WO2018093233A1 - Composition containing adipose stem cell-derived exosomes as active ingredient for preventing or treating liver fibrosis - Google Patents

Abstract

The present invention relates to a composition containing human adipose stem cell-derived exosomes as an active ingredient for preventing, alleviating or treating liver fibrosis. The adipose stem cell-derived exosomes according to the present invention carry genetic information, protein, growth factor and the like for treating liver fibrosis, and have shown excellent results in treating liver fibrosis in animal models therefor, and, being a cell-derived substance, exosomes have the advantages of being highly biocompatible and having a superb absorption rate. Accordingly, the limits of medicine in current use or adverse reactions that can arise due to cancerization of stem cells or cell waste products in treatments using stem cells themselves can be minimized, and the delivery to the liver can be natural via intravenous injection, local injection and the like which can be applied simply without surgery, thus economic cost can be reduced and stress on the patient can also be lessened. In conclusion, the adipose stem cell-derived exosomes according to the present invention are expected to be effectively used for preventing, alleviating and treating liver fibrosis.

Description

Liver fibrosis prevention or treatment composition comprising adipose stem cell-derived exosomes as an active ingredient

The present invention relates to a composition for preventing, improving or treating liver fibrosis comprising fat stem cell-derived exosomes as an active ingredient.

Liver fibrosis is caused by excessive deposition of extracellular matrix due to chronic inflammation, which persists in liver cirrhosis due to alteration of intrahepatic structure and a decrease in the number of liver cells. Proceed. These chronic liver diseases can be seen as a continuous disease that progresses from chronic hepatitis to fibrosis through chronic fibrosis due to chronic hepatitis B or C, continuous heavy drinking and use of liver toxic substances, rather than an independent disease.

Currently, treatment for hepatitis and cirrhosis is to delay the progression of symptoms and the deterioration of human liver function.In the case of cirrhosis, depending on the cause, drugs such as peginterferon or antiviral agent are used and severe liver transplantation is performed. There is a way to cure. However, in the case of drug treatment, treatment resistance may be shown, and liver transplantation has a limitation in donor shortage and risks and costs associated with surgery.

Liver regenerative therapy using stem cells is a treatment that has attracted much attention in recent years, and it is known that the injected stem cells have a therapeutic effect by differentiating and reconstituting functional stem cells from damaged liver. Until now, there have been reports of functional stem cell differentiation of stem cells with high efficiency in vitro (Korea Patent Registration 10-1542849). However, when stem cells are transplanted into animal models or humans, the efficiency of stem cell differentiation is low. Existed. Moreover, there is a disadvantage that the exact marker of liver stem cells is not specified, so that stem cells proliferating and differentiating into hepatocytes are not known exactly.

In addition, the biggest problem of conventional treatment with stem cells is that the in vivo engraftment rate and survival rate of the cells are not constant according to the patients, and thus the accuracy of the cancer cells can not be excluded. In addition, the culture medium containing various growth factors secreted from stem cells may cause potential problems because it contains not only growth factors but also various cell wastes.

Thus, there is a need for a new therapeutic approach to address the limitations of stem cell liver disease treatment.

In order to solve the above problems, the present inventors evaluated the therapeutic effect on liver fibrosis using exosomes isolated from human adipose stem cells, and administered the exosomes in animal models of human hepatic stellate cell line and liver fibrosis. It was confirmed that the excellent therapeutic effect by, the present invention was completed based on this.

Accordingly, an object of the present invention is to provide a composition for the prevention, improvement, or treatment of liver fibrosis comprising fat stem cell-derived exosomes as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis comprising fat stem cell-derived exosomes as an active ingredient.

In addition, the present invention provides a dietary supplement for improving liver fibrosis comprising fat stem cell-derived exosomes as an active ingredient.

In one embodiment of the invention, the adipose stem cells may be human adipose stem cells.

In another embodiment of the invention, the composition is aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin in the blood Total bilirubin (TP), or ratio of liver weight to body weight (LW / BW).

The present invention also provides a method for preventing or treating liver fibrosis, comprising administering to a subject a pharmaceutical composition comprising adipose stem cell-derived exosomes as an active ingredient.

The present invention also provides a fat stem cell-derived exosomes, liver fibrosis prevention or treatment.

