KR20180066816A - Production method of enhanced hepatocyte regenerating mesenchymal stem cell and Composition for treating liver cirrhosis using the same - Google Patents

Abstract

The present invention relates to a method for manufacturing mesenchymal stem cells for increasing regeneration of hepatocytes and a composition for treating liver cirrhosis using the same. A cytokine cocktail according to the present invention contains VEGF, bFGF, EGF and IGF as active ingredients. When the cytokine cocktail containing the above ingredients is applied to a culture of mesenchymal stem cells, the proliferative capacity of the mesenchymal stem cells, the engraftment rate in the liver tissue, and the expression of HGF are increased, and thus an effect of inhibiting hepatic fibrosis is confirmed to be remarkably alleviated. Therefore, the present invention can be utilized as an effective substance which can replace conventional therapeutic agents for liver diseases.

Description

TECHNICAL FIELD The present invention relates to a method for producing mesenchymal stem cells for regeneration of hepatocytes, and a method for treating hepatocyte stem cells using the same,

The present invention relates to a method for producing mesenchymal stem cells for regeneration of hepatocytes and a composition for treating liver hardening using the same.

The liver is a type of organs that perform various functions, and recent deaths from liver disease and liver disease are increasing. In Korea, hepatic diseases tend to be more common than hepatitis B or C. However, as in the case of Gumi, the number of patients with liver disease due to alcohol and the like is increasing more than infectious liver disease. In addition, the number of women suffering from the disease is also increasing.

In general, liver cirrhosis is the final state of chronic liver disease. The main causes of liver cirrhosis are hepatitis virus infection, alcohol poisoning, bile acid secretion disorder, drug poisoning, allergy, excessive deposition of iron Do. Hepatocytes can be regenerated by regeneration even if they are destroyed to some extent because of their strong regeneration ability, but they are not regenerated at any time and become fibrous. If they become fiber components, the liver becomes hardened. The structure of the liver is changed so that it can not go back to its original condition. The process of liver fibrosis leading to liver cirrhosis is a reaction against continuous stimulation, which is similar to the wound healing process: 1) acute inflammation, 2) synthesis of extracellular matrix (ECM) components including collagen, 3 ) Tissue reconstruction (formation of scars). The components of the extracellular matrix are composed of collagen, matrix glycoproteins (fibronectin, laminin, etc.), proteoglycans, etc., but the main component is collagen. In other words, after the liver cells are destroyed, in the process of reconstituting the tissues with new liver cells, only an extracellular matrix, which maintains the skeletal structure of the cells without forming a perfect cell structure such as cytoplasm and extracellular membrane, Collagen, which is the main component of the outer membrane matrix, is excessively deposited and fibrous collagen hardens the liver tissue, resulting in cirrhosis.

In addition, hepatic fibrosis refers to a disease in which liver tissue in chronic inflammation is repeatedly damaged and regenerated, and connective tissues such as collagen accumulate excessively in the tissue, resulting in scarring of liver tissue. Generally, liver fibrosis is reversible, unlike cirrhosis, is composed of thin fibrils and has no nodule formation. In addition, if the cause of liver damage is lost, normal recovery may be possible. However, when such liver fibrosis mechanism is repeatedly performed, crosslinking between connective tissues is increased to accumulate thick fibrils, and the normal structure of hepatic lobules is lost and nodules are formed. It progresses to irreversible cirrhosis.

Currently, studies on liver cirrhosis and the like are actively carried out in various fields such as research on ECM, relationship between cells and various cells involved therein, activation mechanism of cells, various cytokines and antifibrogenic agents have. Conventionally, as an effective substance for hepatic fibrosis or hepatocyte treatment, there have been used various pharmaceutical agents such as penicillamine, 16,16-dimethylproplygladine E2, biphenyldimethyldicarboxylic acid, colchicin, glucocorticoid, malotilate, Interferon, pentoxifylline, pyridine-2,4-dicarboxylic-diethylamide, pyridine-2,4-dicarboxylic-di (2-methoxyethyl) amide, hypothemycin ) Have been developed (Korean Patent Laid-Open Publication No. 10-2012-0062496). However, there is no therapeutic agent for hepatic cirrhosis and hepatic fibrosis, which is currently clinically effective due to insufficient therapeutic effects or severe side effects when applied to clinical applications.

