TWI856252B - Use of secretome of amniotic fluid stem cell in the treatment of dry eye disease - Google Patents

Abstract

Disclosed herein is a method or a pharmaceutical composition for the treatment of dry eye disease or corneal wound healing, comprising administering to a subject in need thereof a therapeutically effective amount of secretome of amniotic fluid stem cells. Also provided is a use of secretome of amniotic fluid stem cells for manufacturing a medicament for the treatment of dry eye disease or corneal wound healing.

Description

Use of amniotic fluid stem cell secretions for the treatment of dry eye syndrome

The present invention relates to the application of amniotic fluid stem cell secretions in treating dry eye and healing corneal wounds.

Amniotic fluid cells are used for routine prenatal diagnosis to detect fetal chromosomal abnormalities. In addition, some small nucleated cells with characteristics of hematopoietic progenitor cells were found in the amniotic fluid before 12 weeks of pregnancy, indicating that these cells may come from the yolk sac. From 2004 to 2006, there were some reports on the successful isolation and identification of another group of stem cells in amniotic fluid. These amniotic fluid stem cells were shown to express specific biomarkers of mesenchymal stem cells and neural stem cells and have the ability to differentiate into bone, cartilage, fat, and nerve cells. It has been confirmed that amniotic fluid stem cells can proliferate in large quantities and stably in vitro, and their proliferation rate is faster than that of mesenchymal stem cells obtained from adult bone marrow and fat, and they do not have the tumorigenic potential of embryonic stem cells. Therefore, amniotic fluid stem cells have the potential to be applied in clinical medicine.

Dry eye is commonly referred to as keratoconjunctivitis sicca. Millions of people suffer from this condition each year, particularly due to the rapid development of electronic products, which are the most common cause of dry eye today. Dry eye symptoms are traditionally managed with eyelid hygiene, topical antibiotics, tetracycline, deoxytetracycline, anti-inflammatory compounds (cyclosporine), or corticosteroids, which are often time-consuming, frustrating, and often ineffective or with variable results.

Therefore, there is a need to develop a new dry eye treatment that can effectively relieve symptoms.

One aspect provided herein is a method for treating dry eye in an individual, comprising administering to the individual a composition comprising amniotic fluid stem cell secretions and a pharmaceutically acceptable carrier; wherein the secretions are prepared by a method comprising the following steps: culturing amniotic fluid stem cells in a basic culture medium for 24-72 hours to obtain a culture medium; collecting the supernatant of the culture medium after centrifugation, and filtering the supernatant to obtain the secretions.

In one embodiment, the supernatant is filtered through a filter having a pore size less than 0.5 μm; preferably, the pore size is less than 0.22 μm.

In one embodiment, the composition is a pharmaceutical composition that is topically applied to the eye of the individual, preferably in the form of an eye gel, eye drops, eye wash, eye drops, eye inserts, eye ointment or eye spray.

Another aspect provided herein is a method for healing corneal epithelial wounds in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of amniotic fluid stem cell secretions as disclosed herein and a pharmaceutically acceptable carrier.

On the other hand, the present invention provides a pharmaceutical composition for treating dry eye, comprising amniotic fluid stem cell secretions and a pharmaceutically acceptable carrier; wherein the secretions are prepared by a method comprising the following steps: culturing amniotic fluid stem cells in a basic culture medium for 24-72 hours to obtain a culture medium; collecting the supernatant of the culture medium after centrifugation, and filtering the supernatant to obtain the secretions.

In another aspect, the present invention provides a pharmaceutical composition for corneal epithelial wound healing, comprising administering to an individual in need thereof a pharmaceutical composition comprising a therapeutically effective amount of amniotic fluid stem cell secretions as disclosed herein and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides the use of the amniotic fluid stem cell secretor in the preparation of a medicament for treating dry eye disease.

In another aspect, the present invention provides the use of the amniotic fluid stem cell secretion body in the preparation of a medicament for corneal epithelial wound healing.

