Composition for preventing or treating ocular diseases containing exosomes derived from amniotic epithelial cells as active ingredient

Abstract

To provide compositions for preventing or treating ocular diseases having excellent wound healing ability and anti-inflammatory effect.SOLUTION: Provided are a pharmaceutical composition for preventing or treating ocular diseases, and a functional health food composition for preventing or improving ocular diseases, which contain exosomes derived from amniotic epithelial cells as an active ingredient. The exosomes derived from amniotic epithelial cells have the ability to heal wounds in ocular tissue, restore lacrimal gland tissue destroyed by ocular diseases, induce tear secretion, and reduce the secretion of various inflammatory cytokines, making it possible to more effectively prevent or treat ocular diseases.SELECTED DRAWING: Figure 9

Description

[Technical field]

[0001] The present invention relates to a method for preventing ocular diseases, comprising administering exosomes derived from amniotic epithelial cells as an active ingredient. Or it relates to a therapeutic composition. [Background technology]

[0002] The amnion is a thin biological membrane that surrounds the fetus and absorbs various nutrients from the mother. The amniotic membrane acts as a barrier to protect the fetus from infection and immune reactions. The amniotic membrane is composed of simple cuboidal epithelium, a thick basement membrane, and avascular mesenchymal epithelium. It has the characteristic that it does not show rejection reaction when transplanted, and amniotic membrane transplantation is performed for ophthalmic diseases. It has also been shown that amniotic membrane can preserve the growth and properties of corneal and conjunctival cells. It has been shown that it inhibits epithelial cell death, promotes epithelialization during wound healing, and reduces inflammation. It reduces the amount of blood vessels in the blood, inhibits the formation of new blood vessels, and inhibits the formation of scars after wound healing. Since the procedure must be performed through a surgical procedure, there are risks such as scar formation, bleeding, and infection. In order to overcome these shortcomings, An eye drop treatment utilizing the components of this part has been proposed.

[0003] Exosomes are lipid bilayers secreted by all cells into the external environment. Exosomes are nano-sized endoplasmic reticulum surrounded by a membrane. During the maturation of multivesicular endosomes, the endosomal membrane is inserted inward to generate They are lumenal vesicles secreted when multivesicular endosomes bind to the cell surface. The endothelial cells contain a variety of biologically active substances, including proteins, lipids, nucleic acids, and metabolic products. Exosomes are intercellular proteins secreted by cells, which reflect the state of the cells from which they originate. They function as signal transmitters for the nervous system, but contain nuclear components as well as receptors and proteins. It is known that it may play a role in intercellular communication. In order to achieve this, bioactive molecules are transferred to the target cells while they are fused with the target cells, and the bioactive effects of the target cells are enhanced. In addition, since exosomes are derived from cells, they can be easily induced by immune cells. They have low virulence, have the same membrane topology as human cells, and easily attach to target cells. Exosomes are known to play important roles in angiogenesis, immunosuppression, and the pathological stages of cancer. It is attracting attention as an excellent candidate for treatment in the early stages of disease.

[0004] This will allow the development of new therapeutic agents for ocular diseases using exosomes derived from human cells. We are trying to develop Summary of the Invention [Problem to be solved by the invention]

[0005] The object of the present invention is to develop an exoblastic graft of human-derived cells having excellent wound healing ability and anti-inflammatory effect. By providing a pharmaceutical composition for preventing or treating ocular diseases, which contains the compound as an active ingredient, be.

[0006] Another object of the present invention is to provide a method for preventing or treating ocular diseases comprising the exosomes as an active ingredient. The present invention provides a functional food composition for improving health. [Means for solving the problem]

[0007] In order to achieve the above object, the present invention provides a method for the treatment of cancer using exosomes derived from amniotic epithelial cells as an active ingredient. The present invention provides a pharmaceutical composition for preventing or treating ocular diseases, which comprises the compound as described above.

[0008] The present invention also relates to a method for treating ocular diseases comprising administering exosomes derived from amniotic epithelial cells as an active ingredient. The present invention provides a functional health food composition for preventing or improving the above. Effect of the Invention

[0009] The exosomes derived from amniotic epithelial cells according to the present invention have the ability to heal wounds in ocular tissues, and It restores the tissue of the lacrimal gland destroyed by disease, induces tear secretion, and reduces various inflammatory sites. A pharmaceutical composition for preventing or treating eye diseases by reducing the secretion of ketone. It can be used as a functional health food composition and can be used to effectively prevent eye diseases. , can be improved or cured. [Brief description of the drawings]