Therefore, it is necessary to develop a new approach that can solve the problems of the conventional treatment methods. Therefore, development of a therapeutic agent using stem cells, which is emerging as a treatment method for various diseases, has been proposed.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a method for inhibiting HGF-overexpressed HGF associated with hepatocyte regeneration ability by using cytokine cocktail (VEGF, bFGF, EGF and IGF) Mesenchymal stem cells were obtained, and the excellent effect of inhibiting the hepatic fibrosis of the mesenchymal stem cells was confirmed, and the present invention was completed based on this finding.

It is an object of the present invention to provide a cytokine cocktail for inducing HGF expression of mesenchymal stem cells, and a medium composition for inducing mesenchymal stem cells expressing HGF.

Another object of the present invention is to provide a method for producing HGF-expressing mesenchymal stem cells, which comprises culturing mesenchymal stem cells isolated from a living body in the medium composition, and a method for producing HGF- Stem cells.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating liver disease comprising the mesenchymal stem cells.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a cytokine cocktail for inducing HGF expression of mesenchymal stem cells, which comprises VEGF, bFGF, EGF, and IGF as an active ingredient, and HGF The present invention provides a medium composition for inducing mesenchymal stem cells.

In one embodiment of the present invention, the concentration of VEGF, EGF, or IGF may be 10 to 200 ng / ml, respectively, and the concentration of bFGF may be 10 to 800 ng / ml.

In another embodiment of the present invention, the medium may be selected from the group consisting of DMEM, MEM, BME, RPMI 1640, DMEM / F-10, DMEM / F-12, a- MEMG-MEM, EGM, have.

The present invention also provides a method for producing HGF-expressing mesenchymal stem cells, comprising culturing mesenchymal stem cells isolated from a living body in the medium composition, and a method for producing HGF-expressing mesenchymal stem cells Provide stem cells.

In one embodiment of the present invention, the mesenchymal stem cells may be separated from umbilical cord, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, or placenta.

In another embodiment of the present invention, the step of culturing may be performed for 2 to 60 days.

In another embodiment of the present invention, the mesenchymal stem cells are capable of expressing CD14 negative, CD34 negative CD45 negative, CD29 positive, CD44 positive, CD73 positive, CD90 positive, and CD105 positive phenotype, The expression of HGF may be 1.2 to 15 times higher than that of human umbilical vein endothelial cells (HUVEC).

The present invention also relates to a pharmaceutical composition for preventing or treating liver disease, comprising mesenchymal stem cells expressing HGF as an active ingredient, the use of mesenchymal stem cells expressing HGF for the treatment of liver disease, And administering to the individual an mesenchymal stem cell expressing an effective amount of the HGF.

In one embodiment of the present invention, the liver disease can be selected from the group consisting of liver cancer, hepatitis, hepatic fibrosis, and liver cirrhosis.

The cytokine cocktail according to the present invention comprises VEGF, bFGF, EGF, and IGF as an active ingredient. When cytokine cocktail containing these components is applied to the culture of mesenchymal stem cells, the cytotoxic activity of the mesenchymal stem cells, The rate of engraftment in the liver tissue and the expression of HGF were increased and thus the hepatic fibrosis inhibiting effect was remarkably improved. As a result, the present invention can be utilized as an effective substance which can replace the conventional liver disease therapeutic agent.