The present invention also provides the use of the amniotic fluid stem cell secretion body in preparing a dry eye medicine.

The present invention also provides the use of the amniotic fluid stem cell secretion body in preparing a corneal epithelial wound healing drug.

The present invention will be further described by the following examples. However, it should be understood that the following examples are only for the purpose of illustration and should not be construed as limiting the present invention in implementation.

In order to clearly demonstrate the above and other technical contents, features and effects of the present invention, the following specific embodiments are read in conjunction with the attached drawings. Through the description of the specific embodiments, ordinary technicians in this field will clearly understand the technical methods and effects adopted by the present invention to achieve the above aspects.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. In this application, "or" or "and" is used to mean "and/or" unless otherwise indicated. In addition, the use of the term "including" and other forms such as "include", "includes", and "included" is not limiting. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. Unless otherwise indicated, the materials used herein are commercially available and readily available.

As used herein, the word "about" or similar terms used herein refers to a measured amount, such as a dosage, including a deviation of ±15% or ±10% relative to a specific amount in one embodiment; a deviation of ±5% relative to a specific amount in a preferred embodiment; a deviation of ±1% relative to a specific amount in another preferred embodiment; or a deviation of ±0.1% relative to a specific amount in a best embodiment; and the properties of the substance to which the amount belongs are not affected.

As used herein, the term "disease" as used herein refers to any symptom, infection, condition, or syndrome that requires medical intervention or a condition requiring medical intervention. Such medical intervention may include treatment, diagnosis, and/or prevention.

The terms "dry eye disease (DED)", "dry eye syndrome (DES)", "keratoconjunctivitis sicca (KCS)", or simply "dry eye" are used interchangeably to refer to eye diseases caused by decreased tear production or increased tear film evaporation. Dry eye may be the result of another underlying condition that causes dry eye, such as Sjogren's syndrome, menopause, or rheumatoid arthritis. Dry eye may also be a complication of an inflammatory response. Dry eye may also be the result of an infection, a side effect of medications, or exposure to toxins, chemicals, or other substances that may cause symptoms or symptoms of dry eye. Dry eye syndrome may manifest as one or more ophthalmic clinical symptoms known in the art, including but not limited to dryness, foreign body sensation, burning, itching, irritation, redness, eye pain, blurred vision and/or decreased vision.

As used herein, the term "secretome", also referred to as "conditioned medium", refers to all (or a collection of) proteins that are secreted from cells into the cell environment (into the culture medium) when the cells are cultured.

As used herein, the term "stem cells" is a general term for undifferentiated cells before differentiation into various cells constituting tissues, and stem cells have the ability to differentiate into specific cells through specific differentiation stimuli. According to the present invention, the cells used to prepare the secretory body of the present invention are amniotic fluid stem cells.

As used herein, the term "combination" means a product resulting from the mixture or combination of more than one active ingredient, and includes fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredient and an adjuvant are both administered to the patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredient and an adjuvant are administered to the patient as separate entities simultaneously, concurrently or sequentially, without specific intervention time restrictions, wherein such administration provides effective levels of both agents in the patient's body. The latter also applies to cocktail therapy, for example, the administration of three or more active ingredients.

According to the present invention, the term "pharmaceutical composition" used herein refers to a therapeutically effective amount of amniotic fluid stem cell secretion or conditioned medium and optionally a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable" means that the drug is suitable for use in contact with the tissues of individuals (such as humans) taking the drug within the scope of reasonable medical judgment, without excessive toxicity, irritation, allergic reaction or other problems or complications, and has a reasonable benefit/risk ratio. Each carrier must be compatible with other ingredients to be "acceptable".

