Application of EPO in preparation of medicine for preventing and treating corneal injury

Abstract

The invention discloses application of EPO (erythropoietin) in preparation of a medicine for preventing and treating corneal injury, belongs to the technical field of medicines, and focuses on innovative application of EPO in preparation of the medicine for preventing and treating corneal injury. The cornea is used as a key structure of eyes and is easily damaged by various factors such as mechanical trauma, chemical injury, infection and diabetes, and more than ten millions of people in the world are troubled by cornea blindness. The invention finds that EPO has a remarkable treatment effect on corneal injury, and can promote proliferation and migration of corneal epithelium and stromal cells, increase the generation of extracellular matrix and enhance the tolerance of corneal epithelium cells and corneal stromal cells to high-level sugar. The medicine can be a self-freeze-drying preparation or a solution preparation, the administration modes are diversified, and various eye local injection, eye drop, gene therapy and the like are covered. Refined treatment schemes are provided for corneal injuries of different pathogenesis, a new way is developed for treatment of the corneal injuries, the corneal blindness rate is expected to be reduced, and the development of ophthalmic medicine is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and specifically relates to an application of EPO in the preparation of a medicine for preventing and treating corneal damage. Background Art

[0002] As a key part of the front structure of the eye, the cornea plays a vital role in the process of vision formation. It is not only an important barrier to the eyeball, effectively blocking the invasion of foreign matter, but also undertakes the vital refractive function, ensuring that light can be accurately focused on the retina, thus laying the foundation for the formation of clear vision. However, because the cornea is at the front of the eye, it is directly exposed to the external environment and lacks sufficient protective barriers. Compared with other parts of the eyeball, it is extremely vulnerable to various factors. In addition, the cornea of ​​diabetic patients in the middle and late stages is often prone to high sugar-induced corneal stromal deposition and slow corneal repair, resulting in frequent corneal damage. According to statistics, more than 10 million people worldwide suffer from corneal blindness, and corneal damage has become an ophthalmic problem that needs to be solved urgently.

[0003] It is currently known that corneal damage refers to a condition in which the tissue structure of the cornea is damaged or its normal function is negatively affected. In this context, studies have found that EPO has the potential to treat corneal damage. It may help the repair process of corneal damage by accelerating the proliferation and migration of corneal epithelial cells, improving the tolerance of cells to high sugar, and promoting the production of extracellular matrix, thus opening up a new direction for the treatment of corneal damage. Summary of the invention

[0004] The purpose of the present invention is to provide an application of EPO in the preparation of a drug for preventing and treating corneal damage, so as to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: an application of EPO in the preparation of a drug for preventing and treating corneal damage, comprising: Corneal damage includes situations where the corneal tissue structure is destroyed or its function is affected due to mechanical trauma, chemical damage, infectious factors, and diabetes; EPO is used to promote the proliferation and migration of corneal epithelial cells and corneal stromal cells, increase the production of extracellular matrix, and thus improve corneal damage; When the drug is a lyophilized preparation, the preparation method thereof comprises the steps of pre-freezing a solution containing EPO, sublimation drying, and analytical drying, and finally obtaining a stable lyophilized product; When the drug is a solution preparation, the solvent of the solution includes but is not limited to physiological saline, phosphate buffer, sodium hyaluronate solution; If the drug is administered as gene therapy, the gene encoding EPO is introduced into the eye cells through a vector, and the vector includes a viral vector and a non-viral vector, wherein the viral vector includes one or more of adenoviral vectors, adeno-associated viral vectors, and lentiviral vectors, and the non-viral vector includes one or more of liposomes and polymer nanoparticles.

[0006] Preferably, the corneal damage caused by mechanical trauma includes corneal abrasions, cuts, and contusions, and when the EPO is used to treat corneal abrasions, the concentration of EPO in the eye drop solution is 500U / mL to 1000U / mL, and the eye drop frequency is 6 to 10 times a day. It can significantly promote the repair of corneal epithelial cells within 48 to 72 hours after the trauma occurs, and reduce the area of ​​corneal epithelial defects to less than 30% of the initial area.