FIG. 1 is a graph showing changes in the proliferative capacity of mesenchymal stem cells according to the present invention. FIG. 1 (a) is a result of observation with an optical microscope, and FIG. 1 (b) is a result of quantitative comparison of the number of proliferated cells (Control: cytokine Untreated group, bFGF: bFGF alone group, Cocktail: cytokine cocktail group, Bar = 100m).
FIG. 2 shows the results of qRT-PCR analysis of HGF expression in mesenchymal stem cells according to the present invention. (Control: cytokine-treated group, bFGF: bFGF alone group, VEGF-A: VEGF-A IGF: IGF alone treatment group, bFGF + EGF: bFGF and EGF treatment group, Cocktail: cytokine cocktail treatment group).
FIG. 3 shows changes in phenotype of mesenchymal stem cells according to the present invention. FIG. 3 (a) is a result of confirming phenotypic changes of CD14, CD34, CD45, CD29, CD44, CD73, CD90 and CD105, HGF expression was compared with that of human endothelial cells.
FIG. 4 is a graph showing changes in engraftment rate in liver tissues of mesenchymal stem cells according to the present invention. FIG. 4A is a result of immunostaining and FIG. 4B is a result of quantitative comparison of changes in engraftment in liver tissue (ASC: Group transplanted with mesothelial stem cells cultured in medium without cytokine cocktail, Group transplanted with mesenchymal stem cells cultured in medium supplemented with cytokine cocktail to ASC-C: mouse, Bar = 50m ).
FIG. 5 shows the result of immunohistochemical staining for a blood vessel marker (isolectin B-4; ILB-4), which confirmed the differentiation of mesenchymal stem cells into vascular cells according to the present invention.
FIG. 6 shows the result of visually observing changes in liver tissues after transplanting the mesenchymal stem cells according to the present invention into an animal model of liver cirrhosis (TAA + ASC: cytokine cocktail was not added to the liver-induced mice Transplantation of mesenchymal stem cells cultured on medium supplemented with cytokine cocktail to mice transfected with TAA + ASC-C: liver cirrhosis).
FIG. 7 shows the result of transplantation of mesenchymal stem cells according to the present invention into an animal model of liver cirrhosis, followed by H & E staining for changes in liver tissue (TAA + ASC: cytokine cocktail TAA + ASC-C: transplantation of mesenchymal stem cells cultured in a medium supplemented with cytokine cocktail to liver-induced mice, Bar = 100m).
FIG. 8 shows the degree of fibrosis after transplantation of the mesenchymal stem cells according to the present invention into an animal model of liver cirrhosis. FIG. 8A shows the result of Masson's trichrome staining, and FIG. 8B shows a quantitative comparison of fibrosis ASC: transplantation of mesenchymal stem cells cultured in culture medium without cytokine cocktail, transplantation of ASC-C (transfected) with ASC-C : A group transplanted with mesenchymal stem cells cultured in medium supplemented with cytokine cocktail, Bar = 100m).
FIG. 9 shows the result of quantifying collagen content in liver tissues after transplantation of mesenchymal stem cells according to the present invention into an animal model of liver cirrhosis (Sham: group treated with 50ul of physiological saline alone, TAA: liver cure group TAA + ASC: transplantation of mesenchymal stem cells cultured in culture medium without cytokine cocktail, TAA + ASC-C: liver Group transplantation of mesenchymal stem cells cultured in medium supplemented with cytokine cocktail to cured mice).
FIG. 10 shows the result of detection of liver damage marker in serum after transplanting the mesenchymal stem cells according to the present invention into an animal model of liver cirrhosis. FIG. 10A shows the result of detecting GPT in serum, FIG. FIG. 10C is a result of detection of ammonia in serum, and FIG. 10D is a result of detecting serum bilirubin. FIG. (Sham: group treated with 50ul of physiological saline alone, PBS: group treated with PBS for hepatocyte-induced mice, ASC: group of mesenchymal stem cultured in medium lacking cytokine cocktail, Cell transplantation group, ASC-C: transplantation of mesenchymal stem cells cultured in medium supplemented with cytokine cocktail to mice induced liver cirrhosis).

Hereinafter, the present invention will be described in detail.