Adipose stem cell-derived exosomes according to the present invention is loaded with genetic information, proteins, growth factors, etc. for the treatment of liver fibrosis can exhibit an excellent liver fibrosis treatment effect in liver fibrosis animal model, the exosomes are cell-derived As it is a substance, it has good merit and good water absorption.

Therefore, it is possible to overcome the limitations of currently used therapeutic drugs and to minimize side effects due to cancer cellization or cellular waste of stem cells that may occur in the treatment using stem cells themselves. In addition, since it can be delivered to the liver naturally through the intravenous therapy or topical administration method that can be easily applied without surgery may have the effect of reducing the cost of treatment.

Therefore, the adipose stem cell-derived exosomes according to the present invention may be usefully used for the prevention, improvement, or treatment of liver fibrosis.

1 is a result of confirming the physical and biochemical properties of exosomes extracted from human adipose stem cells using an electron microscope and a nanoparticle tracking analyzer.

FIG. 2 shows human adipose stem cell-derived exosomes (A-Exosome) to identify α-smooth muscle actin (α-SMA) expressed in LX-2, which is activated hepatic stellate cells induced by TGF-β1. It shows the results confirmed by immunofluorescence to reduce the amount of α-SMA expression by treating by concentration.

3 is a result of real-time polymerase chain reaction evaluation to confirm the quantitative mRNA change of LX-2, a hepatic stellate cell line associated with hepatic fibrosis.

4 is a result of confirming the expression level of α-SMA by performing immunofluorescence evaluation to compare and evaluate the efficacy of human adipose stem cell-derived exosomes and other tissue stem cell-derived exosomes.

5 is a result of confirming the expression level of α-SMA, Collagen 1 by performing a real-time polymerase chain reaction evaluation in order to evaluate the efficacy of human adipose stem cell-derived exosomes and other tissue stem cell-derived exosomes .

FIG. 6 is a fluorescence-labeled human adipose stem cell-derived exosomes intravenously administered to each model to confirm the in vivo behavior of human adipose stem cell-derived exosomes in a normal model and a liver fibrosis-induced model. It is the result of confirming the amount of fluorescence by securing major organs at 24 hours later.

Figure 7 shows an animal experimental schematic established to confirm the antifibrotic effect of a mouse model of hepatic fibrosis of human adipose stem cell-derived exosomes.

FIG. 8 is administered three times of exosomes to a normal model and a mouse model of liver fibrosis, and blood is collected five days after the first administration. Total bilirubin (TP), total protein (TP) and ratio of liver weight to body weight (LW / BW) were measured.

FIG. 9A is an image observed by naked eye after extracting exosomes a certain number of times in a normal model and a mouse model of liver fibrosis 5 days after the first administration.

Figure 9b is the result of observation by optical microscope after performing H & E staining, Masson's trichrome (MT) staining and α-SMA staining for the obtained liver tissue.

Fig. 9C shows a quantitative graph comparing the amount of collagen and α-SMA by MT with the total area.

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The present inventors made a thorough study to overcome the problems of cell therapy using the therapeutic drug or stem cell itself currently used in the treatment of liver fibrosis, and as a result, by confirming the effect of treating liver fibrosis of human adipose stem cell-derived exosomes, The invention has been completed.

Hereinafter, a pharmaceutical composition for preventing or treating liver fibrosis according to a specific embodiment of the present invention will be described in detail.

Specifically, in one embodiment of the present invention, by culturing human adipose stem cells, recovering the cell culture supernatant and isolating and purifying the secreted exosomes from the cells (see Example 1), anti-fibrosis of the exosomes Effects in vitro cell experiments And in vivo mouse models.

In addition, the expression level of α-SMA was confirmed by immunofluorescence evaluation using an optical microscope, and quantitatively verified the amount of fibrosis-related mRNA through real-time polymerase chain reaction, and treated with TGF-β1 to activate hepatic stellate cells. In the present invention, it was confirmed that the expression level of α-SMA was greatly increased, and that the expression level of α-SMA was significantly decreased as the concentration of human adipose stem cell-derived exosomes was increased (see Example 2).

