CN106794223B

CN106794223B - Composition for preventing or treating tissue fibrosis using MFG-E8

Info

Publication number: CN106794223B
Application number: CN201680002518.6A
Authority: CN
Inventors: 金钟埙; 安姝妍; 张有真
Original assignee: Nihil Corp
Current assignee: Nihil Corp
Priority date: 2015-07-28
Filing date: 2016-07-13
Publication date: 2021-07-20
Anticipated expiration: 2036-07-13
Also published as: CN106794223A; WO2017018698A1; KR20170013621A

Abstract

The present invention relates to a composition for preventing or treating tissue fibrosis using milk fat globule epidermal growth factor 8 protein (MFG-E8), and more particularly, MFG-E8 inhibits the expression of collagen induced by TGF β/Smad signaling and inhibits the activity of hepatic stellate cells, thereby acting to improve liver fibrosis, and having the following effects: the degree of fibrosis is reduced in a mouse model having a hepatic fibrosis disease, and if the mouse model is treated with hepatic stellate cells cultured in vitro, the degree of fibrosis can be inhibited, and the mouse model can be used as a prophylactic or therapeutic agent for tissue fibrosis.

Description

Composition for preventing or treating tissue fibrosis using MFG-E8

Technical Field

The present invention relates to a use of MFG-E8 for preventing or treating tissue fibrosis by a fibrosis reduction effect of Milk fat globule epidermal growth factor 8 protein (MFG-E8, Milk fat globule epidermal growth factor 8), which is a protein secreted from mesenchymal stem cells (MFG-E8).

Background

In the case of studies on differentiation of Mesenchymal Stem cells (mesenchyme Stem cells) into hepatocytes, although mesoderm Mesenchymal Stem cells were reported early to exceed the genetic limitations of the germ layer and have the possibility of differentiation transformation (transduction) into endoderm hepatocytes, the following possibilities were recently proposed: according to the accumulated research results, which are not the results of differentiation transformation, the death of hepatocytes in the host tissue is inhibited by active factors secreted after mesenchymal stem cells are transplanted, and the regeneration of damaged host liver tissues itself can be promoted (paracrine effects).

Among indications for cell therapy using stem cells, various diseases such as viral hepatitis including hepatitis C (hepatitis C), fatty liver, alcoholic liver disease, liver cancer, acute/chronic cirrhosis, hereditary metabolic disease, biliary tract disease, and genetic liver function impairment are factors that cause liver function damage.

Recently, evidence is accumulating that proteins secreted from stem cells are involved in regeneration and immunoregulation of various tissues. The protein pharmaceuticals market is now a representative industry in the field of life engineering, and has high therapeutic effects and fewer side effects than chemically synthesized pharmaceuticals that affect the whole body by absorption in the digestive organs, and has the following advantages in terms of market value as compared with conventional chemically synthesized pharmaceuticals: the development period is short while the price is maintained high. Thus, in the case of exploring a new function of a secreted protein that is secreted from stem cells and differentiated cells and plays an important role in tissue regeneration and remodeling together with a cell therapeutic agent, there is a possibility that the protein drug can be developed as a high-value-added protein drug through a protein engineering process for enhancing functionality and stability.

Reports on the paracrine (paracrine) effect of mesenchymal stem cells in various tissues are ongoing. However, the detailed mechanism (mechanism) for secreted substances is not known, and there is still controversy regarding the characteristics of cells differentiated from mesenchymal stem cells, and thus there is a limit to the use of mesenchymal stem cells or differentiated cells as therapeutic agents as they are. This trend is that therapeutic discovery using substances secreted from cells is more attractive than therapeutic discovery using cells as they are.

Hepatic fibrosis is a disease resulting from abnormal accumulation of ECM (extracellular matrix), and may progress to cirrhosis or liver cancer. If hepatic fibrosis occurs, macrophages are aggregated by chemokines (chemokines) secreted from damaged hepatocytes or vascular cells, and hepatic stellate cells (hepatic stellate cells) present in liver tissues become myofibroblast-like cells (myofibroblast-like cells) due to secreted TGF β, thereby performing ECM production, but substances or methods for preventing and treating this are not known.

In addition, renal fibrosis refers to a symptom in which tissues and/or blood vessels of the kidney become hard, and pulmonary fibrosis or pulmonary fibrosis is known as a disease characterized by proliferation of diffuse fibers on the alveolar wall and mainly manifested by dry cough or dyspnea during work.