The present invention provides a cytokine cocktail for inducing HGF expression of mesenchymal stem cells comprising VEGF, bFGF, EGF, and IGF as an active ingredient, and a medium composition for inducing mesenchymal stem cell expressing HGF comprising the same .

As used herein, the term "HGF or hepatocyte growth factor" is one of the multifunctional heterodimeric polypeptides produced by mesenchymal stem cells, which promotes angiogenesis, morphogenesis, and motor production It is also known that there is a close relationship with the regeneration of hepatocytes. Despite the excellent biological efficacy of HGF, however, the amount of HGF production or expression in mesenchymal stem cells cultured by conventional methods is only a very small amount, and thus it has been somewhat difficult to be used clinically. Therefore, the present invention is designed to overcome the above-mentioned problems of the prior art, and it has been found that by adding a cytokine cocktail consisting of VEGF, bFGF, EGF, and IGF to the mesenchymal stem cell culture medium, , And significantly increased the engraftment rate in liver tissue (see Examples 2 to 4).

Examples of media usable in the present invention include DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F- ), DMEM / F-12 (Dulbecco's Modified Eagle's Medium, a-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), and IMDM (Isocove's Modified Dulbecco's Medium) Any cell culture medium commonly used in the art can be used.

In order to improve the HGF expression-enhancing effect of mesenchymal stem cells, the concentration of VEGF, EGF, or IGF contained in the composition of the present invention may be 10 to 40 ng / ml, and the concentration of bFGF may be 100 to 200 ng / ml, but these components may be composed, if necessary, at concentrations outside the above range. In one embodiment of the present invention, the VEGF, EGF, or IGF was added to the culture medium at a concentration of 20 ng / ml and bFGF at a concentration of 100 ng / ml.

In another aspect of the present invention, the present invention provides a method for producing HGF-expressing mesenchymal stem cells, comprising culturing mesenchymal stem cells isolated from a living body in the medium composition, and a method for producing HGF Lt; RTI ID = 0.0 > mesenchymal < / RTI > stem cells.

As used herein, the term "stem cell" refers to a cell that is the basis of a cell or tissue constituting an individual, and is capable of differentiating into a specific or pluripotent functional cell and self-replicating ability , ≪ / RTI > can be referred to as undifferentiated cells. The stem cells may be autologous or allogeneic stem cells, and may be derived from any type of animal including human and non-human mammals. Embryonic stem cells include adult stem cells, but preferably adult stem cells. The adult stem cell may be a mesenchymal stem cell derived from a human tissue, a mesenchymal stem cell derived from a human tissue, a multipotential stem cell or an amniotic epithelial cell, and the mesenchymal stem cell may be a cord, cord blood, bone marrow, , The skin, the amniotic membrane, or the placenta. In one embodiment of the present invention, adipose tissue-derived stem cells were used as the stem cells.

For the purpose of the present invention, the mesenchymal stem cells can express CD14 negative, CD34 negative CD45 negative, CD29 positive, CD44 positive, CD73 positive, CD90 positive, and CD105 positive phenotype, (HUVEC) compared to human umbilical vein endothelial cells (HUVEC). As described above, the mesenchymal stem cells prepared by the method of the present invention, in which the expression of HGF is promoted, have the effect of improving hepatocyte regeneration ability and inhibiting hepatic fibrosis, that is, (See Example 5).

Accordingly, as another embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing or treating liver disease, which comprises the HGF-expressing mesenchymal stem cells as an active ingredient, a pharmaceutical composition for preventing or treating HGF, The use of mesenchymal stem cells, and a method of treating a liver disease comprising administering to a subject a therapeutically effective amount of mesenchymal stem cells expressing said HGF.

As used herein, the term " prophylactic " means any action that inhibits or slows the onset of liver disease by the administration of the pharmaceutical composition according to the present invention.

As used herein, the term " treatment " refers to any action that improves or alters the symptoms of liver disease by the administration of the pharmaceutical composition according to the present invention.