In addition, the amount of RNA of fibrosis-related factors α-SMA, Collagen 1 and MMP-2 increased significantly when TGF-β1 was activated to activate hepatic stellate cells, but the concentration of human adipose stem cell-derived exosomes under the same conditions was When treated, it was found to decrease significantly and this trend was confirmed to be more effective as the concentration of exosomes increased (see Example 2).

On the other hand, in one embodiment of the present invention, the adipose stem cells may be human adipose stem cells. Human adipose stem-derived exosomes can effectively alleviate liver fibrosis by lowering the levels of fibrosis-related factors of hepatic stellate cells activated by fibrosis compared to other tissue stem cell-derived exosomes.

In addition, in order to evaluate the antifibrotic comparison between human adipose stem cell-derived exosomes and other tissue stem cell-derived exosomes used in the present invention, an experiment for comparing the effects through immunofluorescence evaluation and real-time polymerase chain reaction It was observed that the fluorescence of α-SMA was significantly reduced in human adipose stem cell-derived exosomes than umbilical stem cell-derived exosomes or bone marrow stem cell-derived exosomes that are exosomes derived from other tissue stem cells. It was confirmed that the derived exosomes effectively alleviate liver fibrosis by lowering the levels of fibrosis-related factors of activated hepatic stellate cells compared to other tissue stem cell-derived exosomes (see Example 3).

On the other hand, the present invention provides a method for preventing or treating liver fibrosis, comprising administering to a subject a pharmaceutical composition comprising adipose stem cell-derived exosomes as an active ingredient.

Administration of human adipose stem cell-derived exosomes not only effectively inhibits the formation of the fibrous septum, but also maintains the structural form of the hepatocytes, thereby effectively suppressing the fibrosis of the liver tissue, thus maintaining the normal tissue in the form.

On the other hand, human adipose stem cells and human adipose stem cell-derived exosomes have an effect on improving liver function, but when compared with human adipose stem cells-derived exosomes, the liver function is more effectively improved compared to both. (See Example 5).

In addition, the pharmaceutical composition for preventing or treating hepatic fibrosis according to the present invention is aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (Alkaline phosphatase); ALP), total bilirubin (TP) or ratio of liver weight to body weight (LW / BW).

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis comprising fat stem cell-derived exosomes as an active ingredient.

As used herein, the term "prevention" means any action that inhibits or delays the development of liver fibrosis by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action in which symptoms caused by liver fibrosis are improved or beneficially altered by administration of the pharmaceutical composition according to the present invention.

As used herein, the term “stem cell” refers to a cell that is the basis of a cell or tissue constituting an individual, and its feature is capable of repeated division and self-renewal, and has a specific function according to the environment. It means a cell having a multipotent ability to differentiate into cells. It occurs in all tissues during the development of the fetus, and is found in some tissues, such as bone marrow and epithelial tissue, which are actively replaced even in adulthood. Stem cells are totipotent stem cells that form when the fertilized egg begins its first division, depending on the type of cells that can differentiate, and pluripotent stem cells in the endoplasmic linings where these cells continue to divide. It is classified as multipotent stem cells in mature tissues and organs. At this time, pluripotent stem cells are cells that can only differentiate into cells specific to the tissues and organs that contain the cells, and the homeostasis of adult tissues as well as the growth and development of individual tissues and organs in the prenatal, neonatal and adult periods. It is involved in the maintenance of regeneration in maintenance and tissue damage. These tissue specific pluripotent cells are collectively referred to as adult stem cells.

Mesenchymal stem cells, which are classified as adult stem cells, are spotlighted as a material for regenerative medicine. They can be collected from tissues such as bone marrow, umbilical cord blood, adipose tissue, and umbilical cord. Has the ability to differentiate into cells constituting various human tissues such as cells, bone cells, chondrocytes, nerve cells, cardiomyocytes. In the present invention, but used stem cells isolated from human adipose tissue, but is not limited thereto.

As used herein, the term “exosome” refers to cell-derived vesicles present in almost all eukaryotic cell fluids, including blood, urine, and cultures of cells, with an average diameter of 30 to 100 nm. It is known to have. The exosomes have been reported to be clinically because multivesicular bodies of cells are fused to the plasma membrane and secreted or secreted directly from the plasma membrane, and they perform specific functions important in the process of aggregation, intracellular signaling, and cellular waste management. Attention has been drawn to biomarkers, diagnosis, and treatment of specific diseases. In the present invention, the exosomes may be naturally secreted from the cells by culturing human adipose stem cells.