Although the possibility of helping the regeneration and repair of host tissues itself (regeneration of host tissue) is suggested by stimulating the periphery of tissues damaged together with cell replacement effects (replacement by donor cells) with various secretory factors after stem cells or cells differentiated from stem cells are transplanted, there are few studies on such a situation. Further, no therapeutic agent for fibrosis capable of fundamentally treating the disease itself has been known so far.

The MFG-E8 protein is a protein found in mammals and includes not only arginine (arginine) -glycine (glycine) -aspartic acid (aspartic acid) motif (motif) but also phosphatidylserine (phosphatidylserine) binding domain (domain) so as to be able to bind to integrin (integrin). MFG-E8 is well known to have the following characteristics: it has opsonin action of cell death cells by binding to phosphatidylserine exposed on the surface of the cell death cells, has a function of mediating phagocytosis of dead cells by binding to integrin on the surface of phagocytic cells, and helps to remove accumulated collagen (collagen). In addition, studies have been made on the involvement of the growth of new blood vessels while inhibiting the death of intestinal epithelial cells and reducing damage. In addition, it has been reported that hepatocytes induced to differentiate from human embryonic stem cells are used for liver regeneration and improvement of liver diseases by increasing the expression rate of hepatocytes and promoting proliferation of hepatocytes and angiogenesis (see patent document 1). But the reality is that there is no report on what effect is produced in fibrosis stages in liver tissues or in other tissues in addition to liver tissues.

Disclosure of Invention

The present invention is intended to clarify the effect on fibrosis of a secreted proteome (secretome) protein secreted from hepatocytes induced to differentiate in mesenchymal stem cells, and to provide a pharmaceutical use for preventing or treating tissue fibrosis based on the fibrosis-inhibiting effect of MFG-E8 on one of the secreted proteome proteins.

It is another object of the present invention to provide a use of the MFG-E8 and induced-differentiation hepatocytes obtained from mesenchymal stem cells as a cell therapeutic agent for treating tissue fibrosis.

It is still another object of the present invention to provide a method for screening (screening) a medicine for preventing or treating tissue fibrosis by measuring the expression or secretion level of MFG-E8.

It is still another object of the present invention to provide a method for screening mesenchymal stem cells having improved therapeutic efficacy for tissue fibrosis, by measuring or determining the increased expression or secretion level of MFG-E8 in the mesenchymal stem cells.

To achieve the object, the present invention provides a composition for preventing or treating tissue fibrosis, comprising Milk fat globule epidermal growth factor 8 protein (MFG-E8, Milk fat globule-EGF factor 8).

The invention also provides the use of MFG-E8 for the manufacture of a pharmaceutical composition for the prevention or treatment of tissue fibrosis.

The present invention also provides a method of treating tissue fibrosis comprising the step of administering to an individual a pharmaceutically effective amount of MFG-E8.

The present invention also provides a composition for preventing or treating tissue fibrosis, comprising a secreted proteome (secretome) of hepatocytes induced differentiation obtained from mesenchymal stem cells.

The present invention also provides a use of a secreted proteome of induced-differentiation hepatocytes obtained from mesenchymal stem cells for the manufacture of a pharmaceutical composition for the prevention or treatment of tissue fibrosis.

The present invention also provides a method for treating tissue fibrosis, which comprises the step of administering a secretory protein group of hepatocytes obtained by inducing differentiation from mesenchymal stem cells to an individual.

The invention also provides a composition comprising MFG-E8 and

a cell therapeutic agent for liver cells for treating tissue fibrosis.

The present invention also provides a method for screening a drug for preventing or treating tissue fibrosis, comprising the steps of: contacting a secreted proteome of a hepatocyte induced to differentiate derived from a mesenchymal stem cell or MFG-E8 with a drug candidate; and measuring whether the drug candidate results in an increase in expression or secretion levels of MFG-E8, thereby identifying the drug candidate as a medicament for preventing or treating tissue fibrosis.

The present invention also provides a method for screening mesenchymal stem cells with improved tissue fibrosis treatment efficacy, comprising the steps of: whether the expression or secretion level of MFG-E8 is increased for any mesenchymal stem cell is measured or determined in comparison to normal mesenchymal stem cells.

The present invention reduces the expression of collagen induced by the TGF β/Smad signaling pathway, and particularly, inhibits the activity of hepatic stellate cells to improve hepatic fibrosis, and reduces the degree of hepatic fibrosis in a hepatic fibrosis disease model, and if treatment is performed on hepatic stellate cells cultured in vitro, the present invention can be used for the prevention or treatment of tissue fibrosis of the liver, lung, kidney, brain, heart, diaphragm, or the like, based on the characteristics of MFG-E8 protein that inhibits activation.