The term "liver disease" which is a disease to be prevented or treated by the composition of the present invention refers to a state in which one or more functions among the various functions performed by the liver are problematic and the metabolism can not be normally performed. , Hepatitis, liver fibrosis, or liver cirrhosis.

In the present invention, the term "individual" means a subject in need of treatment of a disease, and more specifically refers to a mammal such as a human or non-human primate, mouse, dog, cat, horse and cattle do.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, body weight, absorbency of the active ingredient, inactivation rate and excretion rate, disease type, The severity of obesity, sex, weight, age and the like, the dose is not limited in any way to the scope of the present invention.

In one embodiment, the pharmaceutical composition of the present invention can be prepared in the form of a cell therapeutic agent. The "cell therapeutic agent" is a therapeutic or prophylactic agent for the treatment, diagnosis and prevention of cells and tissues produced by isolation, culture, (FDA regulation) is a set of medicines (US FDA regulations) for the purpose of restoring the function of a cell or tissue, such as living, autologous, allogeneic, or xenogeneic cells in vitro, Refers to medicines used for the purpose of treatment, diagnosis and prevention through conduct.

The pharmaceutical composition of the present invention may be injected into a patient's body in a state of being cultured alone or in an incubator, for example, Lyndval et al. (1989, Arch. Neurol. 46: 615-31) or Douglas conchiolca Douglas Kondziolka, Pittsburgh, 1998). The preparation may contain conventional pharmaceutically acceptable carriers other than the hepatocyte differentiated from the mesenchymal stem cells, and in the case of an injectable preparation, preservatives, analgesic agents, solubilizing agents or stabilizers, An excipient, an excipient, a lubricant or a preservative, and the like.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components.

The pharmaceutical composition of the present invention may be formulated into a unit dosage form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in an oil or aqueous medium, and may additionally contain dispersing or stabilizing agents.

The composition of the present invention may be administered parenterally, intravenously, subcutaneously, intraperitoneally or topically, preferably intracardiac injection. Compositions for parenteral administration (e. G., Injections) of the compositions according to the present invention can be prepared by dispersing and / or dissolving a pharmaceutically acceptable carrier, e. G., Sterile purified water, buffer at about pH 7, or physiological saline, And if necessary, may contain conventional additives such as preservatives, stabilizers and the like.

In addition, although the amount of mesenchymal stem cells injected in the present invention is not limited thereto, it may be 10 ¹ to 10 ¹⁰ cells / cycle, preferably 10 ⁵ to 10 ⁷ cells / cycle, It is not. The dose may be variously prescribed depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1. Using cytokine cocktail Intermediate lobe  Culture of stem cells

Human adipose-derived mesenchymal stem cells were purchased from ATCC (Manassas, VA, USA). First, the mesenchymal stem cells were cultured in a-MEM medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 mg / ml streptomycin under 37 and 5% CO ₂ , The medium was supplemented with 20 ng / ml human vascular endothelial growth factor (VEGF), 100 ng / ml basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), 20 ng / ml human epidermal growth factor ml of human insulin-like growth factor-1 (IGF), and the mesenchymal stem cells were further cultured for 5 days.

On the other hand, all the experimental data in the following examples are shown as the mean ± standard deviation. Statistical analysis was performed using Student's T test for comparison between groups, and variance analysis (ANOVA) according to Bonferroni's multiple compraison test using SPSS v11.0 Data at significance level P <0.05 Respectively.

Example  2. Following cytokine cocktail addition Intermediate lobe  Stem cell Proliferative ability  change

In this Example, in order to confirm the change in the proliferative capacity of the mesenchymal stem cells according to Example 1, the cultured mesenchymal stem cells were observed under an optical microscope, and the number of cells under proliferation was calculated based on the observation. In the present study, bFGF alone (bFGF, n = 5) was used as a control group (Control, n = 5) as a control group and bFGF alone treatment group was used as a comparative group.