[0007] Preferably, the chemical damage includes acid and alkali burns to the cornea. For the case of alkali burns to the cornea, when subconjunctival injection is used, the drug injection dose is 200U to 500U each time, and the injection interval is 24 hours. It can inhibit corneal inflammatory response, reduce the number of inflammatory cell infiltration in the corneal stroma to less than 50% of the normal level, and promote corneal stromal cell proliferation. The arrangement of corneal stromal collagen fibers tends to be regular 7 to 14 days after treatment.

[0008] Preferably, the corneal damage caused by the infectious factor is caused by bacterial, viral or fungal infection. In the treatment of corneal damage caused by viral infection, if local eye drops are chosen, the concentration of EPO in the eye drop solution is 300U / mL to 800U / mL, and the frequency of eye drops is 8 to 12 times a day, which can effectively enhance the antiviral ability of the corneal epithelium and reduce the risk of corneal ulcer formation.

[0009] Preferably, for diabetic corneal damage, sodium hyaluronate and an antioxidant are further added to the solution preparation of the drug, and the amount of sodium hyaluronate added is 0.1%-0.5% of the total mass of the solution; the antioxidant is selected from one or more of vitamin C, vitamin E, and glutathione, and its amount added is 0.5%-2% of the total mass of the solution. When treating diabetic corneal damage, the solution preparation can be treated by eye drops at a concentration of 600U / mL to 1200U / mL and an eye drop frequency of 5 to 9 times a day to alleviate oxidative stress damage to corneal cells in a high-sugar environment. After continuous treatment for 14 to 28 days, the survival rate of corneal epithelial cells is increased to more than 70%, the migration ability of corneal cells is enhanced, and the corneal damage repair rate is increased by 30% compared with when no antioxidant is added.

[0010] Preferably, when the drug is a lyophilized preparation, the pre-freezing temperature range is -40°C to -50°C, and the rate is 1°C / min to 3°C / min; the sublimation drying temperature range is -20°C to -30°C, and the vacuum degree is 10Pa to 50Pa; the analytical drying temperature range is 0°C to 20°C, and the vacuum degree is 1Pa to 10Pa. The lyophilized preparation prepared in this way has a storage stability of 1 to 2 years at room temperature, and the activity retention rate of EPO after reconstitution is above 80%.

[0011] Preferably, if the drug is administered as gene therapy, the gene sequence encoding EPO is codon-optimized to increase its expression efficiency in ocular cells by 30%, and the transfection efficiency of the vector introduced into ocular cells reaches more than 50%. Within 3 to 7 days after introduction, the expression level of EPO in ocular cells is sufficient to promote the proliferation of corneal epithelial cells, increasing the proliferation rate by 25% compared to when the gene sequence is not optimized.

[0012] Compared with the prior art, the present invention provides an application of EPO in the preparation of a drug for preventing and treating corneal damage, which has the following beneficial effects: Through a large number of experimental verifications, EPO can effectively improve the problem of sugar-induced weakened proliferation and migration ability of corneal stroma and corneal epithelial cells. In cell experiments, EPO showed significant effects in promoting cell viability, proliferation and migration for HCSC (corneal stromal cells) and HCEC (corneal epithelial cells) after normal culture and high sugar treatment. For example, after specific treatment of HCSC and HCEC cells in good condition, different concentrations of EPO were added for culture, and cell viability was detected by CCK8 reagent, cell proliferation and migration were analyzed by Edu staining and image J software, and RNAseq sequencing technology was used to analyze the RNA expression of cells. The results showed that EPO can significantly enhance the relevant physiological activity of cells.

[0013] In the animal experiment, a corneal injury mouse model was constructed. After mechanical wound treatment on the mouse cornea, 1500U / mL EPO eye drops were given once every 8 hours. After 48 hours of treatment, the corneal injury was labeled with sodium fluorescein and observed under a slit lamp. It was found that the corneal injury in the EPO treatment group was significantly improved, which strongly proved the effectiveness of EPO in treating corneal injury in vivo.