[0010] [Figure 1] 1 is an image of exosomes extracted from amniotic epithelial cells according to one embodiment of the present invention, observed using a transmission electron microscope (TEM). [Diagram 2] FIG. 2 shows the results of measuring the size and polydispersity of exosomes shown in FIG. 1. [Diagram 3] This is a diagram confirming exosomes using Western blotting. [Figure 4] These results confirm the effect of exosome treatment on the proliferation of a human conjunctival cell line. [Diagram 5] This is a diagram confirming that exosomes are invaginated into human conjunctival epithelium. [Figure 6] These are the results of confirming changes in IL-8 concentration due to exosome treatment. [Figure 7] These results confirm the wound-healing power of exosome treatment. [Figure 8] These results confirm the change in tear secretion due to exosome treatment in an animal model of dry eye syndrome. [Figure 9]These are the results of corneal Lissamine green staining in an animal model of xerophthalmia. [Figure 10] These are the results of PAS (periodic acid Schiff) staining of conjunctival tissue in an animal model of xerophthalmia. [Figure 11] These are the results of Alcian Blue staining of lacrimal gland tissue in an animal model of xerophthalmia. [Figure 12] 13 is a graph showing the results of confirming changes in cytokine secretion in corneal tissue of an animal model of xerophthalmia. [Figure 13] 1 is a graph comparing the concentration of IL-8 following treatment with exosomes derived from amniotic epithelial cells and exosomes derived from mesenchymal stem cells. [Figure 14] 1 is a graph comparing the wound-healing ability of amniotic epithelial cell-derived exosomes and mesenchymal stem cell-derived exosomes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in detail below.

[0012] The present inventors obtained exosomes derived from amniotic epithelial cells from amniotic membranes donated by women and administered them to the eyes. Exosomes were administered to mice with xerostomia induced to observe the therapeutic effect on eye diseases. Therefore, exosomes derived from human amniotic epithelial cells may be a new therapeutic strategy for ocular diseases. The present invention was completed by confirming that the above-mentioned

[0013] As used herein, "exosomes" refer to the endosomal compartment of most eukaryotic cells. Any kind of extracellular vesicle (EV) that is produced outside the cell and surrounded by a membrane cullar vesicles, EVs).

[0014] In the present specification, "prevention" refers to the use of a pharmaceutical composition or a functional health food composition according to the present invention. suppresses the occurrence of an ocular disease or at least one of its symptoms by administering the means any action that delays the onset of the disease, or prevents or averts its recurrence. This includes treating a subject who is suffering from said disease.

[0015] As used herein, "treatment" refers to the treatment of ocular diseases, or alleviating, reducing, or eliminating at least one or more of the symptoms thereof. means any action that changes or alters something for the better.

[0016] In the present specification, "improvement" means the improvement of the eyes by taking the functional health food composition of the present invention. alleviating, reducing, or eliminating a pulmonary disease or at least one symptom thereof, It means any action that improves or favorably alters the condition.

[0017] As used herein, a "pharmaceutical composition" refers to a composition administered for a specific purpose. For purposes of the present invention, the present invention relates to a method for preventing or treating an ocular disease or at least one or more symptoms thereof. This means that the drug is administered to treat

[0018] In this specification, "health functional foods" refers to foods that meet the requirements of the Health Functional Foods Act No. 6727. This includes foods manufactured and processed using raw materials and ingredients that have beneficial functions for the human body, In addition to providing nutrients, the present invention also aims to prevent eye diseases, improve biological functions such as defense, immunity, and recovery. It refers to foods with high medical and therapeutic effects that have been processed to efficiently express the functions of the joints.

[0019] The present invention relates to a method for preventing ocular diseases, comprising administering exosomes derived from amniotic epithelial cells as an active ingredient. Alternatively, a pharmaceutical composition for treatment is provided.

[0020] The "amnion epithelial cell" refers to a cell that is capable of absorbing the placenta. They are isolated from the constituent amniotic epithelial tissue and may have the properties of pluripotent stem cells.

[0021] The "amniotic epithelial cell-derived exosomes" can be obtained from amniotic epithelial cells. Exosomes may be included without limitation.

[0022] In the pharmaceutical composition according to the present invention, the exosomes are effective in healing wounds in the cornea or conjunctival tissue. It can have healing powers.

[0023] The exosomes circulate lacrimal tissue destroyed by eye diseases such as dry eye. It can restore the function of the goblet cells, which are involved in stabilizing the tear film, and regulate tear secretion. It can be induced.

[0024] The "goblet cells" secrete mucus from the conjunctiva to keep the tear film stable.

[0025] In addition, the exosomes contain IL-1β (Interleukin 1 beta), IL-8 (Interleukin 8), IL-6 (Interleukin 6), IFNγ (Interferon gamma) and TNFα (Tumor necr inflammatory cytokines selected from the group consisting of It can reduce secretion.

[0026] In the pharmaceutical composition according to the present invention, the ocular disease includes ocular surface diseases and intraocular diseases. More specifically, dry eye syndrome, corneal epithelial damage, keratitis, conjunctivitis, and keratoconjunctivitis. An ocular surface disease selected from the group consisting of endophthalmitis, uveitis, and macular degeneration. The intraocular disease is selected from, but is not limited to, the following:

[0027] The pharmaceutical composition according to the present invention can be prepared by conventional methods in the pharmaceutical field. The pharmaceutical composition is mixed with a suitable pharma- ceutically acceptable carrier depending on the dosage form, and, if necessary, an excipient is added. It may further include excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. The suitable carriers can be prepared by the method of the present invention. The activity and properties of the drug are not affected by the dosage form and dosage form. can be selected.