In addition, hepatocytes induced to differentiate obtained from mesenchymal stem cells, together with the MFG-E8, may be used as a cell therapeutic agent for treating tissue fibrosis.

Drawings

Fig. 1a is a photograph showing the results of comparing the structure and fibrosis degree of liver tissues by H & E, Sirius Red (Sirius Red) and MT staining by treating a mouse model for chronic liver disease with a hpUCMSC secretory proteome, a secretory proteome after treating the hpUCMSC secretory proteome with an MFG-E8 antibody, and a MFG-E8 synthetic protein, respectively, fig. 1b is a photograph showing the results confirmed in fig. 1a by a graph, fig. 1c is a photograph showing the results of treating a mouse model for chronic liver disease with different concentrations of MFG-E8 protein and confirming the accumulation of collagen by Sirius Red, fig. 1d is a photograph showing the results of observing the results of fig. 1d by an optical microscope in the mouse model for liver fibrosis as in fig. 1 c.

FIG. 2a is a photograph showing the expression of α -SMA (α -smooth muscle actin), which is obtained by activating hTERT-HSCs as hepatic stellate cell lines using TGF- β 1 (transforming growth factor- β), and then treating 3 synthetic proteins of decorin, PEDF (pigment epithelium-derived factor), and MFG-E8, FIG. 2b is a photograph showing the results of FIG. 2a using Western blot (Western blot), FIG. 2c is a photograph showing the results of HSCs after reducing the activity of Hepatic Stellate Cells (HSCs) obtained by treating a hpUCHC protein group with 3 protein antibodies, and FIG. 2d is a photograph showing the results of HSCs cultured in human primary hepatocytes (human primary hepatocytes, human primary HSCs), fig. 2E is the result of treatment of hTert-HSCs with different concentrations of MFG-E8, fig. 2f is the result of treatment of HSCs in Human primary culture with different concentrations of MFG-E8, fig. 2g is the result of confirmation of expression of MFG-E8 protein in various mesenchymal stem cell secretory proteomes, and fig. 2h is the result of confirmation of the result of fig. 2g by ELISA.

Detailed Description

Hereinafter, the structure of the present invention will be specifically described.

The present invention relates to a composition for preventing or treating tissue fibrosis comprising milk fat globule epidermal growth factor 8 protein (MFG-E8).

Furthermore, the present invention provides the use of MFG-E8 for the manufacture 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 β/Smad signaling pathway in lung, kidney, liver, brain, heart, diaphragm tissue or the like, and MFG-E8 inhibits expression of collagen induced through TGF β/Smad signaling pathway or inhibits activation of hepatic stellate cells, thereby reducing hepatic fibrosis.

More specifically, according to one embodiment of the present invention, when a secretory proteome of hepatocytes induced to differentiate from mesenchymal stem cells is introduced into a mouse model of chronic liver disease, the expression of α -SMA (Smooth Muscle Actin), which is a liver tissue marker, is reduced, and fibrosis is reduced. In order to confirm the hepatic fibrosis-inhibiting effect of the secretory proteome of hepatocytes, the secretory proteome was treated with an antibody against MFG-E8 protein and then administered to a mouse model for chronic liver disease, and as a result, no effect of reducing α -SMA expression was produced. Based on this, as a result of administering the MFG-E8 synthetic protein to the mouse model of chronic liver disease, it was confirmed that the effect of reducing the expression of α -SMA similar to the secretory proteome of hepatocytes was exhibited. Furthermore, the dosing of MFG-E8 protein resulted in a reduction in collagen accumulation, the reducing effect being exhibited by a concentration-dependent manner of MFG-E8. Furthermore, the MFG-E8 protein also exhibits a fibrosis-reducing effect in a mouse model of liver fibrosis.

Next, as a result of treatment with decorin, PEDF (Pigment Epithelium-Derived Factor) and MFG-E8 protein after activating hepatic stellate cell line with TGF β 1, expression of α -SMA was decreased, and as a result of treatment with MFG-E8 protein antibody, secretory proteome of hepatic cells induced to differentiate from mesenchymal stem cells was decreased, Hepatic Stellate Cells (HSCs) were treated, and activation of HSCs was confirmed, expression of α -SMA was increased, and in the case of treatment with MFG-E8 protein, expression of α -SMA in HSCs was decreased in a concentration-dependent manner, and it was found that MFG-E8 protein inhibited hepatic fibrosis.

