WO2017018698A1 - Composition pour la prévention ou le traitement d'une fibrose tissulaire utilisant le facteur egf de globule gras du lait (mfg-e8) - Google Patents

Composition pour la prévention ou le traitement d'une fibrose tissulaire utilisant le facteur egf de globule gras du lait (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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English (en)
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/zh
Publication of WO2017018698A1 publication Critical patent/WO2017018698A1/fr

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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

La présente invention concerne une composition pour la prévention ou le traitement d'une fibrose tissulaire le facteur EGF 8 de globule gras du lait (MFG-E8). Plus spécifiquement, MFG-E8 a un rôle dans la suppression de l'expression de collagène induite par la transmission de signal TGFβ/Smad et dans la suppression de l'activité de cellules stellaires hépatiques et donc l'amélioration de la fibrose hépatique, et réduit l'ampleur de la fibrose dans un modèle de souris ayant une maladie de fibrose hépatique, et supprime l'activation lorsqu'il est utilisé pour traiter des cellules stellaires hépatiques cultivées in vitro, de manière à présenter l'avantage de pouvoir être utilisé en tant qu'agent pour la prévention ou le traitement d'une fibrose tissulaire.
PCT/KR2016/007611 2015-07-28 2016-07-13 Composition pour la prévention ou le traitement d'une fibrose tissulaire utilisant le facteur egf de globule gras du lait (mfg-e8) WO2017018698A1 (fr)

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CN201680002518.6A CN106794223B (zh) 2015-07-28 2016-07-13 利用mfg-e8的用于预防或者治疗组织纤维化的组合物

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WO2020084344A3 (fr) * 2018-10-25 2020-07-16 Nexel Co., Ltd. Compositions et procédés de traitement ou de prévention de la fibrose
WO2021044360A1 (fr) 2019-09-06 2021-03-11 Novartis Ag Protéines de fusion thérapeutiques
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WO2018212372A1 (fr) * 2017-05-17 2018-11-22 (주)넥셀 Protéine recombinante pour la prévention ou le traitement de la fibrose tissulaire et composition pour la prévention ou le traitement de la fibrose tissulaire comprenant celle-ci
CN109661405A (zh) * 2017-05-17 2019-04-19 尼希尔株式会社 组织纤维化预防或者治疗用重组蛋白质以及包含该蛋白质的组织纤维化预防或者治疗用组合物
US11028139B2 (en) 2017-05-17 2021-06-08 Nexel Co., Ltd. Recombinant protein for preventing or treating tissue fibrosis and composition for preventing or treating tissue fibrosis comprising the same
CN109661405B (zh) * 2017-05-17 2022-05-13 尼希尔株式会社 一种重组蛋白质以及包含该蛋白质的组合物
WO2020084344A3 (fr) * 2018-10-25 2020-07-16 Nexel Co., Ltd. Compositions et procédés de traitement ou de prévention de la fibrose
EP3870210A4 (fr) * 2018-10-25 2022-10-19 Nexel Co., Ltd. Compositions et procédés de traitement ou de prévention de la fibrose
WO2021044360A1 (fr) 2019-09-06 2021-03-11 Novartis Ag Protéines de fusion thérapeutiques
WO2021044362A1 (fr) 2019-09-06 2021-03-11 Novartis Ag Protéines de fusion thérapeutiques
WO2021044361A1 (fr) 2019-09-06 2021-03-11 Novartis Ag Protéines de fusion thérapeutiques

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