[0028] The pharmaceutical composition according to the present invention can be applied in any dosage form, more specifically, in the conventional Depending on the method, it is formulated into oral dosage forms, external preparations, suppositories, and parenteral dosage forms such as sterile injection solutions. It can be done.

[0029] Preferably, the composition is in the form of eye drops, injections, granules, tablets, pills, capsules, gels, etc. Any one selected from the group consisting of syrup, suspension, emulsion, drop, and liquid. It is a type.

[0030] Among the oral dosage forms, the solid dosage forms include granules, tablets, pills, capsules, powders, etc. In one embodiment, the composition further comprises at least one excipient, such as starch, calcium carbonate, sucrose, etc. Mix and prepare lactose, sorbitol, mannitol, cellulose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can be used. In the case of capsule dosage forms, in addition to the above-mentioned substances, liquids such as fatty oils may also be included. The device may further include a body carrier.

[0031] Among the oral dosage forms, liquid dosage forms include suspensions, oral liquids, emulsions, syrups, etc. However, in addition to the commonly used simple diluents water and liquid paraffin, various excipients are used. For example, wetting agents, sweetening agents, flavoring agents, preservatives, and the like may be included.

[0032] The parenteral dosage form may be a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a lyophilized preparation, or a soluble form. Examples of non-aqueous solvents and suspensions include propylene glycol. Recall, polyethylene glycol, vegetable oils such as olive oil, ethyl oleate Injectable esters such as terpenes are used. As a base for suppositories, witepsol ( witepsol), macrogol, Tween 61, cocoa butter, lauric butter, glycerol Gelatin, etc., may be used, without being limited thereto, and any suitable formulation known in the art may be used. This can also be used.

[0033] In the pharmaceutical composition according to the present invention, the pharmaceutical composition is administered in a pharma- ceutical effective amount. .

[0034] As used herein, a "therapeutically effective amount" refers to a reasonable amount applicable to medical treatment. "Amount" means an amount that is sufficient to treat a disease at a reasonable risk and that does not cause side effects.

[0035] The effective dosage level of the pharmaceutical composition depends on the purpose of use, the age, sex, weight and health condition of the patient. , type of disease, severity, drug activity, drug sensitivity, administration method, administration time, administration history Factors including pathways and excretion rates, duration of treatment, combinations or concurrent drugs, and other medical It is determined differently depending on factors well known in the field. For example, Generally, it is 0.001 to 100 mg / kg, and preferably, it is 0.01 to 10 mg / kg. The dosage is administered once or several times a day. The above dosage amounts are not intended to limit the scope of the present invention in any way. It is not something that can be achieved.

[0036] The pharmaceutical composition according to the present invention can be administered to any animal in which an ocular disease occurs, The animals include, for example, not only humans and primates, but also livestock such as cows, pigs, horses, and dogs. Ugh.

[0037] The pharmaceutical composition according to the present invention is administered via a suitable administration route depending on the formulation, and reaches the target tissue. As long as the dosage is accessible, it may be administered via a variety of routes, including oral and parenteral. The administration method may be, for example, oral, intraocular, rectal, intravenous, intramuscular, or skin application, without being particularly limited thereto. , intrarespiratory inhalation, intrauterine dura or intracerebrovascular (intracere-broventri The drug is administered in the usual manner, such as by injection.

[0038] The pharmaceutical composition according to the present invention can be used alone or after surgery for the prevention or treatment of ocular diseases. Or it may be used in combination with other drug treatments.

[0039] The present invention relates to a method for preventing ocular diseases, comprising administering exosomes derived from amniotic epithelial cells as an active ingredient. Or, a functional health food composition for improving the condition is provided.

[0040] The exosomes have the ability to heal wounds in corneal or conjunctival tissues and to repair damaged lacrimal gland tissues. It can restore the function of goblet cells involved in stabilizing the tear film and induce tear secretion. In addition, the exosomes can express IL-1β, IL-8, IL-6, IFNγ, and TNFα, thereby reducing the secretion of inflammatory cytokines selected from the group consisting of ocular It can be used as a functional health food composition for preventing or improving diseases.

[0041] Corresponding features may be substituted in the above-mentioned portions.

[0042] In the functional health food composition according to the present invention, the functional health food is effective in preventing or treating eye diseases. For improvement purposes, it can be manufactured as powder, granules, tablets, capsules, syrup or beverage. The health functional food may take any form without limitation, and may be used in the same manner as the pharmaceutical composition. They are either formulated in the same way and used as functional foods or added to various foods.

[0043] In the functional health food composition according to the present invention, the functional health food does not mean food in the usual sense. For example, beverages and various drinks, fruits and their processed foods (fruits, etc.) canned foods, jams, etc.), fish, meat and its processed foods (ham, bacon, etc.), bread and Noodles, cookies and snacks, dairy products (butter, cheese, etc.) are possible, and usually Also, any food used as feed for animals may be included. It may also include products.