The pharmaceutical composition according to the present invention includes adipose stem cell-derived exosomes as an active ingredient, and may include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, external preparation for skin, oral ingestion, and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, skin, nasal, airways) according to the desired method, and the dosage is determined by the condition and weight of the patient, disease Depending on the degree, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The composition according to the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses.

Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and generally 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight is administered daily or every other day or 1 It can be administered in 1 to 3 times a day. However, since the dose may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., the above dosage does not limit the scope of the present invention by any method.

In another aspect of the present invention, the present invention provides a dietary supplement for improving liver fibrosis comprising fat stem cell-derived exosomes as an active ingredient.

As used herein, the term " improvement " means any action that at least reduces the parameters associated with the condition being treated, such as the extent of symptoms.

In the nutraceutical composition of the present invention, the active ingredient may be added to the food as it is, or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed may be appropriately determined depending on the purpose of use thereof. In general, in the manufacture of food or beverages the compositions of the invention are added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw materials. However, in the case of prolonged intake for health and hygiene purposes or health control purposes, the amount may be below the above range.

The health functional food composition of the present invention, in addition to containing the active ingredient as an essential ingredient in the indicated ratio, is not particularly limited to other ingredients, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate can be appropriately determined by the choice of those skilled in the art.

In addition to the above, the nutraceutical composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. These components can be used independently or in combination. The proportion of such additives may also be appropriately selected by those skilled in the art.

As another aspect of the present invention, the present invention provides a method for preventing or treating liver fibrosis comprising administering to a subject a composition comprising adipose stem cell-derived exosomes as an active ingredient.

In the present invention, "individual" means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, and the like. Means mammals.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example  1: human Derived from adipose stem cells Exosome  Manufacturing and physical and biochemical characterization evaluation

To extract exosomes from human adipose stem cells, the human adipose stem cells are cultured in Dulbecco's Modified Eagle Medium (DMEM) medium (Gibco, Cat #: 11995065), and serum, antibiotics, And replaced with DMEM medium (Gibco Cat #: 31053028) not containing phenol red (phenol red) was incubated for 24 hours.

Thereafter, the cell culture supernatant was recovered and centrifuged at 10 ° C. for 10 minutes at 4 ° C. and 3000 × g. Then, cell debris and wastes were removed from the culture medium using a 0.2 μm filter. Next, the culture solution was extracted and purified using the Tangential Flow Filtration System (TFF System). On the other hand, after recovering the culture supernatant from human adipose stem cells, normal culture medium DMEM was added again and the cells were cultured, and this process was repeated up to 8 passages to extract the exosomes.

Through the above process, the physical and biochemical properties of the exosomes derived from human adipose stem cells (A-Exo) were confirmed by electron microscopy and nanoparticle tracking analyzer .

As a result, as shown in Figure 1, the exosomes were confirmed to have a spherical average size of 200 nm or less, through which the exosomes were successfully extracted from human adipose stem cells.

Example  2: human Hepatic phase  Humans targeting cell lines Derived from adipose stem cells Exosome  Immunofluorescence And real-time polymerase chain reaction evaluation

To determine the effect of the exosomes extracted in Example 1 in human hepatic stellate cells known to be activated by the liver damage in the actual liver fibrosis process and to be involved in the release of collagen and liver fibrosis factor, etc. For this purpose, anti-fibrotic efficacy evaluation was performed using the relevant cell line LX-2.

Specifically, to determine the effect of human adipose stem cell-derived exosomes on the expression level of α-Smooth Muscle Actin (α-SMA), a fibrosis factor generated from hepatic stellate cells activated by hepatic fibrosis, immunofluorescence The degree was evaluated using the method.

α-SMA is one of the factors expressed in activated hepatic stellate cells, and it is known that a greater amount is expressed as hepatic fibrosis is induced. In this experiment, LGF-2, a hepatic stellate cell line, treated 1 ng / mL of TGF-β1. 2 x 10 ⁵ cells / well of cells were planted in one DMEM medium, incubated for 24 hours, and then washed 1 or 2 times with DPBS.