The term "carrier" as used herein refers to a non-toxic compound or agent that has the function of assisting cells or tissues to absorb active ingredients. The carrier is selected from, for example, excipients, adjuvants, diluents, fillers, or bulking agents, granulating agents, coating agents, release control agents, adhesives, disintegrants, lubricants, preservatives, surfactants, antioxidants, buffers, suspending agents, thickeners, stabilizers or other carriers used in pharmaceutical compositions. Examples of carriers include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropylmethylcellulose, poloxamer, hydroxyethylcellulose cyclodextrin, carboxymethylcellulose (CMC), phosphate buffered saline (PBS), water, emulsifiers (e.g., oil and water emulsions), or wetting agents. Tonicity modifiers may be added as needed or convenient. They include, but are not limited to, salts, particularly sodium chloride, potassium chloride, mannitol, and glycerol, or any other suitable ophthalmologically acceptable tonicity modifier. A variety of buffers and methods of adjusting pH may be used, as long as the resulting formulation is ophthalmologically acceptable. Thus, buffers include acetate buffers, citrate buffers, phosphate buffers, and borate buffers. Acids or bases may be used to adjust the pH of these formulations as needed. Similarly, ophthalmologically acceptable antioxidants for use in the present invention include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene. Other excipient components that may be included in ophthalmic formulations are chelating agents and antibiotics. A preferred chelating agent is disodium ethylenediaminetetraacetate, although other chelating agents may be used in place of or in combination with it. Non-limiting examples of antibiotics that can be used in the present invention include trimethoprim sulfate/polymyxin B sulfate, gatifloxacin, moxifloxacin hydrochloride, tobramycin, teicoplanin, vancomycin, azithromycin, clarithromycin, amoxicillin, moxicillin, penicillin, ampicillin, carbenicillin, ciprofloxacin, levofloxacin, amikacin, gentamicin, kanamycin, neomycin, and streptomycin.

As used herein, the terms "effective amount" or "therapeutically effective amount" refer to an amount of an agent or compound administered that is sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated. The result may be a reduction and/or alleviation of the signs, symptoms, or causes of the disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a peptide or protein disclosed herein that is required to provide a clinically significant reduction in the symptoms of the disease. In any individual case, the appropriate "effective" amount can be determined using techniques such as dose escalation studies.

As used herein, the terms "treat," "treating," or "treatment" include reducing, alleviating, or ameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting a disease or condition, such as arresting the development of a disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a symptom caused by the disease or condition, or prophylactically and/or therapeutically stopping symptoms of the disease or condition.

The present invention provides a composition comprising amniotic fluid stem cell secretions; wherein the secretions are prepared by a method comprising the following steps: culturing amniotic fluid stem cells in a basal medium for 24-72 hours; collecting and centrifuging the supernatant of the basal medium, and filtering the supernatant to obtain the secretions.

In one embodiment, the supernatant is filtered through a filter having a pore size less than 0.5 μm. Preferably, the pore size of the filter is 0.45 μm, 0.3 μm, or 0.22 μm.

In a specific embodiment, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier.

According to the present invention, a method for treating dry eye disease comprises administering a pharmaceutical composition comprising amniotic fluid stem cell secretions to an individual in need thereof.

According to the present invention, a method for healing corneal epithelial wounds comprises administering a pharmaceutical composition comprising amniotic fluid stem cell secretions to an individual in need thereof.

In one embodiment, the pharmaceutical composition is topically administered to the eye of the individual.

In one embodiment, the pharmaceutical composition is in the form of an ophthalmic preparation, such as an eye gel, eye drops, eye wash, eye drops, eye inserts, eye ointment or eye spray.

The present invention also provides the use of amniotic fluid stem cell secretions in the preparation of dry eye drugs.

The present invention also provides the use of amniotic fluid stem cell secretions in the preparation of corneal epithelial wound healing drugs.

The present invention is further described by the following examples, which are provided for purposes of illustration and not limitation.