Furthermore, MFG-E8 is expressed in a secreted proteome of various Mesenchymal Stem cells, for example, Umbilical Cord Mesenchymal Stem Cells (UCMSCs), Deciduous tooth pulp Stem cells (Stem cells from Human infected cells; SHED), Bone Marrow Stem cells (Bone Marrow Stem cells; BMSC), or the like, or in hepatocytes induced to differentiate from such Stem cells, but is not expressed in Human embryonic Stem cells.

Thus, MFG-E8 may be used for the purpose of preventing or treating tissue fibrosis through a fibrosis-inhibiting function.

The MFG-E8 includes natural or recombinant MFG-E8, or proteins having substantially equivalent physiological activity thereto. The proteins having substantially the same physiological activity include natural/recombinant MFG-E8, functional equivalents (functional equivalents) and functional derivatives (functional derivatives).

The term "functional equivalent" refers to a variant of an amino acid sequence in which a part or all of the amino acids in a natural protein are substituted, or a part of the amino acids is deleted or added, and which has substantially the same physiological activity as natural MFG-E8.

The term "functional derivative" means a protein which is modified to increase or decrease the physicochemical properties of the above-mentioned MFG-E8 protein and has substantially the same physiological activity as that of natural MFG-E8.

The MFG-E8 may be a mammalian protein derived from human, mouse, rat (rat), etc.

The MFG-E8 can be produced by a genetic engineering method known to practitioners based on a known sequence, for example, the sequence of human MFG-E8 disclosed in the gene bank (GenBank) NM _ 005928.

The recombinant MFG-E8 can be isolated by general column chromatography (column chromatography) or the like, and the degree of purification of the protein can be confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) or the like.

The present invention also relates to a composition for preventing or treating tissue fibrosis comprising a secretory proteome of hepatocytes induced to differentiate obtained from mesenchymal stem cells.

Further, the present invention provides a use of a secretory proteome of hepatocytes obtained from mesenchymal stem cells for inducing differentiation for the manufacture of a pharmaceutical composition for the prevention or treatment of tissue fibrosis.

According to the present invention, the secreted proteome of induced-differentiation hepatocytes obtained from mesenchymal stem cells includes the MFG-E8, so that the secreted proteome of induced-differentiation hepatocytes obtained from mesenchymal stem cells can be used for the prevention or treatment of tissue fibrosis.

The "differentiation" is a general term for a process of changing the structure and morphology from stem cells to specific cells, and refers to a process of changing the structure and morphology suitable for performing each function. The differentiation includes spontaneous differentiation and induced differentiation. The induced differentiation from the stem cells into specific cells can be performed using various methods known in the art or by applying the methods.

The "secretory proteome" generally means a mixture of organic and inorganic elements secreted from cells, tissues, organs, organisms, and more particularly, refers to secreted proteins, and the secretory proteome of the hepatic cells induced to differentiate from mesenchymal stem cells of the present invention comprises MFG-E8.

The secretory proteome may be obtained by differentiating the mesenchymal stem cells in a serum medium and culturing for a certain time, and then concentrating the culture, but is not limited thereto.

The cell therapeutic agent for treating fibrosis of the liver, lung or kidney using the MFG-E8 protein and hepatocytes is caused by astrocytes (myofibroblasts) activated by TGF- β, in addition to fibrosis of the liver, lung or kidney, and thus a secretory proteome of hepatocytes induced to differentiate from the mesenchymal stem cells can be used for fibrosis treatment of the brain, heart, diaphragm, and the like.

Thus, the tissue fibrosis may be a fibrosis generated in any one of lung, kidney, liver, brain, heart or diaphragm.

The composition for preventing or treating tissue fibrosis of the present invention may further comprise a pharmaceutically acceptable carrier.

Carriers which are generally used in the pharmaceutical field include carriers and vehicles (vitamins) which include, in particular, ion exchange resins (ion exchange resin), aluminum oxides (aluminum), aluminum stearates (aluminum stearate), lecithins (lecithin), serum proteins (e.g., human serum albumin (albumin)), buffer substances (e.g., various phosphates (phosphate), glycine (glycine), sorbic acid (sorbic acid), potassium sorbate (potassium sorbate), partial glyceride (glyceride) mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g., protamine sulfate, Disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride (sodium chloride) and magnesium silicate (zinc silicate)), colloidal silica (colloidal silica), magnesium stearate, polyvinyl pyrrolidone (polyvinylpyrrolidone), colloidal silica (sodium stearate), polyvinyl pyrrolidone (polyvinylpyrrolidone), and mixtures thereof), Cellulose (cellulose) base, polyethylene glycol (polyethylene glycol), sodium carboxymethylcellulose (sodium carboxymethyl cellulose), polyarylate (polyarylate), wax (wax), polyethylene glycol (polyethylene glycol), lanolin (wool grease), or the like, but is not limited thereto.