As a result, as shown in Fig. 1, when cytokine cocktail consisting of VEGF, bFGF, EGF, and IGF was added (Cocktail), it was confirmed that the cell number increased by the proliferation of mesenchymal stem cells, (BFGF) treated group was about 1.5 times higher than control group (control) and about 4 times higher than control group (control). These results indicate that the cytotoxic cocktail according to the present invention can be used for culturing mesenchymal stem cells to enhance the proliferative capacity of mesenchymal stem cells.

Example  3. Following cytokine cocktail addition Intermediate lobe  Changes in expression of stem cells

In this example, qRT-PCR was performed as follows to confirm the HGF expression of the mesenchymal stem cells according to Example 1. RNA-stat protocol (Iso-Tex Diagnostics, Friendswood, TX) (Total RNA) was isolated from the cells. The same amount of RNA extracted from the cells was reverse-transcribed using Taqman reverse transcription reagent (Applied Biosystems, Foster City, Calif.) According to the manufacturer's instructions. The synthesized cDNA was used for qRT-PCR or reverse-transcription-PCR using human / mouse-specific primers and probes. The amount of RNA was quantitated using the ABI PRISM 7000 Sequence Detection System (Applied Biosysytems) and the relative expression level of mRNA was normalized by the antagonistic gene GAPDH. The primers used in the qRT-PCR were HGF (Hs00300159_m1) and GAPDH (Hs99999905_m1). In contrast, in the present study, no treatment with cytokine was used as a control (Control), BFGF alone (bFGF), VEGF-A alone (VEGF-A), EGF alone (EGF) , IGF alone treatment group (IGF), BFGF and EGF treatment group (bFGF + EGF).

In addition, phenotypic changes of mesenchymal stem cells according to Example 1 were confirmed, and the expression pattern of HGF was compared with human umbilical vein endothelial cells (HUVEC). Specifically, for fluorescence-activated cell sorting (FACS) analysis, the mesenchymal stem cells were suspended in PBS (Cellgro, Manassas, MD) and incubated with PE- or FITC-conjugated antibodies directly at 4 ° C for 20 minutes . The antibodies used were anti-CD14, anti-CD29, anti-CD34, anti-CD44, anti-CD45, anti-CD73, anti-CD90 anti-CD105 and the corresponding isotype-identical IgG was used as a negative control . After cell staining, FACS was performed using a flow cytometer (BD).

As a result, as shown in Fig. 2, it was confirmed that HGF expression of mesenchymal stem cells cultured by treating with one or more cytokines was increased as compared with the control group without cytokine treatment. Particularly, in the case of addition of cytokine cocktail consisting of VEGF, bFGF, EGF, and IGF (Cocktail), the increase of HGF expression in mesenchymal stem cells was the most excellent. Specifically, BFGF alone treatment group (bFGF) It was confirmed that the amount of HGF expressed by BFGF and EGF treated group (bFGF + EGF) was increased about 5 times. These results indicate that by using the cytokine cocktail according to the present invention for culturing mesenchymal stem cells, mesenchymal stem cells with enhanced HGF expression, that is, enhanced mesenchymal regeneration ability, can be obtained more efficiently . As shown in Fig. 3, the mesenchymal stem cells of the present invention showed an immunophenotype negative for CD14, CD34 and CD45, positive for CD29, CD44, CD73, CD90 and CD105, The expression of HGF was about 20 times higher than that of the endothelial cells.

Example  4. Implanted Intermediate lobe  Evaluation of engraftment rate of stem cells in liver tissue

In this Example, the transplantation of mesenchymal stem cells according to Example 1 was performed to evaluate the engraftment rate and differentiation in liver tissue. Specifically, mesenchymal stem cells stained with CM-Dil (invitrogen) or not stained were suspended in physiological saline at a concentration of 5 × 10 ⁵ cells, and then administered to liver portal vein. After transplanting mesenchymal stem cells into mice After 3 weeks from the day, the mice were sacrificed and liver tissue was extracted therefrom. Subsequently, the prepared liver tissue sections were subjected to immunostaining (primary antibody: anti-HLA-1 antibody (Sigma), secondary antibody: Alexa Flour 555 (1: 400, invitrogen), nuclear staining: DAPI). In the present experiment, as a comparative group, mesenchymal stem cell transplantation group (ASC) cultured in a medium without cytokine cocktail was used.