Embodiment

Embodiment 1

Therapeutic amniotic fluid stem cells (AFSC) are obtained by a method comprising the following steps: thawing cells from a qualified cell bank, such as a working cell bank (WCB) and culturing the cells in MSC NutriStem® XF medium (serum-free/xeno-free). AFSCs are further cultured in a base medium (α-minimum essential medium) for 48 hours to collect the conditioned medium of AFSCs.

Subsequently, the conditioned medium collected from the cell culture was centrifuged at 2,000 rpm for about 10 minutes, filtered through a 0.22 μm filter membrane to remove cell residues, and the AFSC secretome was obtained and stored in a -20°C refrigerator for future use.

Embodiment 2

This example uses UVB irradiation to induce dry eye animal models, and evaluates the effect of amniotic fluid stem cell secretions on treating dry eye based on the experimental results.

Materials and methods

In this example, female ICR albino mice (BioLASCO) were used as animal models. The experiment was started when the mice were 5 to 6 weeks old. ICR mice were divided into 6 groups, each group included 6 mice (n=6): 1.         Healthy control group (blank group): treated with 0.9% saline eye drops; 2.         Positive control group (injury group): treated with 0.9% saline eye drops and UVB to cause injury; 3.         1/5 amniotic fluid stem cell secretion (1/5D group): treated with UVB to cause injury, and treated with 1/5 diluted amniotic fluid stem cell (AFSC) secretion; 4.         1/5 amniotic fluid stem cell secretion (1/5D group): treated with UVB to cause injury, and treated with 1/5 diluted AFSC secretion by eye drops; 5.        1/10 amniotic fluid stem cell secretion (1/10D group): treated with UVB to cause damage, and treated with 1/10 diluted AFSC secretion by eye drops; 6.         1/20 amniotic fluid stem cell secretion (1/20D group): treated with UVB to cause damage, and treated with 1/20 diluted AFSC secretion by eye drops; 7.         Artificial tear control group (Artificial tear, AFT group): treated with 0.9% artificial tears (ALLERGAN) by eye drops, and treated with UVB to cause damage.

Eye drops were administered to all mice at 10:00 and 17:00 daily according to the proposed protocol starting 3 days before the start of dry eye induction. UVB irradiation was performed continuously from day 1 to day 7, with the intensity of UVB being 0.72 J/cm ² .

The schematic timeline of the experiment is shown in Figure 1. Tears were collected on days 0, 4, and 7 to measure the changes in tear volume; the tear break up time (TBUT) of each mouse was also measured on days 0, 4, and 7.

The mice were sacrificed on day 8 for further histopathological analysis.

result

The amount of tears collected from each mouse was measured, and the results are shown in Figure 2. As shown in Figure 2, the tear secretion efficiency of the groups treated with AFSC secretion body was improved, especially in the group treated with 1/10D on day 7. Compared with the positive control group (injury group), the improvement of the groups treated with AFSC secretion body (1/5D, 1/10D, and 1/20D) on days 4 and 7 showed statistical significance (p < 0.05). In contrast, although the mice treated with artificial tear solution (AFT) showed significant improvement on day 7, the efficacy of artificial tear solution in improving tear secretion was still lower than that of AFSC secretion body.

The tear membrane break-up time (TBUT) of each mouse was measured, and the results are shown in Figure 3. As shown in Figure 3, except for the healthy control group, the TBUT of each group gradually decreased until the 7th day. However, on the 4th and 7th days, the TBUT of the groups treated with AFSC secretion (1/5D, 1/10D, and 1/20D) was improved, showing statistical significance compared with the positive control group (p < 0.05). Although the results did not show an obvious dose-dependent effect, the tear membrane stability maintenance effect of the group treated with AFSC secretion was higher than that of the AFT group.

Corneal surface photography includes assessment of corneal opacity, corneal smoothness, corneal mapping, and corneal staining (Lissamine Green Stain) examination, where higher assessment scores represent higher severity of damage. The photographic results are shown in Figure 4.