Thereafter, human adipose stem cell-derived exosomes were dispersed at concentrations of 1 × 10 ⁶ cells / mL and 1 × 10 ⁷ cells / mL, respectively, treated with 2 mL / well, and cultured for 48 hours. Then, after finally reacting with Anti-actin α-Smooth Muscle (α-SMA, A2547, Sigma aldrich, USA) for 12 hours, the secondary fluorescent antibody Anti-Cyanine2 was reacted to complete the tissue slide. Morphology was confirmed by confocal laser microscopy.

As a result, as shown in FIG. 2, it was confirmed that the expression level of α-SMA was significantly increased in TGF-β1-activated hepatic stellate cells. As the concentration of human adipose stem cell-derived exosomes increased, α- It was found that the expression level of SMA was significantly reduced (TFG-β1: TGF-β1 treated group, A-Exosome (1 x 10 ⁶ ) and A-Exosome (1 x 10 ⁷ ): human stem cell-derived exosomes 1 x 10 ^{6-10 7} groups / ml).

In addition, in order to quantitatively confirm the antifibrotic effect of human adipose stem cell-derived exosomes on activated hepatic stellate cell LX2, real-time polymerase chain reaction was performed on α-SMA, Collagen 1 and MMP-2, which are related to fibrosis. Evaluation was performed and mRNA expression changes were observed after treatment with exosomes against fibrosis factors α-SMA, Collagen 1, and Matrix Metalloproteinase-2 (MMP-2).

As a result, as shown in Figure 3, when the TGF-β1 treatment to activate hepatic stellate cells, the RNA amount of the fibrosis-related factors α-SMA, Collagen 1, MMP-2 significantly increased, but derived from human fat under the same conditions Stem cell exosomes were treated with different concentrations (10 ⁶ -10 ⁷ ), and significantly decreased, and this trend was confirmed to be more effective as the concentration of exosomes increased (Control: PBS treated Group, TFG-β1: TGF-β1 treated group, A-Exosome (1 x 10 ⁶ ) to A-Exosome (1 x 10 ⁷ ): human stem cell-derived exosomes 1 x 10 ^{6-10 7} / group treated with ml).

Example  3: human Derived from adipose stem cells Exosomes  human Derived from other tissue stem cells Exosomes  in vitro Antifibrotic  Efficacy comparison evaluation

In order to compare and compare the human adipose stem cell-derived exosomes according to the present invention with superior antifibrotic effect compared to human other tissue stem cell-derived exosomes, Bone-marrow stem cell exosome as a test group Exosome) and exosomes extracted from human umbilical cord stem cell (Umbilical-cord stem cell exosome: U-Exosome), respectively, were prepared and evaluated by immunofluorescence and real-time polymerase chain reaction experiment of Example 2.

As a result, as shown in Figure 4, it was observed that the amount of fluorescence of α-SMA is significantly reduced in human adipose stem cell-derived exosomes than human umbilical stem cell-derived exosomes or human bone marrow stem cell-derived exosomes. It was confirmed that the decrease was apparent as the concentration of exosomes increased (A-Exosome (1 x 10 ⁶ ) to A-Exosome (1 x 10 ⁷ ): 1 x 10 ^6-10 to human adipose stem cell-derived exosomes. ⁷ -ml / ml group, B-Exosome (1 x 10 ⁶ ) to B-Exosome (1 x 10 ⁷ ): Human bone marrow stem cell-derived exosomes were treated with 1 x 10 ^{6-10 7} / ml Group, U-Exosome (1 × 10 ⁶ ) to U-Exosome (1 × 10 ⁷ ): group treated with human umbilical cord stem-derived exosomes at 1 × 10 ⁶ -10 ⁷ / ml).

In addition, this trend was further verified by quantitative RNA amount comparison. In particular, as shown in Figure 5, it is confirmed that the RNA amount of α-SMA and Collagen 1 is more effectively reduced when compared with human other tissue stem cell-derived exosomes when treated with human fat stem cell-derived exosomes (10 ⁷ ). (A-Exosome (1 x 10 ⁶ ) to A-Exosome (1 x 10 ⁷ ): Group treated with human adipose stem cell-derived exosomes at 1 x 10 ^{6-10 7} / ml, B-Exosome (1 x 10 ⁶ ) -B-Exosome (1 x 10 ⁷ ): group treated with human bone marrow stem cell-derived exosomes at 1 x 10 ^{6-10 7} / ml, U-Exosome (1 x 10 ⁶ ) -U-Exosome (1 × 10 ⁷ ): group treated with human umbilical cord stem-derived exosomes at 1 × 10 ^{6-10 7} / ml).