[0044] The health functional food composition according to the present invention is a food-scientifically acceptable food commonly used by those skilled in the art. It can be manufactured further containing additives (food additives) and other auxiliary ingredients as appropriate. The suitability of a substance as an additive is based on the food additives approved by the Food and Drug Administration, unless otherwise specified. Judging based on the specifications and standards for the item in question based on the general provisions of the official code and general test methods The items listed in the "Food Additives Code" include, for example, ketones, Chemical compounds such as glycine, calcium citrate, nicotinic acid, and cinnamic acid; blue dye, sweetener Natural additives such as grass extracts, crystalline cellulose, sorghum pigment, and guar gum; L-glutamate A mixture of sodium phosphate preparations, noodle additive alkali agents, preservative preparations, and tar color preparations. Examples of such agents include

[0045] The other supplementary ingredients include, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, etc. , coloring agent, pectinic acid, alginic acid, organic acid, protective colloid thickener, pH adjuster, stabilizer The composition may further contain antioxidants, preservatives, glycerin, alcohol, carbonation agents, and the like. In particular, the natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as sucrose and dextrins and cyclodextrins polysaccharides, sugar alcohols such as xylitol, sorbitol, and erythritol As sweeteners, natural sweeteners such as thaumatin and stevia extract can be used. Sweeteners such as saccharin and synthetic sweeteners such as aspartame can be used.

[0046] The effective volume of the exosomes derived from the amniotic epithelial cells contained in the health functional food according to the present invention The amount is appropriately adjusted depending on the purpose of use, such as prevention or amelioration of ocular diseases.

[0047] The health functional food composition uses food as a raw material and reduces side effects that occur when taking general medicines for a long period of time. It has the advantage of being portable and can be used as an adjunct for the prevention or treatment of eye diseases. It is ingested.

[0048] In order to facilitate understanding of the present invention, the present invention will be described in detail below with reference to examples. The examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples. The present invention is not intended to be limited to the specific embodiments described herein. It is something that can be done.

[0049] <Example 1> Isolation and culture of human amniotic epithelial cells and exosome extraction

[0050] The placenta was removed by Caesarean section from an uncomplicated pregnant woman and placed in a sterile container. The amniotic membrane was removed by washing with saline. (Amniotic membrane) is peeled off little by little from the chorion by inserting a forceps. The separated amniotic membrane was spread on a stainless steel plate with the epithelium facing downwards, and then rubbed with hands. The remaining chorion and blood were removed by rinsing with saline five times. The membrane was spread on a stainless steel plate with the epithelium facing downwards, and then NC paper (nitro The amnion was attached to the NC paper by covering it with cellulose paper (0.45 μm). After the adhesion, the specimen was cut into square pieces of about 5-10 cm and then soaked in HBSS (Hank The experiment was carried out by placing the sample in a container containing 's balanced salt solution. Moved it to the room.

[0051] The separated amniotic membrane was transferred to a sterile beaker and washed several times with HBSS to remove any remaining blood. After completely removing the tissue debris, the amniotic membrane pieces were soaked in 0.5 mM EDTA (ethylenediaminetetraacetic acid). aminetetraacetic acid and penicillin - 10 minutes at 37°C in PBS containing streptomycin After that, the plates were incubated with 0.05% trypsin. After leaving the mixture in the n / EDTA solution at 37°C for 5 minutes, the solution was discarded and a new The cells were incubated in a new 0.05% trypsin / EDTA solution at 37°C for 10 min with intermittent shaking. After the reaction, the culture medium was collected. Four volumes of DMEM / F12 culture medium were added to the cells. After inactivating rypsin / EDTA, the cells were centrifuged at 300 × g for 10 minutes and resuspended. Then, the cells were strained through a cell strainer with a pore size of 100 μm. The cells were filtered through a trainer, centrifuged, and counted.

[0052] The isolated cells were cultured in DMEM / F12 (10% FBS, 1% P / S) medium. 1.2×10 5 / cm 2 Poly-L-lysine copolymer with a density of The culture plates were seeded and incubated at 37°C under 5% CO2. When necessary, 10 ng / ml epidermal growth factor (EGF) was added. rowth Factor, EGF), ROCK(Rho-Associated Co il Kinase inhibitor, leukemia inhibitor The cells were cultured with the addition of LIF (LIF-lysine activator factor).

[0053] The exosomes can be extracted by various methods, for example, by phosphatidylserine Extraction can be performed using binding separation, antibody binding separation, and ultra-high speed centrifugation. .