As shown in Figure 4, corneal damage was reduced in the groups treated with AFSC secretions. Specifically, corneal opacity was reduced in the group treated with AFSC secretions, which also showed statistical significance (p < 0.05). In addition, corneal smoothness, corneal map, and corneal staining (Lissamine Green Stain) examinations also showed improvement after treatment with AFSC secretions.

In addition, although AFT was used as a comparison in this article and also showed an improvement effect, as shown in Figure 4, the improvement effect of AFT treatment was poor, so the results showed that AFSC secretome is a potential substitute for AFT.

Ocular tissues including cornea, lacrimal glands, and leucoderma (also called palpebral glands) were subjected to tissue staining and IHC staining.

H&E staining shows the morphology of cells and indicates the integrity of tissues. IHC targets include Cox-2, p63, PCNA, NFκ-B, p53, and 4-HNE. Cox-2 and NFκ-B are regulators of immune and inflammatory responses. P63 and PCNA are regulators of cell proliferation and reproduction. p53 is an apoptotic factor. 4-HNE is associated with oxidative stress.

Table 1 shows the quantitative results of H&E staining and IHC staining of Cox-2, p63, and PCNA in corneal tissue. (The mark "+" indicates the degree of health.) As shown in Table 1, treatment with AFSC secretion can effectively alleviate the inflammatory response of corneal tissue after UVB damage and enhance cell proliferation, among which the expression of p63 and PCNA increased but the expression of Cox-2 decreased. The scores of each group are evaluated as follows: blank group > 1/10 D group > 1/5 D group > 1/20 D group > AFT group > damage group. Table 1 organization dyeing Blank group Damage Group 1/5D Group 1/10D Group 1/20D group AFT Group cornea H&E ++++++ + ++++ +++++ +++ ++ Cox-2 ++++++ + +++ +++++ ++++ ++ p63 ++++++ + +++++ ++++ +++ ++ PCNA ++++++ + +++++ ++++ +++ ++

Table 2 shows the quantitative results of H&E staining and IHC staining of Cox-2, p53 and NFκ-B in lacrimal gland tissue. As shown in Table 2, treatment with AFSC secretion can effectively alleviate the inflammatory response and inhibit cell apoptosis in lacrimal gland tissue after UVB injury, among which the expression of Cox-2, p53, and NFκ-B is reduced. The scores of each group are evaluated as follows: blank group > 1/10 D group > 1/5 D group = 1/20 D group > AFT group > injury group.

Table 2 organization dyeing Blank group Damage Group 1/5D Group 1/10D Group 1/20D group AFT Group Tear glands H&E ++++++ + ++++ +++++ +++ ++ Cox-2 ++++++ + +++ +++++ ++++ ++ NFκB ++++++ + +++++ ++++ +++ ++ p53 ++++++ + +++++ ++++ +++ ++

Table 3 shows the quantitative results of H&E staining and IHC staining of Cox-2, 4-HNE, and NFκ-B in the optic gland tissue. As shown in Table 3, treatment with AFSC secretion can effectively alleviate the inflammatory response and reduce oxidative stress in the optic gland tissue after UVB injury, among which the expression of Cox-2, 4-HNE, and NFκ-B is reduced. The scores of each group are evaluated as follows: blank group > 1/10 D group > 1/5 D group > 1/20 D group > AFT group > injury group.

Table 3 organization dyeing Blank group Damage Group 1/5D Group 1/10D Group 1/20D group AFT Group Pachypaedic gland H&E ++++++ + ++++ +++ +++++ ++ Cox-2 ++++++ + ++++ +++++ +++ ++ NFκB ++++++ + ++++ +++++ +++ ++ 4-HNE ++++++ + ++++ +++++ +++ ++

As shown in Tables 1 to 3, treatment with AFSC secretions can maintain the integrity of cell tissues, relieve inflammatory responses, rescue cell apoptosis, and enhance proliferation after UVB damage, indicating that AFSC secretions should be an effective agent for restoring and repairing ocular tissues, such as the cornea.