Through the experiments, human adipose stem cell-derived exosomes can be found to effectively alleviate liver fibrosis by lowering the levels of fibrosis-related factors of activated hepatic stellate cells due to fibrosis compared to human other tissue stem cell-derived exosomes. there was.

Example  4: Human in Liver Fibrosis Animal Model Derived from adipose stem cells Exosome  In vivo distribution behavior assessment

To evaluate the in vivo distribution behavior of human adipose stem cell-derived exosomes, DiR; 1,1'-diotadecyl-3,3,3 ', 3'-tetramethylindotricarbocyanine rodide (1,1'-Dioctadecyl-3,3,3 ', 3'-Tetramethylindotricarbocyanine lodide) Fluorescence Substance-labeled human adipose stem cell-derived exosomes were dispersed in 100 μL of the culture medium and tail vein injection. Then, after 24 hours, the mice of each group were dissected to cut out major organs and check their fluorescence.

As a result, as shown in Figure 6, both the normal model and the liver fibrosis-induced model was found to accumulate the largest amount of exosomes per unit area in the liver, especially in the hepatic fibrosis model compared to the normal amount of exosomes The amount was confirmed. This is believed to be due to the large number of hepatic stellate cells differentiated during the activation process due to fibrosis (Hien T. T et al., Small 2015; 19: 2291-2304).

Example  5: Human in Liver Fibrosis Animal Model Derived from adipose stem cells Exosome  Treatment efficacy assessment

Using the human adipose stem cell-derived exosomes of Example 1 was confirmed whether the antifibrotic effect in the actual liver fibrosis animal model, human adipose stem-derived exosomes rather than human Adipose Derived Stem Cells (ADSC) When (A-Exosome) was administered, it was confirmed that liver function was improved more effectively.

Specifically, hepatic fibrosis model by intraperitoneally administering normal C57BL / 6 mice and TAA (Thioacetamine), a drug known to induce liver fibrosis, at a concentration of 200 mg / kg three times a week for 12 weeks, as shown in the animal experimental schematic of FIG. 7. Was produced. After 12 weeks, exosome samples were administered intravenously three times with concentrations of 1 × 10 ⁷ and 1 × 10 ⁸ , and then the liver fibrosis treatment effects were verified.

First, in order to conduct liver function test according to exosome administration, 1 x 10 ⁷ and 1 x 10 ⁸ exosomes were administered three times to the liver fibrosis mouse model, and blood was collected 5 days later, followed by blood biochemical analysis. Proceeded. The collected blood was centrifuged at 4500rpm for 15 minutes at 4, and then the supernatant was collected.

More specifically, biochemical markers such as AST (Aspartate aminotransferase), ALT (Alanine aminotransferase), ALP (Alkaline phosphatase), TBIL (Total bilirubin) and TP (Total protein) using an automated blood analyzer (ADVIA2120, SIEMENS, USA) The numerical value was measured. In addition, the weight of the collected liver tissue was measured to calculate the ratio of weight of liver tissue to the total body weight (Ratio of LW / BW) to illustrate the efficacy.

As a result of measuring the levels of the enzymes in the blood, as shown in FIG. 8, when human adipose stem cells and human adipose stem cell-derived exosomes were administered to a normal model, as in the control group, it did not significantly affect liver function. The above enzymes were identified (Normal: normal mouse model, Fibrosis: liver fibrosis-induced model, PBS: PBS group, ADSC (1 x 10 ⁵ ): human adipose stem cells 1 x 10 ⁵ administration One group, A-Exosome (1 × 10 ⁷ ) to A-Exosome (1 × 10 ⁸ ): a group in which 1 × 10 ^{7-10 8} exosomes were derived from human adipose stem cell-derived exosomes).

In the liver fibrosis model, liver function was improved by administration of human adipose derived stem cells (ADSC) and human adipose stem cell-derived exosomes (A-Exosome), compared with the group administered with PBS alone. In particular, when A-Exosome was administered 1 x 10 ⁸ times three times than ADSC (1x10 ⁵ ), it was confirmed that liver function was more effectively improved. In addition, it was confirmed that the weight of the liver compared to the total weight also has a value of the ratio close to the normal animal model, effectively inhibiting the fibrosis of liver tissue.