In addition, the composition of the present invention may additionally include a lubricant, a wetting agent, an emulsifier, a suspending agent, a preservative, or the like, in addition to the ingredients.

As one aspect, the composition according to the present invention may be manufactured by a water-soluble solution for parenteral administration (partial administration), and preferably, a Hank's solution (Hank's solution), a Ringer's solution (Ringer's solution), or a buffer solution such as saline buffered in a physical form may be used. Water-soluble injection (injection) suspending agents it is possible to add a matrix which, like sodium carboxymethylcellulose, sorbitol or dextran, can bring about an increase in the viscosity of the suspension.

The composition of the present invention can be administered systemically or locally, and for the purpose of administration as described above, it can be formulated into an appropriate dosage form by a known technique. For example, in oral administration (oral administration), the administration may be by mixing with an inert diluent or an edible carrier, or sealing in a hard or soft capsule (gelatin capsule) or compression molding in the form of a tablet. For oral administration, the active compound may be combined with an excipient (excipient) and used in the form of an absorptive lozenge, buccal lozenge, tablet (troche), capsule (capsule), elixir (elixir), suspension (suspension), syrup (syrup), or tablet.

Various dosage forms for injection, parenteral administration and the like can be manufactured according to a method known in the art or a general method. Since MFG-E8 is easily dissolved in saline or a buffer solution, MFG-E8 may be administered as a solution after storage in a freeze-dried state, before an effective dose of the solution is administered in saline or a buffer solution in a form suitable for intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.

The appropriate dosage of the composition of the present invention may be variously prescribed depending on such factors as formulation method, administration mode, patient age, body weight, sex, disease state, diet, administration time, administration route, excretion rate and reaction sensitivity. For example, in terms of the amount of the composition of the present invention administered, an adult may administer from 0.1 to 1000 mg/kg per day, preferably from 10 to 100 mg/kg per day over several times.

The present invention also provides a method of treating tissue fibrosis comprising the step of administering a pharmaceutically effective dose of MFG-E8 to an individual.

Further, the present invention provides a method for treating tissue fibrosis, comprising the step of administering to an individual a secreted protein group of hepatocytes induced to differentiate obtained from mesenchymal stem cells.

MFG-E8 used in the method for treating tissue fibrosis or the secretory proteome of hepatocytes induced differentiation obtained from mesenchymal stem cells and the administration method follow the reference and administration method of the pharmaceutical composition as described above, and thus the common contents between the two are omitted in order to avoid unnecessary complexity of the present specification.

The subject may be, but is not limited to, a dog, cat, mouse, human, etc.

The tissue fibrosis may be a fibrosis generated in any one of lung, kidney, liver, brain, heart or diaphragm.

The invention also relates to a composition comprising MFG-E8 and

a cell therapeutic agent for liver cells for treating tissue fibrosis.

The cell therapeutic agent can further improve the therapeutic effect when treating tissue fibrosis by cell transplantation of hepatocytes.

In this regard, the hepatocyte may be a hepatocyte induced to differentiate from a mesenchymal stem cell, but is not particularly limited thereto.

The hepatocyte induced to differentiate from the mesenchymal stem cell may be used in admixture with the term "hepatocyte-like cell" or "hepatocyte-like cell".

Thus, it is useful as a cell therapeutic agent for treating liver, lung or kidney fibrosis by cell therapy using MFG-E8 and liver cells. Furthermore, since fibrosis is also caused in the brain, heart, diaphragm, and the like by myofibroblast (myofibroblast) activated by TGF- β, it can be used for fibrosis treatment of the brain, heart, diaphragm, and the like by cell therapy using MFG-E8 and hepatocytes.

The invention also relates to a medical screening method for preventing or treating tissue fibrosis, which comprises the following steps: contacting a secreted proteome of a hepatocyte induced to differentiate derived from a mesenchymal stem cell or MFG-E8 with a drug candidate; and measuring whether the drug candidate results in an increase in expression or secretion levels of MFG-E8, thereby identifying the drug candidate as a medicament for preventing or treating tissue fibrosis.

The drug candidates may be assumed to be substances that promote or inhibit transcription and translation to mRNA (Messenger RNA), protein, or have a possibility as a medicine for increasing or inhibiting the function or activity of MFG-E8 protein in the MFG-E8 gene sequence in accordance with a generally selected manner, or may be any selected individual nucleic acid, protein, peptide (peptide), other extract or natural product, compound, or the like.