As a result, as shown in Fig. 4, As compared with the group (ASC) transplanted with the mesenchymal stem cells cultured in the medium without the cytokine cocktail, the engraftment rate of the mesenchymal stem cells according to the present invention (ASC-C) was about 3 times As shown in Fig. As shown in FIG. 5, when mesenchymal stem cells were transfected into vascular cells, some of the mesenchymal stem cells (ASC-C) were transfected with isolectin B-4 (ILB-4) Expression in the liver tissue. These results suggest that the mesenchymal stem cells according to the present invention can be expected to have effective therapeutic effects as a cell therapy agent for the treatment of liver disease.

Example  5. Verification of liver cure treatment effect using liver cure animal model

In this Example, the effects of the HGF-induced mesenchymal stem cells according to Example 1 on liver cure were specifically confirmed. For this purpose, we analyzed the histopathological changes of liver tissue and the changes of collagen content synthesized by liver fibrosis according to the transplantation of stem cells in liver cirrhotic animal model.

5-1. The manufacture of liver cure animal models and Intermediate lobe  Transplantation of stem cells

All experimental protocols were carried out with the approval of the Catholic Animal Care and Use Committee of KCTU. Six-week-old female BALB / c nude mice were purchased from Koatech (Pyeongtaek, Korea), and the mice received 250 mg / kg of thioacetamide (TAA, Sigma Chemical, St. Louis, Mo., USA) Liver cirrhosis was induced by intraperitoneal administration for 6 weeks. In addition, mesenchymal stem cells were suspended in physiological saline at a concentration of 5 × 10 ⁵ cells, and then injected into the liver-induced mice through the portal vein.

5-2. Histopathological analysis

After 6 weeks from the day when the mesenchymal stem cells were transplanted into the mice as described above, the mice were sacrificed and liver tissues were extracted therefrom. Mouse liver tissue was fixed with 4% paraformaldehyde for 1 day and embedded in a Tissue-TEK-OCT compound (Sakura Finetechnical Co. Ltd. Japan), and liver tissue sections were prepared. Tissue samples were cut into 5 μm thickness and stained with Harris Hematoxylin solution (sigma) for 2 minutes and again with eosin Y (sigma) for 20 seconds. In contrast, in the present study, the transplantation of mesenchymal stem cells (TAA + ASC, n = 10) cultured in a culture medium without cytokine cocktail was used as a comparative group.

In addition, staining was performed with Masson's trichrome to observe the lesions in the liver tissue, and the fibrotic area in liver tissue was measured with software MetaMorph (Downingtown, PA, USA). In the present experiment, mice treated with PBS (n = 10) and mice treated with liver cirrhosis as a control group were compared with those of mice treated with a medium without cytokine cocktail The stem cell transplantation group (ASC, n = 10) was used.

As a result, as shown in Fig. 6 and Fig. 7, the hepatic tissue induced by hepatic cirrhosis by TAA treatment had a surface irregularly formed in the surface of the mesenchymal stem cells (ASC) (ASC-C) in the case of the mesenchymal stem cells according to the present invention, remarkably alleviates these liver-specific findings.

In addition, as shown in Fig. 8, in the group transplanted with the mesenchymal stem cells (ASC) cultured in the PBS-treated group (PBS) or the medium supplemented with the cytokine cocktail, necrosis of parenchymal cells , Fibrosis and vacuolar degeneration. On the other hand, when the mesenchymal stem cells according to the present invention are transplanted (ASC-C), the fibrotic area is greatly reduced as well as these pathological symptoms I could confirm.