[0054] 1) Exosome extraction using ultra-high speed centrifugation

[0055] Amniotic epithelial cells were cultured in DMEM / F12 (10% FBS, 10ng / ml EGF, 1% P / S) 80% confluence on poly-L-lysine coated culture dishes fluence) and wash the cells three times with PBS to remove any remaining medium. The medium was changed to DMEM / F12 (1% Exosome Free FBS, 10 ng / ml l EGF, 1% P / S medium was replaced, and after 72 hours, the conditioned medium (conditioned medium) was The endothelial cells were collected from the endothelial cells. To do this, the mixture was centrifuged at 300 × g for 10 minutes, and the resulting supernatant was collected and stirred for 30 minutes. After centrifugation, the supernatant was collected and concentrated in a centrifugal concentrator ( centrifugal concentrator, 100,000 MWCO me The conditioned medium was concentrated using mebrane (Corning). The mixture was ultracentrifuged at 100,000 × g for 2 hours. The pellet was resuspended in PBS and then centrifuged at 100,000 × g for 2 hours. The final pellet contained exosomes, and the pellet was washed with PBS. The mixture was resuspended in 0.5% CO and used immediately or stored at 4°C until use.

[0056] 2) Separation method via phosphatidylserine binding

[0057] Amniotic epithelial cells were cultured in DMEM / F12 (10% FBS, 10ng / ml EGF, 1% P / S) Grown to 80% confluence on poly-L-lysine-coated culture dishes. The cells were washed three times with PBS to remove any remaining medium. The medium was replaced with DMEM / F1 2(1% Exosome Free FBS, 10ng / ml EGF, 1% P / S ) medium, and after 72 hours, the conditioned medium was collected. To remove any remaining cell debris, centrifuge at 300 × g for 5 min and collect the supernatant. The supernatant was centrifuged at 1,200×g for 20 minutes to remove the lysates. Large extracellular vesicles were isolated by centrifugation at 1 × g for 30 min. The final centrifuged preparation was separated from cellular vesicles and exosomes. The supernatant of the conditioned medium may contain exosomes.

[0058] The collected supernatant was concentrated in a centrifugal concentrator (100,000 MWCO memebrane Corning) and stored at 4°C until use. The concentrated supernatant was purified using a phosphatidylcholine binding isolation kit (MagCapture TM E Exosomes were isolated using the Xosome Isolation Kit PS (Wako). Separated.

[0059] The MagCapture method uses phosphatidylserine (phosphatidylserine This is a method for isolating exosomes through binding with a magnetron sp. (PS) without using an antibody. Exosome capture with magnetic beads (exosome capture) to isolate exosomes that bind to the exosomes. The streptavidin magnetic beads contained in the Exosome capture immobilization buffer The mixture was diluted in lizing buffer and placed on a magnetic stand. The beads are reacted with the biotin-labeled tand for 1 minute to leave only the beads. Add the identified exosome capture solution and rotate the mixture at 4℃ using a rotator. After 10 minutes of treatment, the cells were placed on a magnetic stand for 1 minute to capture exosomes. The beads were fixed to the gel.

[0060] The concentrated conditioned medium was mixed with exosome-binding enhancer (exosome-binding enhancer) at a volume ratio of 1:500. The samples were prepared by adding exonuclease binding enhancer (×500). After reacting with the chromatin-immobilized beads at 4°C for more than 3 hours using a rotator, Only the EVs-bound beads were separated using a magnetic stand.

[0061] Wash the EVs-bound beads using a washing buffer containing an exosome-binding enhancer. Washing and use a magnetic stand to separate only the EVs-bound beads. The EVs-bound beads were added with exosome elution buffer and then eluted with magnetic beads. The exosomes were finally obtained by eluting the exosomes from the IgG. The mixture was resuspended in PBS and used immediately or stored at -80°C until use.

[0062] FIG. 1 is a transmission electron microscope image of exosomes extracted from amniotic epithelial cells according to one embodiment of the present invention. Figure 2 shows the image of exosomes observed using a microscope (TEM). Size and polydispersity were measured using a NanoSight (Malvern zetasizer, Wo The results are from measurements using a 50-200 nm The size of exosomes was measured.

[0063] Figure 3 shows the results of Western blotting to confirm exosomes in sheep. The exosome pellet derived from amniotic epithelial cells was suspended in cell lysis buffer and then subjected to amniotic epithelial cell lysis. The lysate was used as a control. decyl sulfate, SDS) loading dye for 5 minutes, and then simmered in 10% SDS S-Polyacrylamide gel electrophoresis After electrophoresis on a polyacrylamide gel electrophoresis (PAGE), the NC membrane The protein was transferred to 5% skim milk for 1 hour. After incubation, exosomes derived from amniotic epithelial cells were confirmed by CD63 antibody treatment.

[0064] Through this, we were able to confirm stable isolation of exosomes derived from human amniotic epithelial cells. Done.

[0065] <Experimental Example 1> Cell proliferation analysis

[0066] Human conjunctival cell lines were distributed into 96-well plates and allowed to grow to 60% of the plate area. After 16 hours of growth factor and FBS starvation, the cells were treated with 70 mM NaCl. An environment similar to xerophthalmia was induced.