Thereafter, the expression level of the MFG-E8 gene, the amount of protein or the activity of the protein in the cells treated with the candidate drug can be measured, and as a result of the measurement, if the expression level of the MFG-E8 gene, the amount of the protein or the activity of the protein is measured to be increased, it is judged that the candidate drug is 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 as described above may be performed by various methods known in the art, but for example, it is not limited thereto, and may be performed by reverse transcription-polymerase chain reaction (reverse transcriptase-polymerase chain reaction), real-time polymerase chain reaction (real time-polymerase chain reaction), western blot (western blot), northern blot (northern blot), enzyme linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), radioimmunoassay (radioimmunodification), immunoprecipitation (immunoprecipitation), and the like.

The candidate substance for a therapeutic agent for tissue fibrosis as described above functions as a lead substance (lead compound) in the subsequent development of a therapeutic agent for tissue fibrosis, and the lead substance deforms the structure and optimizes the structure so as to have an effect of promoting the function of the MFG-E8 gene or a protein expressed therefrom, thereby enabling the development of a novel therapeutic agent for tissue fibrosis.

In the present invention, the matters related to genetic engineering techniques have become clearer from the disclosures of Sambrook et al (Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)) and Frederick et al (Frederick M. Ausubel et al, Current protocols in Molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994)).

The invention also relates to a method for screening mesenchymal stem cells with improved tissue fibrosis treatment efficacy, which comprises the following steps: the presence or absence of an increase in the expression or secretion level of MFG-E8 is measured or determined for any mesenchymal stem cell as compared to a normal mesenchymal stem cell.

The invention discloses the mesenchymal stem cells for treating the tissue fibrosis for the first time, wherein the effective components of the mesenchymal stem cells are MFG-E8 secreted from the mesenchymal stem cells, and the mesenchymal stem cells with increased expression or secretion level of MFG-E8 can be regarded as that the treatment efficiency of the tissue fibrosis is improved.

Thus, the expression or secretion level of MFG-E8 within mesenchymal stem cells may act on a basis for improving the pharmacological therapeutic effect of mesenchymal stem cells for the treatment of tissue fibrosis.

Hereinafter, the present invention will be described in more detail by way of examples according to the present invention, but the scope of the present invention is not limited by the examples set forth below.

< example 1> production of culture solution for differentiated hepatocytes from mesenchymal Stem cells

The mesenchymal stem cells used in this experiment were derived from umbilical cord blood and differentiated into hepatocytes at 3X 10⁴cell/cm²Attached to a collagen-coated culture dish.

MesenPro RSTM medium (medium) (Gibco) was used as a minimal medium. If the culture dish is filled with 70-80%, Epidermal growth factor (Epidermal growth factor) (EGF; Peprotech EC Ltd, London, England, 20ng/mL) and basic fibroblast growth factor (bFGF; Peprotech EC Ltd, 10ng/mL) are placed in Iscove's modified Dulbecco's medium (IMDM; Invitrogen, Carlsbad, CA, USA) as a basic differentiation medium and cultured for two days.

Thereafter, hepatocyte growth factor (HGF; Peprotech EC Ltd, 20ng/mL), bFGF (10ng/mL), nicotinamide (nicotinamide) (Sigma, 0.61g/L), 1% insulin-transferrin-selenium (ITS) premix (Invitrogen) was added to the basal medium IMDM as a second differentiation medium, and cultured for ten days.

Finally, oncostatin M (oncostatin M) (Peprotech EC Ltd, 20ng/mL), dexamethasone (dexamethasone) (Sigma, 1. mu. mol/L), 1% ITS were placed in basal medium IMDM and re-cultured for ten days.

To obtain the secreted proteome of differentiated mesenchymal stem cells, after 0.05% FBS (fetal bovine serum, total calf serum) was added to the basal medium IMDM and cultured for 24 hours, it was concentrated 25-fold using a 3-kDa cut-off filter ultrafiltration unit (3-kDa cut-off filter ultrafiltration units) (Millipore).

< example 2> experiment of Effect of MFG-E8 in model of chronic liver disease and in model of hepatic fibrosis

In order to induce chronic liver disease, Thioacetamide (TAA) was diluted in saline at 200mg/kg body weight and then intraperitoneally injected into 5-6 weeks old mice (C57 BL/C). Intraperitoneal injections were repeated for 8 weeks, 3 times in 1 week. After 8 weeks, the proteins of the concentrated secreted proteome were quantified by Bradford assay (Bradford assay), after which a 500 μ g quantity was intraperitoneally injected. After the injection of the secretory proteome, the degree of hepatic fibrosis of the mice was analyzed after 3 days and 7 days. MFG-E8 protein (R & D systems, kit) was injected into 160. mu.g/kg of body weight as a base, and 32. mu.g/kg of body weight was set to a low concentration, 160. mu.g/kg of body weight was set to an intermediate concentration, and 800. mu.g/kg of body weight was set to a high concentration in order to confirm the effects of the different concentrations, and then injected into the abdominal cavity. After the administration of the MFG-E8 protein, the degree of hepatic fibrosis was analyzed 3 days later.

