.
  11. 제10항에 있어서,The method of claim 10,
    간세포는 중간엽 줄기세포에서 유도 분화된 간세포인, 조직섬유화 치료용 세포치료제.Hepatocytes are hepatic cells induced and differentiated from mesenchymal stem cells.
  12. 중간엽 줄기세포에서 유도 분화된 간세포의 세크레톰(secretome) 또는 Milk fat globule-EGF factor 8 (MFG-E8)을 후보약물과 접촉시키는 단계; 및Contacting a secretory of secreted hepatocytes or Milk fat globule-EGF factor 8 (MFG-E8) derived from mesenchymal stem cells with a candidate drug; And
    상기 후보약물이 MFG-E8의 발현 또는 분비 수준을 증가시키는지 측정하여 상기 후보약물을 조직섬유화의 예방 또는 치료용 의약으로 판정하는 단계를 포함하는 조직섬유화의 예방 또는 치료용 의약의 스크리닝 방법.A method for screening a medicament for preventing or treating tissue fibrosis, comprising: determining whether the candidate medicament increases the expression or secretion level of MFG-E8 and determines the candidate drug as a medicament for preventing or treating tissue fibrosis.
  13. 정상 중간엽 줄기세포 대비 임의의 중간엽 줄기세포의 Milk fat globule-EGF factor 8 (MFG-E8)의 발현 또는 분비 수준의 증가 여부를 측정 또는 결정하는 단계를 포함하는 조직섬유화 치료능이 개선된 중간엽 줄기세포의 선별방법.Mesenchyme with improved tissue fibrosis therapeutic ability, comprising measuring or determining whether the expression or secretion level of milk fat globule-EGF factor 8 (MFG-E8) of any mesenchymal stem cells is increased compared to normal mesenchymal stem cells Stem cell selection method.
PCT/KR2016/007611 2015-07-28 2016-07-13 Composition for preventing or treating tissue fibrosis using milk fat globule-egf factor (mfg-e8) WO2017018698A1 (en)

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