[0067] Amniotic epithelial cell-derived exosomes were cultured at 0, 300, 600, and 3,000 percent per cell. After treating the particles and culturing for 24 hours, the medium was removed and 20 μl L-Methylthiazol tetrazolium MTT (microtubule, MTT) was added to each well and incubated at 37°C for 3 hours to obtain the cells. Growth was confirmed.

[0068] As a result, as shown in Figure 4, stable cell proliferation was observed in human conjunctival cell lines after treatment with exosomes at various concentrations. This indicates that the exosome treatment suppresses cell proliferation or No cytotoxicity was observed.

[0069] <Experimental Example 2> Intracellular migration of exosomes into human conjunctival epithelial cells (endocytosin Analysis of s)

[0070] Confirming endocytosis of exosomes in epithelial cells To detect exosomal fragments, the exosomal fragments were stained with a red fluorescent dye (Dil, Thermo Fisher). 1×10 -3 The mixture was incubated with 10 mM Dil for 30 minutes at 37°C and then diluted in 100 μL PBS. The cells were diluted and used immediately. When the cells reached 0% confluence, the medium was replaced with 1% exosome-free medium. The Dil-stained exosomes were treated and grown for 4 hours at 37°C in a 5% CO2 environment. I did.

[0071] As a result, as shown in Figure 5, Dil-labeled exosomes were transferred to human conjunctival epithelial cells. You can see that it works.

[0072] <Experimental Example 3> Analysis of changes in IL-8 concentration

[0073] Human conjunctival cell lines were distributed into 96-well plates and allowed to grow to 80% of the plate area. , 70 mM NaCl was treated to induce an environment similar to xerophthalmia.

[0074] Amniotic epithelial cell-derived exosomes were cultured at 60, 300, and 600 particles per cell. After 24 hours of incubation, the change in the concentration of IL-8 secreted outside the cells was confirmed. The control group was not treated with the exosomes. After collecting the medium used for cell culture, EGEND MAX TM Human IL-8 ELISA kit with Pr e-coated Plates (Biolegend, San Diego, CA, U ELISA was performed using the SA according to the manufacturer's protocol.

[0075] As a result, as shown in Figure 6, exosomes were cultured in a cell culture environment that induced a dry eye syndrome-like environment. When treated with exosomes, the concentration of IL-8 was reduced compared to the control group that did not receive exosomes. We were able to confirm this.

[0076] <Experimental Example 4> Analysis of wound healing

[0077] Human conjunctival cell lines were cultured to examine the wound healing ability of exosomes derived from amniotic epithelial cells in vitro. Check whether it has any effect.

[0078] Specifically, human conjunctival cell lines were cultured in 96-well cell culture plates in an incubator for 24 hours. If the cells are cultured at 4 °C to form a confluent monolayer, After starvation, the medium was replaced with 1% exosome-free serum for 2 h, and the wound formation tool ( WoundMaker (Essen BioScience, Inc., USA) was used. This caused a wound to form.

[0079] Amniotic epithelial cell-derived exosomes were treated at 300 particles per cell, Inc uCyte(Essen BioScience, Inc., Michigan, USA ) and observed for 48 hours to measure the cells' wound healing ability.

[0080] As a result, as shown in Figure 7, the difference between the group treated with exosomes derived from amniotic epithelial cells and the control group was A significant difference was observed between the groups treated with exosomes derived from amniotic epithelial cells. It was confirmed that the wound healing ability was increased compared to the control group that was not treated with the treatment.

[0081] <Experimental Example 5> Measurement of tear secretion in an animal model of xerophthalmia

[0082] 1. Animal Model of Xerophthalmia

[0083] Male BABL / c mice aged 8–12 weeks were fed filter-top, pathogen-free, The mice were housed in a room where they had free access to food. mine) patch (Kimite, Myungmoon Pharmaceutical Co., Ltd., Korea) for 9 days. Mice were housed in an environmentally controlled room during exposure to scopolamine at a concentration of 0.25 mg / day. The mice were kept in a dry chamber (humidity 18.5±5%, 23°C±2.5°C, air speed 20L / min). After the scopulavine exposure, the mice were placed in a normal environment (humidity 0.5-1.0 mm) and placed in a 10-story room. The temperature was controlled at 60±10%, 23°C±2.5°C.

[0084] 2. Processing of exosomes derived from amniotic epithelial cells

[0085] 0.1 μg of scopolamine was administered to mice that had developed corneal wounds after exposure to a scopolamine patch and desiccation stress. (Exo.1), 1μg (Exo.2) of amniotic epithelial cell-derived exosomes, 10μ M dexamethasone (Dex) was administered. The mice were treated with 5 μl of saline three times a day for up to 14 days. The mice were treated with 5 μl of 10 ...

[0086] 3. Experiment to measure tear secretion

[0087] Mice exposed to scopolamine patch and desiccation stress for 10 days were treated with NS, DEX, or Ex. Exo.1 and Exo.2 were administered for up to 14 days, and tear production was measured on days 0, 3, 7, and 14. It was determined.