Secondly, to induce hepatic fibrosis, carbon tetrachloride (CCl) is added₄) After 10% dilution in olive oil, 5-6 weeks old mice (BALB/c) were injected intraperitoneally at 100. mu.g/20 g body weight. Intraperitoneal injections were repeated 6 weeks after 2 times in 1 week. Thereafter, secretory proteome injection and MFG-E8 protein input were performed using the same method as the model for chronic liver disease established by TAA.

In addition, in order to confirm the degree of fibrosis of the liver tissue, the liver tissue was fixed to 4% paraformaldehyde (paraformelhyde). After the liver tissues were fixed to paraffin (paraffin) and obtained in the form of tissue sections, H & E (hematoxylin-eosin staining), Masson's trichrome (Masson's trichrome), sirius red (sirius red) staining was performed and observed under an optical microscope.

For Masson's trichrome staining, observation was performed after staining with wight hematoxylin/pbutyrin-acid fuchsin-aniline blue (Weigert hematoxylin/biedrich-acid fuchsin-aniline blue) or 2% light green (light green) after re-fixing the tissue section obtained by paraffin (paraffin) fixation to the buuin (Bouin) solution through a paraffin (paraffin) process. The fibrotic tissue appears blue, the cytoplasm, muscle, keratin (keratin) red, and the nucleus black-brown.

For sirius red staining, tissue sections obtained by paraffin fixation were subjected to a paraffin removal process and stained for nuclei with hematoxylin (hematoxylin), followed by staining for 1 hour with a picric-sirius red stain. The portion where hepatic fibrosis is induced is shown in red.

For immunofluorescent staining, tissue sections obtained by paraffin fixation were subjected to a paraffin removal process and blocking with 10% donkey serum (donkey serum), and then reacted with α -SMA (α -smooth muscle actin) and F4/80 antibody. Thereafter, the reaction mixture was reacted with a secondary antibody having a fluorescent label attached thereto, and then observed with a fluorescence microscope.

As a result of the staining, fibrosis due to collagen accumulation was observed in the tissue.

In addition, a mouse model of chronic liver disease made using TAA was treated with the hpUCMSC secretory proteome, the hpUCMSC secretory proteome treated with the MFG-E8 antibody, and the MFG-E8 synthetic protein, respectively, and the structure and fibrosis degree of liver tissue were compared by staining with H & E, Sirius Red (Sirius Red), and MT, and as a result, fibrosis was significantly reduced in the group treated with the hpUCMSC secretory proteome and the MFG-E8 protein. The group treated with the MFG-E8 antibody underwent more severe fibrosis than the sham (sham). Expression of α -SMA was also reduced in the group treated with the hpcmcs secreted proteome and MFG-E8 protein compared to the sham group (fig. 1a and 1 b). At this time, F4/80 as a marker (maker) of macrophages was confirmed by the control group, but no difference was shown for each group.

The mouse model of chronic liver disease was treated with MFG-E8 protein at various concentrations and collagen accumulation was confirmed with sirius red, with the result that fibrosis was reduced compared to the sham model (FIG. 1 c).

Furthermore, as shown in FIGS. 1d and 1E, the results of experiments in which the secretory proteome and the MFG-E8 protein were introduced into the mouse model of hepatic fibrosis reduced fibrosis compared to the sham model.

< example 3> Effect test of MFG-E8 in hepatic stellate cell lines (hTERT-HSCs) and Primary cultured hepatic stellate cells (primary HSCs)

In vitro (In-vitro) effects of secreted proteomes were tested using hepatic stellate cell lines and primary cultured hepatic stellate cells. For this purpose, the hepatic stellate cell line was cultured in a culture medium of DMEM (Dulbecco's modified eagle medium) supplemented with 10% FBS, 100 units of penicillin (penicillin) and 100. mu.g of streptomycin (streptomycin). In the experiment, in order to prepare a deactivated hepatic stellate cell line, the hepatic stellate cell line was activated by culturing the cell line in a culture medium of DMEM supplemented with 0.2% FBS for 24 hours and then treating the cell line with TGF β 1 protein in the same medium at a concentration of 10 ng/mL. The MFG-E8 protein used in the experiments was treated with TGF β 1 protein in a form substantially up to 500ng/mL and cultured for 48 hours. When the cells were treated at different concentrations, the cells were cultured in such a manner that they became 100ng/mL, 250ng/mL, 500ng/mL, 1. mu.g/mL, or 5. mu.g/mL. For neutralizing proteins in the secretory proteome, the mixture was mixed with the secretory proteome so that the concentration of the antibody became 20. mu.g/mL, and then allowed to stand at room temperature for 1 hour.