[0014] In summary, EPO is expected to become a new and effective drug for the treatment of corneal damage and related eye diseases, providing more options for clinical ophthalmic treatment, alleviating patients' pain and reducing the risk of corneal blindness. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The effect of different concentrations of EPO on the cell viability of HCSC and HCEC was demonstrated. The experimental procedure was as follows: HCSC and HCEC cells with a density of about 80% - 90% in good condition were digested with 0.25% trypsin, and 1×10 4 Cells were plated in a 96-well plate. After the cells adhered to the wall, they were starved for 12 hours with DMEM / F12 containing 1% FBS. After that, different concentrations of EPO were added and cultured for 24 hours. CCK8 reagent was diluted with DMEM / F12 to prepare the detection working solution (10 microliters of CCK8 reagent was added to every 100 microliters of culture medium). The culture medium was removed and the detection working solution was added. After incubation in the incubator for 2 hours, the absorbance at 450nm was measured using an ELISA reader. The absorbance data intuitively reflected the regulatory effect of different concentrations of EPO on cell viability.

[0016] Figure 2 The effect of EPO on the proliferation of HCSC and HCEC treated with high glucose was presented. Specific operation: HCSC and HCEC cells with a density of about 80% - 90% in good condition were digested with 0.25% trypsin, and 2×10 5 10 cells were plated in a 12-well plate. After the cells adhered, they were starved for 12 hours with DMEM / F12 containing 1% FBS, and then 800UI of EPO or 50mM glucose was added to continue incubation for 24 hours. Edu was diluted with complete medium to a final concentration of about 10μM and added to the well plate for further incubation for 4 hours. The cell culture medium was removed and 4% paraformaldehyde was added to fix the cells at room temperature for about 15 minutes. The fixative was removed and the cells were washed with PBS 3 times, 5 minutes each time. Permeabilization solution (0.3% Triton X-100 in PBS) was added and incubated at room temperature for 10 minutes. The permeabilization solution was removed and the cells were washed with PBS 1-2 times, 5 minutes each time, and the Edu reaction solution was added and incubated at room temperature for 30 minutes in the dark. The reaction solution was removed and the cells were washed with PBS 3 times, 5 minutes each time. After staining the nucleus with DAPI for 5 minutes, the cells were washed with PBS 3 times, 5 minutes each time. Pictures were taken under a fluorescence microscope and then calculated using the formula: proliferation capacity = number of cells stained with Edu / number of cells stained with DAPI, in order to quantify the data to clarify the promoting effect of EPO on cell proliferation after high glucose treatment.

[0017] Figure 3The experimental steps are as follows: HCSC and HCEC cells with a density of about 80% - 90% in good condition were digested with 0.25% trypsin, and 2×10 5 12 cells were plated in a 12-well plate. After the cells adhered to the wall, they were starved for 12 hours with DMEM / F12 containing 1% FBS. When the cell confluence reached about 70% - 80%, a suitable sterile pipette tip was selected, the pipette tip was placed perpendicular to the 12-well plate, and the pipette tip was gently pressed with a pipette to draw a straight line smoothly on the cell layer. After washing with PBS, 800UI of EPO or 50mM glucose was dissolved in 1% FBS DMEM / F12. After taking pictures with a microscope, the cells were placed in an incubator for further culture for 24 hours. Then the microscope was used to take pictures again. Finally, the scratch area was counted by using image J software. The migration rate of the cells was calculated by the formula: migration rate = (initial scratch area - scratch area after 24 hours) / initial scratch area × 100%, and the improvement effect of EPO on cell migration ability was shown with intuitive migration rate data.

[0018] Figure 4 To reflect the therapeutic effect of EPO in the corneal injury mouse model, the corneal injury was labeled with sodium fluorescein and the results were collected under the slit lamp. Specifically, after the mouse cornea was mechanically wounded, 1500U / mL of EPO eye drops were given once every 8 hours. After 48 hours of treatment, the corneal injury in the EPO treatment group was significantly improved. The image contrast under the slit lamp clearly demonstrated the therapeutic advantages of EPO, providing a strong in vivo experimental basis for its clinical application.