5-3. Analysis of changes in content of collagen components synthesized by liver fibrosis

Collagen detection in liver tissue was measured using a Sirius Red / Fast Green collagen staining kit (chondrex, Inc, Redmond, WA). A tissue sample sectioned to a thickness of 10 mu m was immobilized in a Kahle solution for 30 minutes and stained in the staining solution for 30 minutes. The stained tissue samples were then washed with purified water and then mixed with Dye Extraction Buffer. The eluted dyeing solution was obtained and OD values at wavelengths of 540 nm and 620 nm were measured using a spectrophotometer. In the present study, the control group was treated with 50ul of physiological saline only (sham, n = 10), the control group was treated with heparin-induced mice (TAA, n = 10) Transplantation of mesenchymal stem cells (TAA + ASC, n = 10) cultured in a culture medium without cytokine cocktail was used in mice in which hepatic cirrhosis was induced.

As a result, as shown in Fig. 9, the content of collagen increased by the induction of liver hardening tended to decrease to some extent (TAA + ASC) when transplanted mesenchymal stem cells were cultured in a medium without cytokine cocktail , But the effect was remarkable in the case of transplanting the mesenchymal stem cells cultured in the medium supplemented with the cytokine cocktail (TAA + ASC-C). From this result, it was found that the superiority of the mesenchymal stem cells according to the present invention Inhibition of liver fibrosis and mitigation of liver cirrhosis.

5-4. Liver function assessment

Two weeks after the transplantation of mesenchymal stem cells as described above, a blood sample was taken from a mouse and centrifuged to obtain a serum sample. Using the FUJIFILM DRI-CHEM 3500 machine (Tokyo, Japan), the detection and comparison of GPT, GOP, ammonia, and bilirubin, markers related to liver damage from serum samples, The change was confirmed. In the present study, mice treated with 50 ul of physiological saline alone (sham, n = 10) as a control group, mice treated with PBS (PBS, n = 10) (ASC, n = 10) transplanted with cultured mesenchymal stem cells in a culture medium to which no cytokine cocktail was added.

As a result, as shown in FIG. 10, when the mesenchymal stem cells according to the present invention were transplanted (ASC-C), the amount of GPT, GOP, ammonia, and bilirubin in the serum (ASC) cultured in the culture medium without cytokine cocktail. From this, it was confirmed that the excellent effect of the present invention on the recovery of liver damage from the mesenchymal stem cells.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (11)

A mesenchymal stem cell isolated from a living body in a medium containing VEGF (vascular endothelial growth factor), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor- . The mesenchymal stem cells express HGF.
The mesenchymal stem cell according to claim 1, wherein the concentration of VEGF is 10 to 800 ng / ml.
The mesenchymal stem cell according to claim 1, wherein the bFGF concentration is 10 to 1600 ng / ml.
The mesenchymal stem cell according to claim 1, wherein the EGF concentration is 10 to 800 ng / ml.
The mesenchymal stem cell according to claim 1, wherein the concentration of IGF is 10 to 800 ng / ml.
2. The method of claim 1, wherein the medium is selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, Dulbecco's Modified Eagle's Medium ), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), a-Minimal Essential Medium (G-MEM), Endothelial Growth Medium (EGM) Endothelial Basal Medium) and IMDM (Isocove's Modified Dulbecco's Medium).
The method of claim 1, wherein the mesenchymal stem cells have an immunophenotypic characteristic that is negative for CD14, CD34, and CD45; And
CD29, CD90, CD90, CD105, and CD105. &Lt; / RTI &gt;
The mesenchymal stem cell according to claim 1, wherein the mesenchymal stem cell is 1.2 to 25 times more expressed than the human umbilical vein endothelial cells (HUVEC).
The mesenchymal stem cell according to claim 1, wherein the mesenchymal stem cell is isolated from cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, or placenta.
A pharmaceutical composition for preventing or treating liver disease, which comprises the HGF-expressing mesenchymal stem cell of claim 1 as an active ingredient.
[Claim 11] The pharmaceutical composition according to claim 10, wherein the liver disease is any one selected from the group consisting of liver cancer, hepatitis, hepatic fibrosis, and liver cirrhosis.

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