In the case of primary cultured hepatic stellate cells, the primary culture was performed in a stecm (sciencell) culture medium, and thereafter, experiments were performed in which the primary culture was performed under the same conditions as those of the hepatic stellate cell line.

FIG. 2a shows that hTERT-HSCs were activated with TGF β 1 and then treated with three synthetic proteins, and as a result, hepatic stellate cell lines were inactivated in a serum-free state, and thin cells were observed, whereas cells were activated in a wide and flat shape by treatment with TGF β 1, and α -SMA expression was increased. At this time, the treatment with decorin, PEDF and MFG-E8 all resulted in a decrease in the expression of α -SMA. Similar results were also confirmed in western blot results (fig. 2 b).

In addition, after the hpcmcs secretory proteome was treated with the antibody for each protein to decrease the activity of the protein, the activity of HSCs was confirmed, and as a result, the expression of α -SMA was increased as compared with the hpcmcs secretory proteome that was not treated with the antibody (fig. 2 c). It was confirmed whether or not HSCs cultured in human primary cultures exhibited the same effect as 3 proteins, and as a result, the expression of α -SMA was significantly reduced in the group treated with MFG-E8 (fig. 2 d).

The concentration-dependent decrease of hTERT-HSCs was observed when different concentrations of hTERT-HSCs were treated with MFG-E8 (FIG. 2E), and when different concentrations of HSCs primarily cultured in humans were treated with MFG-E8 (FIG. 2 f).

< example 4> confirmation of MFG-E8 protein expression Using Western blotting and ELISA (enzyme-linked immunosorbent assay)

To confirm the degree of activation of the hepatic stellate cell line, protein was extracted and western blotting was performed. The proteins of the cells were extracted using RIPA buffer containing protease inhibitor (protease inhibitor). After 40. mu.g of each group of proteins was separated by SDS-PAGE gel (gel), the separated proteins were transferred to a polyvinylidene fluoride transfer membrane (polyvinylidene fluoride transfer membrane). Ponceau s (ponceau s) solution was used to confirm whether the protein was successfully transferred to the membrane. After that, after a retardation at room temperature in a TBS-T buffer solution to which 5% skim milk (skimmilk) was added, the membrane was refrigerated in a solution containing α -SMA and each of the antibodies to MFG-E8 and GAPDH for one night. Thereafter, the secondary antibodies corresponding to the respective antibodies were reacted at room temperature for 1 hour, and then the expression of the corresponding proteins was confirmed using a chemiluminescence kit (chemiluminiscence kit).

In the case of ELISA experiments for the MFG-E8 protein, the human MFG-E8 enzyme linked immunosorbent assay kit (ELISA kit) marketed by Cusabio was used.

As shown in fig. 2g, the MFG-E8 protein was expressed in various stem cells (umbilical cord blood mesenchymal stem cells (UCMSC), exfoliated deciduous tooth stem cells (SHED), Bone Marrow Stem Cells (BMSC)) and the secretory proteome of induced-differentiation hepatocytes (hpcumscs, hpSHEDs, hppmscs) obtained from the stem cells, but not in embryonic stem cells. The results of enzyme-linked immunosorbent (ELISA) analysis also showed the same results (FIG. 2 h).

Possibility of industrial utilization

The present invention may be used in the field of preventing or treating tissue fibrosis.

Claims

1. Use of a secreted proteome of hepatocytes induced to differentiate obtained from umbilical cord mesenchymal stem cells for the manufacture of a medicament for preventing or treating liver tissue fibrosis;

the secreted proteome includes milk fat globule epidermal growth factor 8 protein (MFG-E8).

2. A screening method for a drug for preventing or treating liver tissue fibrosis, comprising the steps of:

contacting a secreted proteome of hepatocytes induced to differentiate obtained from umbilical cord mesenchymal stem cells with a drug candidate, the secreted proteome including milk fat globule epidermal growth factor 8 protein (MFG-E8); and

measuring whether the drug candidate causes an increase in expression or secretion level of MFG-E8, thereby identifying the drug candidate as a medicament for preventing or treating liver tissue fibrosis.