[0019] Figure 5 This reflects that EPO enhances the tolerance of human corneal epithelial cells to high glucose levels. Specifically, the distribution of corneal epithelial cells was treated with EPO, high glucose, and high glucose combined with EPO. Cluster analysis in Figure A showed that the RNA expression profile of the high glucose group treated with EPO was similar to that of the control group and the EPO treatment group. Figure B showed that EPO treatment significantly increased the gene level of the cell's oxidative phosphorylation process, and Figure C showed that EPO treatment reduced the cell's gene function. The cell biological process analysis proved that EPO enhanced the corneal epithelial cell's ability to handle sugar and protected the corneal epithelium from sugar damage.

[0020] Figure 6This reflects that EPO enhances the tolerance of human corneal stromal cells to high glucose levels. Specifically, the distribution of corneal stromal cells was treated with EPO, high glucose, and high glucose combined with EPO. Cluster analysis in Figure A showed that the RNA expression profile of the high glucose group treated with EPO was similar to that of the control group and the EPO treatment group. Figures B and C show the effect of EPO treatment on the corneal stromal cell process, proving that EPO enhances the tolerance of human corneal stromal cells to sugar and protects sugar from damage to the corneal stroma. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The present invention provides Figure 1-6 shown A use of EPO in the preparation of a drug for preventing and treating corneal damage, comprising: Corneal damage includes situations where the corneal tissue structure is destroyed or its function is affected due to mechanical trauma, chemical damage, infectious factors, and diabetes; EPO is used to promote the proliferation and migration of corneal epithelial cells and corneal stromal cells, increase the production of extracellular matrix, and thus improve corneal damage; When the drug is a lyophilized preparation, the preparation method thereof comprises the steps of pre-freezing a solution containing EPO, sublimation drying, and analytical drying, and finally obtaining a stable lyophilized product; When the drug is a solution preparation, the solvent of the solution includes but is not limited to physiological saline and phosphate buffer; If the drug is administered as gene therapy, the gene encoding EPO is introduced into the eye cells through a vector, and the vector includes a viral vector and a non-viral vector, wherein the viral vector includes one or more of adenoviral vectors, adeno-associated viral vectors, and lentiviral vectors, and the non-viral vector includes one or more of liposomes and polymer nanoparticles.

[0023] The corneal damage caused by mechanical trauma includes corneal abrasions, cuts, and contusions. When the EPO is used to treat corneal abrasions, the concentration of EPO in the eye drop solution is 500U / mL to 1000U / mL, and the frequency of eye drops is 6 to 10 times a day. It can significantly promote the repair of corneal epithelial cells within 48 to 72 hours after the trauma occurs, and reduce the area of ​​corneal epithelial defects to less than 30% of the initial area.

[0024] The chemical damage includes acid and alkali burns to the cornea. For the case of alkali burns to the cornea, when the drug is injected subconjunctivally, the injection dose is 200U to 500U each time, and the injection interval is 24 hours. It can inhibit the corneal inflammatory response and reduce the number of inflammatory cell infiltration in the corneal stroma to less than 50% of the normal level. At the same time, it promotes the proliferation of corneal stromal cells. The arrangement of corneal stromal collagen fibers tends to be regular 7 to 14 days after treatment.

[0025] The infectious factors causing corneal damage are caused by bacterial, viral or fungal infections. In the treatment of corneal damage caused by viral infections, if intravenous injection is chosen, the injection dose is 100U / kg to 300U / kg body weight each time, combined with local eye drops treatment, the concentration of EPO in the eye drops solution is 300U / mL to 800U / mL, and the frequency of eye drops is 8 to 12 times a day, which can effectively enhance the antiviral ability of the corneal epithelium and reduce the risk of corneal ulcer formation.

[0026] For diabetic corneal damage, the solution preparation of the drug is also added with antioxidants and sodium hyaluronate, and the amount of sodium hyaluronate added is 0.1%-0.5% of the total mass of the solution; the antioxidant is selected from one or more of vitamin C, vitamin E, and glutathione, and its amount of addition is 0.5%-2% of the total mass of the solution. When treating diabetic corneal damage, the solution preparation can be treated by eye drops, with a concentration of 600U / mL to 1200U / mL and an eye drop frequency of 5 to 9 times a day, to relieve oxidative stress damage of corneal cells in a high-sugar environment. After continuous treatment for 14 to 28 days, the survival rate of corneal epithelial cells is increased to more than 70%, the migration ability of corneal cells is enhanced, and the corneal damage repair rate is increased by 30% compared with when no antioxidants are added.