[0088] Phenol red-impregnated cotton thread The thread was placed on the cantus lateralis of the mouse eye and measurements were taken for 60 seconds. turned red and was evaluated by measuring the changed length.

[0089] As a result, as shown in FIG. 8, the xerophthalmia-induced mice showed a decreased tear secretion. Furthermore, exosome-treated mice with xerophthalmia showed increased tear secretion. When compared with Dex, which is used as a treatment for dry eye syndrome, when administered for 14 days or more, , and the effect of the exosome treatment group was observed to be better.

[0090] <Experimental Example 6> Analysis of corneal Lissamine Green staining in an animal model of xerophthalmia

[0091] Mice exposed to scopolamine patch and desiccation stress for 10 days were treated with NS, DEX, or Ex. Exo.1 and Exo.2 were administered for up to 14 days, and the incidence of dry eye syndrome was measured on days 0, 3, 7, and 14. Corneal staining was performed with Lissamine green, which is used for diagnosis.

[0092] Lissamine green ophthalmic strips(optit The staining was performed using a slit lamp microscope (Model 900 BQ; Lissamine green staining of the cornea was performed according to the Oxford School of Medicine. me grading system (2003, Grading of Corneal and Con junctival Staining in the Context of Oth The ocular dry eye tests were used to score the eyes from 0 to 5. A grade of 0 is awarded and the maximum score is 5.

[0093] As a result, as shown in Figure 9, in the case of normal mice, there was almost no staining with Lissamine Green. However, the corneal surface of mice with xerophthalmia induced showed a high degree of Lissamine Green staining. After induction of xerophthalmia, in the mice treated with exosomes, the experimental group treated only with NS showed It showed a lower degree of Lissamine Green staining than that of cerebrospinal fluid. It is used as a treatment for dry eye syndrome. When compared with Dex, the exosome-treated mice showed better efficacy when administered for 7 days or more. It was noticed.

[0094] <Experimental Example 7> Analysis of PAS staining of conjunctival tissue in an animal model of xerophthalmia

[0095] Mice exposed to scopolamine patch and desiccation stress for 10 days were treated with NS, DEX, or Ex. Exo.1 and Exo.2 were treated for up to 14 days, and the mice were sacrificed on days 0, 3, 7, and 14. The conjunctival tissue was then fixed in 10% formalin and immersed in paraffin. The tissue was cut to a thickness of 7 μm and then unstained. The slides were prepared and incubated with periodic acid Schiff (PA S) staining was performed using a commercial kit (Merck, Darmstadt, Germany) according to the manufacturer's instructions. Conjunctival goblet cells were stained with PAS using a fluoroscopy kit (Germany). The stained area was photographed using an optical microscope. The glycoproteins and glycogen that are present in the cells are stained red by PAS staining, and the cells are counted. The goblet cell density was measured as the number of goblet cells per 100 μm.

[0096] As a result, as shown in Figure 10, the main component of mucin synthesis in the conjunctival goblet cells of the xerophthalmia-induced mice was It was shown that the distribution of glycoproteins and glycogen, which are major components of the glycoproteins, was affected. The number of goblet cells detected by PAS staining in mice was more than twice that of normal mice. After induction of xerophthalmia, the number of goblet cells was reduced in exosome-treated mice. When compared with Dex, which is used as a treatment for dry eye syndrome, The isosome (Exo.2) was found to be as effective as Dex.

[0097] <Experimental Example 8> Alcian blue staining analysis of lacrimal gland tissue in an animal model of xerophthalmia

[0098] Mice exposed to scopolamine patch and desiccation stress for 10 days were treated with NS, DEX, or Ex. The mice were treated with Exo.1 and Exo.2 for up to 14 days and sacrificed on days 0, 3, 7, and 14. The gland tissue was then collected. The collected lacrimal gland tissue was fixed in 10% formalin and paraffin-embedded. The tissue was cut to a thickness of 7 μm to prepare unstained slides, and the After Lucian blue staining, the stained sections were photographed using an optical microscope.

[0099] As a result, as shown in Figure 11, the lacrimal glands of dry eye-induced mice showed increased lacrimal acini. ) structure was significantly destroyed, and it was restored by Dex and exosome treatment. It was confirmed that exosomes are able to maintain the lacrimal gland structure and secretory function. This is thought to affect retention of

[0100] <Experimental Example 9> Confirmation of changes in cytokine secretion in corneal tissue of an animal model of xerophthalmia

[0101] Reliaprep TM RNA extraction system (Promega, Fitchburg Total RNA was extracted from mouse corneal tissue using a 30-mL PBS (Wisconsin, USA). After washing with PBS, RNA extraction was performed according to the manufacturer's instructions. Using a transcription kit (Applied Biosystems, Waltham, MA), To determine the expression of cytokines in mouse corneas, Power SYB R(R) Green PCR Master Mix (Applied Biosyst EMS, CA, USA) and StepOnePlus TM Real-time PCR The Real Time Imaging System (Applied Biosystems, CA, USA) was used. -time PCR was performed according to the manufacturer's protocol.