Embodiment 3

In this example, human corneal epithelial cells were treated with AFSC secretions and their effects were observed.

Materials and methods

The method for obtaining the AFSC conditioned medium is the same as that in Example 1 and will not be described again here.

Human corneal epithelial cells (HCEC) (Thermo Fisher) were cultured in serum free medium (SFM) (Thermo Fisher) at 37°C and 5% CO ₂ .

To obtain a cell suspension, remove the medium and wash the cells with 3 ml DPBS (Dulbecco's phosphate-buffered saline). After removing the DPBS, add 1 ml 0.05% trypsin and incubate at 37ºC for 5-7 minutes.

After confirming that the cells were detached in a round shape under a microscope, the cells were suspended in 2 ml of culture medium to weaken the trypsin reaction.

Take out 20 μl of the suspension and add an equal volume of trypan blue to mix with the suspension. After that, take out 10 μl of the mixture and count the number of cells on a cell counter.

MTT assay was performed to evaluate the effect of AFSC secretome on corneal cells. MTT assay is a colorimetric assay used to evaluate cell metabolic activity and viability. Under specific conditions, NAD(P)H-dependent cellular oxidoreductases can reflect the number of viable cells present. These enzymes are able to reduce the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to an insoluble formazan, which is purple in color.

HCEC were plated in a 96-well plate, with 8,000 cells per well. 100 μl of culture medium was added, and the cells were cultured for 24 hours. Afterwards, the cells were treated with 100 μl of AFSC secretion solution at different concentrations (1/2, 1/8, and 1/32 dilutions) and cultured for 24, 48, and 72 hours, respectively.

After the incubation period is completed, the medium is replaced with 200 μl of MTT solution and allowed to react for 3 hours. The MTT solution is further replaced with DMSO to dissolve the crystals. The cells are analyzed under an ELISA reader to detect the absorbance at a wavelength of 570 nm.

On the other hand, a wound healing test was performed. 1.6 x 10 ⁵ cells were cultured in a 12-well plate for 24 hours, with each well containing 1 ml of medium. After the culture was completed, 3 lines were drawn on the bottom of the well with a 200 μl pipette tip. Then the medium was removed, the cells were washed with DPBS, and then 500 μl of medium was added. The initial state of cell migration (0 hours) was observed and photographed under a 100X microscope.

Subsequently, 500 μl of AFSC secretion solution of different concentrations (1/2, 1/8, and 1/32 dilutions) were added. Further observation and photography were performed 6, 12, 24, and 48 hours after the start of cell migration. The migration distance was calculated according to the following formula: Distance = [(D0-Dn)/D0]x100% Where D0 is the scratch width at 0 hours, and Dn is the scratch width after 6, 12, 24, and 48 hours.

All experimental results are expressed as mean ± SD (standard deviation). One-way ANOVA was used for the homogeneity test, and the significance of each time point was tested by Scheffe's post hoc test. P < 0.05 indicated statistical significance; p < 0.001 indicated high statistical significance.

result

The results of the MTT test showed the effect of AFSC secretions on human corneal cells as shown in Figure 5, which shows the correlation between treatment with different concentrations of AFSC secretions and cell viability at 24, 48, and 72 hours.

As shown in Figure 5, the absorbance of OD ₅₇₀ (cell viability) increased with the increase of AFSC secretome content. The efficiency of improving viability also increased with the content of AFSC secretome. Among all the groups, the group treated with 1/2 diluted AFSC secretome showed the most significant improvement.

The results of the wound healing test showed the effect of AFSC secretions on corneal cell migration, as shown in Figure 6, which shows the migration efficiency after 6, 12, 24, and 48 hours of treatment with AFSC secretions.