[0027] When the drug is a lyophilized preparation, the pre-freezing temperature range is -40°C to -50°C, and the rate is 1°C / min to 3°C / min; the sublimation drying temperature range is -20°C to -30°C, and the vacuum degree is 10Pa to 50Pa; the analytical drying temperature range is 0°C to 20°C, and the vacuum degree is 1Pa to 10Pa. The lyophilized preparation prepared in this way has a storage stability of 1 to 2 years at room temperature, and the activity retention rate of EPO after reconstitution is above 80%.

[0028] If the drug is administered as gene therapy, the gene sequence encoding EPO is codon-optimized to increase its expression efficiency in ocular cells by 30%, and the transfection efficiency of the vector introduced into ocular cells is more than 50%. Within 3 to 7 days after introduction, the expression level of EPO in ocular cells is sufficient to promote the proliferation of corneal epithelial cells, increasing the proliferation rate by 25% compared to when the gene sequence is not optimized.

[0029] Embodiment 1: Verify the effect of EPO on the cell viability of normally cultured HCSC and HCEC; Cell preparation: HCSC and HCEC cells with a density of about 80%-90% were selected and digested with 0.25% trypsin. Then 1×10 4 The cells were evenly plated in a 96-well plate to ensure uniform cell distribution and provide a reliable cell sample basis for subsequent experiments.

[0030] Pretreatment: After the cells adhered to the wall, the cells were starved for 12 hours using DMEM / F12 medium containing 1% FBS to keep the cells in a relatively synchronous growth state and reduce the interference of different cell growth cycles on the experimental results.

[0031] Drug treatment: Add different concentration gradients of EPO to the well plate, continue culturing for 24 hours, and set up multiple concentration groups to fully explore the dose-effect relationship between EPO concentration and cell viability.

[0032] Detection: After the culture is completed, dilute the CCK8 reagent with DMEM / F12 according to the established process to prepare the detection working solution, carefully remove the culture medium, add the detection working solution, place the well plate in the incubator for 2 hours, and finally use an enzyme reader to measure the absorbance at 450nm. Through the statistics and analysis of the absorbance data of different concentration groups, the EPO concentration and cell viability change curve is accurately drawn to clarify the influence of EPO on the viability of normal cultured cells.

[0033] Embodiment 2: To explore the effect of EPO on the proliferation capacity of HCSC and HCEC after high glucose treatment; Cell plating: HCSC and HCEC cells with a density of about 80%-90% were selected in good condition and digested with 0.25% trypsin. 2×10 5 The cells were plated in a 12-well plate to ensure that the cells had enough growth space for subsequent proliferation experiments.

[0034] Starvation and treatment: After the cells adhered, they were starved with DMEM / F12 containing 1% FBS for 12 hours, and then 800UI of EPO or 50mM glucose were added for further culture for 24 hours. A control group (only glucose) and an experimental group (EPO and glucose) were set up to compare and analyze the intervention effect of EPO on cell proliferation in a high-sugar environment.

[0035] Edu staining: Edu was diluted with complete culture medium to an appropriate final concentration (about 10 μM) and then added to the well plate and incubated for 4 hours to allow Edu to be effectively incorporated into the DNA of proliferating cells as a marker of cell proliferation.

[0036] Subsequent processing: Strictly follow the standard procedures for cell fixation, washing, permeabilization, Edu reaction solution incubation, DAPI nuclear staining, and multiple PBS washing operations to ensure clear and accurate cell staining and reduce nonspecific staining interference.

[0037] Result calculation: High-quality photos were taken under a fluorescence microscope. According to the established formula: proliferation capacity = number of cells stained with Edu / number of cells stained with DAPI, the cells in the photos were accurately counted and calculated to quantitatively demonstrate the effect of EPO on improving cell proliferation capacity after high glucose treatment.

[0038] Embodiment 3: To study the effect of EPO on the migration ability of HCSC and HCEC after high glucose treatment; Preliminary preparation: Select HCSC and HCEC cells with a density of about 80%-90% in good condition, digest them with 0.25% trypsin and take 2×10 5 The cells were plated in a 12-well plate and starved for 12 hours with DMEM / F12 containing 1% FBS after the cells adhered to the plate to keep the cells in a stable growth starting state.