[0102] As a result, as shown in Figure 12, cytokines (cytokines) that were highly expressed in the xerophthalmia-induced mice were We were able to confirm that the concentration of IL-1, IL-2, and IL-1, IL-1, was reduced by Dex and exosome treatment. Through this, exosome treatment reduced cytokine secretion in xerophthalmia-induced mice. is expected to have an impact on

[0103] <Comparative Example 1> Exosomes derived from mesenchymal stem cells

[0104] 1.ELISA analysis

[0105] Human conjunctival cell lines were cultured to separate exosomes derived from amniotic epithelial cells and exosomes derived from mesenchymal stem cells. We confirmed whether it affects the expression of cytokines associated with xerophthalmia.

[0106] Specifically, human conjunctival cell lines cultured in serum-free medium were starved for 12 hours. After this, an environment similar to xerophthalmia was induced using 70 mM NaCl. Two hours before the treatment, the amniotic epithelial cell-derived exosomes and mesenchymal stem cell-derived exosomes were treated. After 24 hours of incubation, the concentration of IL-8 secreted from the cells was monitored. After collecting the medium used for cultivation, TM Human IL-8 E LISA kit with Pre-coated Plates(Biolegen ELISA was performed using the ELISA kit (San Diego, CA, USA) according to the manufacturer's protocol. I did A.

[0107] As a result, as shown in Figure 13, exosomes derived from amniotic epithelial cells and exosomes derived from mesenchymal stem cells were In a xerosom-treated xerophthalmia model, the expression of inflammatory cytokine IL-8 was increased. It was confirmed that there was no significant difference between the doses.

[0108] 2. Analysis of Wound Healing

[0109] Human conjunctival cell lines were cultured to separate exosomes derived from amniotic epithelial cells and exosomes derived from mesenchymal stem cells. We then examined whether lysosomes could affect wound healing in vitro.

[0110] Specifically, human conjunctival cell lines were cultured in 96-well cell culture plates in an incubator for 24 hours. After culturing in 1% serum medium for 4 hours, the cells were starved to form a confluent cell layer. After holding the wound, a wound formation tool (WoundMaker; Essen BioScience, The amniotic membrane isolated by the above method was then wounded using a suction tube (Sample Tube, Inc., Michigan, USA). Exosomes derived from epithelial cells and exosomes derived from mesenchymal stem cells were treated and then incubated with IncuC yte (Essen BioScience, Inc., Michigan, USA) The cells were then observed for 48 hours to measure their wound healing ability.

[0111] As a result, as shown in Figure 14, the group treated with exosomes derived from amniotic epithelial cells and the group treated with mesenchymal stem cells showed A significant difference was observed between the group treated with exosomes derived from amniotic epithelial cells and the control group. Exosomes were observed to have the greatest wound healing potential.

[0112] Through this, amniotic epithelial cell-derived exosomes were compared with mesenchymal stem cell-derived exosomes. It can be confirmed that it is more effective in treating eye diseases than

[0113] As described above, certain parts of the present invention have been described in detail. The specific description is merely a preferred embodiment, and is not intended to limit the scope of the present invention. It is clear that the scope of the present invention is not limited to the claims. and their equivalents.

Claims

1. A method for preventing or treating eye diseases that contains exosomes derived from amniotic epithelial cells as an active ingredient Therapeutic pharmaceutical compositions.

2. The exosomes are The eye disease according to claim 1, characterized in that it has the ability to heal wounds in the cornea or conjunctival tissue. A pharmaceutical composition for the prevention or treatment of a disease.

3. The exosomes are It restores the tissue of the lacrimal gland destroyed by the above-mentioned eye disease, and restores the goblet cells involved in stabilizing the tear film. The method for preventing or preventing eye diseases according to claim 1, characterized in that it regulates the secretion of tears and induces tear production. or a therapeutic pharmaceutical composition.

4. The exosomes are Selected from the group consisting of IL-1β, IL-8, IL-6, IFNγ, and TNFα. The method of claim 1, characterized in that it reduces the secretion of inflammatory cytokines. Prophylactic or therapeutic pharmaceutical compositions.

5. The eye disease is Selected from the group consisting of xerophthalmia, corneal epithelial damage, keratitis, conjunctivitis, and keratoconjunctivitis. Ocular surface disease; or an ocular disease selected from the group consisting of endophthalmitis, uveitis, and macular degeneration. The pharmaceutical composition for preventing or treating eye diseases according to claim 1, characterized in that the eye diseases are internal diseases. composition.

6. The composition comprises: Eye drops, injections, granules, tablets, pills, capsules, gels, syrups, suspensions, emulsions, The pharmaceutical composition is characterized in that it is in any one dosage form selected from the group consisting of drops and liquids. The pharmaceutical composition for preventing or treating an ocular disease according to claim 1.

7. A drug for preventing or improving eye diseases that contains exosomes derived from amniotic epithelial cells as an active ingredient. Good health functional food composition.

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