On the other hand, in order to proceed with the liver tissue gross examination and pathological analysis, the liver of the mouse was extracted after the experiment, fixed with 4% neutral buffered formalin, embedded with paraffin, and tissue sections having a thickness of 4 μm were prepared to produce H & E (Hematoxilin-Eosin). ) Staining, α-Smooth Muscle Actin (α-SMA), and Masson's trichrome staining were observed using an optical microscope.

As a result, as shown in FIG. 9A, when the extracted liver was visually observed, human adipose stem cells (ADSC (1 × 10 ⁵ )) and human adipose stem cell-derived exosomes (A-Exosome) administered to a normal model were observed. 1 x 10 ⁷ -10 ⁸ )) did not affect the tissue shape of the liver. In addition, in the liver fibrosis model, the surface color and morphological changes observed in the group treated with PBS alone were 1 x 10 ⁸ human adipose stem cell-derived exosomes (A-Exosome (1 x 10 ⁸ ) ) Effectively remained similar to normal liver tissue.

On the other hand, α-SMA for detecting myofibroblasts found during H & E staining, Masson's Trichrome (MT) staining, and fibrosis, which can detect the structural characteristics of cells in liver tissues of liver fibrosis model. Staining was performed.

As a result, as shown in Figure 9b, when hepatic fibrosis was induced, it was confirmed that necrosis proceeds as the cell membrane of the structure loses shape or collapses, as shown in Figure 9c, increased by liver fibrosis The expression of collagen and α-SMA expression were almost unrecognized in the normal model, and α-SMA and α-SMA were not detected in the group treated with 1 × 10 ⁸ exosomes derived from human adipose stem cells (A-Exosome (1 × 10 ⁸ )). A decrease in the amount of collagen was observed.

This suggests that the administration of human adipose stem cell-derived exosomes not only effectively suppresses the formation of fibrous septa but also maintains the structural form of hepatocytes, thereby effectively suppressing the fibrosis of liver tissue, thus maintaining the normal tissue in the form of normal tissue. Could.

Through the above experiments, the adipose stem cell-derived exosomes according to the present invention is loaded with genetic information, proteins, and growth factors for the treatment of liver fibrosis, and can exhibit an excellent liver fibrosis treatment effect in an animal model of liver fibrosis. Since the exosomes are cell-derived materials, it has been found that the biocompatibility is good and the absorption rate is also excellent, and the adipose stem cell-derived exosomes according to the present invention may be usefully used for the prevention, improvement, or treatment of liver fibrosis. I could see that.

The description of the present invention set forth above is for illustrative purposes, and one of ordinary skill in the art may understand that the present invention may be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Adipose stem cell-derived exosomes according to the present invention can overcome the limitations of currently used therapeutic drugs, and can minimize side effects due to cancer cellization or cellular waste of stem cells that may occur in the treatment using stem cells themselves. have.

In addition, since it can be delivered to the liver naturally through intravenous injection or topical administration method that can be easily applied without surgery, it can also reduce the cost of treatment, fat stem cell-derived exosomes according to the present invention can prevent liver fibrosis It can be usefully used for, improving, or treating.

Claims (7)

1. Fat stem cell-derived exosomes comprising as an active ingredient, liver fibrosis prevention or treatment pharmaceutical composition.
2. The method of claim 1,

   The fat stem cells are human fat stem cells, characterized in that the pharmaceutical composition.
3. The method of claim 1,

   The pharmaceutical composition is aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (TP) in blood Or reducing the ratio of liver weight to body weight (LW / BW).
4. A method of preventing or treating liver fibrosis, comprising administering to a subject a pharmaceutical composition comprising adipose stem cell-derived exosomes as an active ingredient.
5. Use of adipose stem cell-derived exosomes for the prevention or treatment of liver fibrosis.
6. Fatty stem cell-derived exosomes comprising as an active ingredient, health functional food composition for improving liver fibrosis.
7. The method of claim 6,

   The fat stem cells are human-derived fat stem cells, characterized in that the health functional food composition.

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