[0039] Scratch operation: When the cell confluence reaches about 70%-80%, select a sterile pipette tip of appropriate specifications that has been sterilized, place the pipette tip perpendicular to the 12-well plate, and use a pipette to gently press the pipette tip to smoothly draw a straight line of uniform width on the cell layer to simulate the initial traumatic environment of cell migration. Then use PBS to wash and remove the scratched cell fragments to avoid interference with subsequent experiments.

[0040] Drug addition and culture: Add 800UI of EPO or 50mM glucose dissolved in 1% FBS DMEM / F12, take a photo of the initial scratch using a microscope as a control, and then place the well plate in the incubator for a further 24 hours to ensure that the cells have enough time to migrate.

[0041] Result statistics: After the culture was completed, photos were taken again using a microscope, and the scratch area was accurately counted using professional image J software. The migration rate of the cells was calculated using the formula: migration rate = (initial scratch area - scratch area after 4 hours) / initial scratch area × 100%. The intuitive comparison of migration rate data clearly showed the promoting effect of EPO on the migration ability of cells after high sugar treatment.

[0042] Embodiment 4: Verify that EPO can treat corneal injuries through eye drops and promote the healing process of corneal epithelium; Establishment of animal model: Healthy mice were selected and professional instruments were used to perform precise mechanical wound treatment on the mouse cornea to ensure that the wound size and depth were relatively consistent, and a standardized corneal injury mouse model was constructed to provide a reliable animal model basis for the subsequent evaluation of drug treatment effects.

[0043] Drug administration: 1500U / mL EPO eye drops were administered to mice with corneal injury once every 8 hours, strictly following the set dosing time interval and dosage to ensure the stability and continuity of the drug effect.

[0044] Effect evaluation: After 48 hours of treatment, the corneal damage was marked by dripping sodium fluorescein into the mouse eyes, and then the corneal damage was carefully observed and images were collected under a slit lamp. The sodium fluorescein staining in the corneal damage area and the corneal morphology changes in the treated and untreated groups were compared, and the improvement effect of EPO eye drops on corneal damage was evaluated intuitively and quantitatively, providing a strong in vivo experimental basis for its clinical eye drop treatment of corneal damage.

[0045] Embodiment 5: Verify the mechanism by which EPO promotes corneal repair in diabetic patients by improving the glucose tolerance of human corneal epithelial cells and human corneal stromal cells; Cell treatment: Human corneal epithelial cells and human corneal stromal cells were starved with DMEM / F12 containing 1% FBS for 12 hours, and then 800UI of EPO or 50mM glucose were added for further culture for 24 hours. A control group (normal culture medium), a high glucose group (with 50mM glucose), an EPO group (with 800UI of EPO), and an experimental group (with EPO (800UI) and 50mM glucose) were set up to compare and analyze the effect of EPO on the cellular RNA expression level under high glucose environment.

[0046] Data analysis: RNA from each group of cells after treatment was collected and subjected to high-throughput RNA sequencing. The sequencing results were subjected to cluster analysis and differential gene biological cluster analysis.

[0047] Result evaluation: EPO enhances the tolerance of human corneal epithelial cells and human corneal stromal cells to high glucose levels, improves the effect of high glucose on the expression of RNA by corneal epithelial cells and corneal stromal cells, and reveals the mechanism of using EPO to treat diabetic corneal damage.

[0048] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A use of EPO in the preparation of a drug for preventing and treating corneal damage, characterized in that: include: Corneal damage includes situations where the corneal tissue structure is destroyed or its function is affected due to mechanical trauma, chemical damage, infectious factors, and diabetes; EPO is used to promote the proliferation and migration of corneal epithelial cells and corneal stromal cells, increase the production of extracellular matrix, and thus improve corneal damage; When the drug is a lyophilized preparation, the preparation method thereof comprises the steps of pre-freezing a solution containing EPO, sublimation drying, and analytical drying, and finally obtaining a stable lyophilized product; When the drug is a solution preparation, the solvent of the solution includes but is not limited to physiological saline, phosphate buffer, sodium hyaluronate solution; If the drug is administered as gene therapy, the gene encoding EPO is introduced into the eye cells through a vector, and the vector includes a viral vector and a non-viral vector, wherein the viral vector includes one or more of adenoviral vectors, adeno-associated viral vectors, and lentiviral vectors, and the non-viral vector includes one or more of liposomes and polymer nanoparticles.