As shown in Figure 6, the group not treated with AFSC secretion (negative control group) had a slight and insignificant migration from 0 to 48 hours. In contrast, AFSC secretion migrated significantly from 24 to 48 hours. In addition, the groups treated with 1/2 and 1/8 diluted AFSC secretion showed about 100% migration at 48 hours, indicating that the width of the scratch was almost completely restored, and the group treated with 1/32 diluted AFSC secretion showed about 80% migration.

From the results of this example, it can be seen that the secretion of AFSC can improve the survival rate and vitality of human corneal cells and improve the migration efficiency of human corneal cells. Therefore, the secretion of AFSC shows potential therapeutic efficacy as a drug for dry eye disease.

Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only and may be implemented in combination. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. The following claims are intended to define the scope of the present invention and to cover methods and structures within the scope of these claims and their equivalents.

without

The foregoing summary of the present invention as well as the following detailed description will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, there are shown in the accompanying drawings specific embodiments which are currently preferred.

In the diagram:

FIG. 1 is a schematic timeline of the experiment of Example 1.

FIG2 shows the distribution of tear volume collected from each mouse in Example 1 on day 0, day 4, and day 7 according to FIG1 (*p<0.05, compared with the injury group).

FIG3 shows the curve of tear break up time (TBUT) measured from the eyes of each mouse in Example 1 on day 0, day 4, and day 7 according to FIG1 (*p<0.05, compared with the injury group).

FIG4 shows the photographs of the corneal surface of the mouse eyes damaged by UVB in Example 1, including (A) assessment of corneal opacity, (B) corneal smoothness, (C) corneal map, and (D) corneal staining (Lissamine Green Stain) examination; wherein a higher assessment score represents a higher degree of damage (*p < 0.05, compared with the damaged group).

FIG5 shows the results of MTT assay, which illustrates the correlation between different concentrations of AFSC secretion treatment in Example 2 and the number of viable cells of human corneal epithelial cells at 24, 48, and 72 hours (*p < 0.05, compared with concentration 0).

FIG6 shows the results of the wound healing experiment, which illustrates the migration efficiency of human corneal epithelial cells after 6, 12, 24, and 48 hours of AFSC secretion treatment (*p<0.05, **p<0.001, compared with concentration 0).

without

Claims (9)

A use of a secretion of amniotic fluid stem cells in the preparation of a drug for treating dry eye, wherein the secretion is prepared by a method comprising the following steps: culturing amniotic fluid stem cells in a basic culture medium for 24-72 hours to obtain a culture medium; collecting the supernatant of the culture medium after centrifugation; and filtering the supernatant to obtain the secretion, wherein the supernatant is filtered through a filter with a pore size of 0.22μm to less than 0.5μm. The use as described in claim 1, wherein the drug is suitable for topical administration to an eye of a subject. The use as described in claim 2, wherein the drug is in the form of eye gel, eye drops, eye wash, eye drops, eye inserts, eye ointment or eye spray. A use of a secretion prepared by the method described in claim 1 in the preparation of a drug for corneal epithelial wound healing. The use as described in claim 4, wherein the drug is suitable for topical administration to an eye of a subject. The use as described in claim 5, wherein the drug is in the form of eye gel, eye drops, eye wash, eye drops, eye inserts, eye ointment or eye spray. A pharmaceutical composition for treating dry eye or healing corneal epithelial wounds, comprising secretions of amniotic fluid stem cells and a pharmaceutically acceptable carrier; wherein the secretions are prepared by a method comprising the following steps: culturing amniotic fluid stem cells in a basic culture medium for 24-72 hours to obtain a culture medium; collecting the supernatant of the culture medium after centrifugation; and filtering the supernatant to obtain the secretions, wherein the supernatant is filtered through a filter with a pore size of 0.22 μm to less than 0.5 μm. A pharmaceutical composition as described in claim 7, which is suitable for topical administration to the eye of a subject. The pharmaceutical composition as described in claim 8, which is in the form of eye gel, eye drops, eye wash, eye drops, eye inserts, eye ointment or eye spray.