2. The use of EPO in the preparation of a drug for preventing and treating corneal damage according to claim 1, characterized in that: The corneal damage caused by mechanical trauma includes corneal abrasions, cuts, and contusions. When the EPO is used to treat corneal abrasions, the concentration of EPO in the eye drop solution is 500U / mL to 1000U / mL, and the frequency of eye drops is 3 to 10 times a day. It can significantly promote the repair of corneal epithelial cells within 48 to 72 hours after the trauma occurs, and reduce the area of ​​corneal epithelial defects to less than 30% of the initial area.

3. The use of EPO in the preparation of a drug for preventing and treating corneal damage according to claim 1, characterized in that: The chemical damage includes acid and alkali burns to the cornea. For the case of alkali burns to the cornea, when the drug is injected subconjunctivally, the injection dose is 200U to 500U each time, and the injection interval is 24 hours. It can inhibit the corneal inflammatory response and reduce the number of inflammatory cell infiltration in the corneal stroma to less than 50% of the normal level. At the same time, it promotes the proliferation of corneal stromal cells. The arrangement of corneal stromal collagen fibers tends to be regular 7 to 14 days after treatment.

4. The use of EPO in the preparation of a drug for preventing and treating corneal damage according to claim 1, characterized in that: The infectious factors that cause corneal damage are caused by bacterial, viral or fungal infections. In the treatment of corneal damage caused by viral infections, if local eye drops are chosen, the concentration of EPO in the eye drops solution is 300U / mL to 800U / mL, and the frequency of eye drops is 8 to 12 times a day, which can effectively enhance the antiviral ability of the corneal epithelium and reduce the risk of corneal ulcer formation.

5. The use of EPO in the preparation of a drug for preventing and treating corneal damage according to claim 1, characterized in that: For diabetic corneal damage, sodium hyaluronate and antioxidants are also added to the solution preparation of the drug, and the amount of sodium hyaluronate added is 0.1%-0.5% of the total mass of the solution; the antioxidant is selected from one or more of vitamin C, vitamin E, and glutathione, and its amount is 0.5%-2% of the total mass of the solution. When treating diabetic corneal damage, the solution preparation can be treated by eye drops, with a concentration of 600U / mL to 1200U / mL and an eye drop frequency of 5 to 9 times a day, to relieve oxidative stress damage to corneal cells in a high-sugar environment. After continuous treatment for 14 to 28 days, the survival rate of corneal epithelial cells is increased to more than 70%, the migration ability of corneal cells is enhanced, and the corneal damage repair rate is increased by 30% compared to when no antioxidants are added.

6. The use of EPO in the preparation of a drug for preventing and treating corneal damage according to claim 1, characterized in that: When the drug is a lyophilized preparation, the pre-freezing temperature range is -40°C to -50°C, and the rate is 1°C / min to 3°C / min; the sublimation drying temperature range is -20°C to -30°C, and the vacuum degree is 10Pa to 50Pa; the analytical drying temperature range is 0°C to 20°C, and the vacuum degree is 1Pa to 10Pa. The lyophilized preparation prepared in this way has a storage stability of 1 to 2 years at room temperature, and the activity retention rate of EPO after reconstitution is above 80%.

7. The use of EPO in the preparation of a drug for preventing and treating corneal damage according to claim 1, characterized in that: If the drug is administered as gene therapy, the gene sequence encoding EPO is codon-optimized to increase its expression efficiency in ocular cells by 30%, and the transfection efficiency of the vector introduced into ocular cells reaches more than 50%. Within 3 to 7 days after introduction, the expression level of EPO in ocular cells is sufficient to promote the proliferation of corneal epithelial cells, increasing the proliferation rate by 25% compared to when the gene sequence is not optimized.

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