Use of EPO in preparation of drug for preventing and treating corneal injury
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-01-19
- Publication Date
- 2026-08-13
Smart Images
Figure CN2026073314_13082026_PF_FP_ABST
Abstract
Description
Application of EPO in the preparation of drugs for the prevention and treatment of corneal damage Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of EPO in the preparation of drugs for the prevention and treatment of corneal damage (A). Background Technology
[0002] The cornea, a crucial component of the anterior structure of the eye, plays a vital role in the formation of vision. It is not only an important barrier for the eyeball, effectively blocking the intrusion of foreign objects, but also performs essential refractive functions, ensuring that light is precisely focused on the retina, thus laying the foundation for clear vision. However, because the cornea is located at the very front of the eye, it is directly exposed to the external environment and lacks sufficient protective barriers, making it more vulnerable to damage from various factors compared to other parts of the eyeball. Furthermore, diabetic patients in the middle and late stages are prone to hyperglycemic-induced corneal stromal deposition and slow corneal repair, leading to frequent corneal damage. It is estimated that more than 10 million people worldwide suffer from corneal blindness, making corneal damage a pressing ophthalmic challenge that needs to be addressed.
[0003] Currently, corneal injury is known to refer to a condition in which the structure of the cornea is damaged or its normal function is negatively affected. Against this backdrop, research has found that EPO possesses the potential to treat corneal injury. It may assist in the repair process of corneal injury by accelerating the proliferation and migration of corneal epithelial cells, improving cellular tolerance to high glucose, and promoting the production of extracellular matrix, thus opening up new avenues for the treatment of corneal injury. Summary of the Invention
[0004] The purpose of this invention is to provide an application of EPO in the preparation of drugs for preventing and treating corneal damage, so as to solve the problems mentioned in the background art.
[0005] To achieve the above objectives, the present invention provides the following technical solution: the application of EPO in the preparation of a drug for preventing and treating corneal damage, comprising:
[0006] Corneal injury includes damage to the corneal tissue structure or impairment of function caused by mechanical trauma, chemical damage, infectious factors, or diabetes.
[0007] 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;
[0008] When the drug is a self-lyophilized formulation, its preparation method includes the steps of pre-freezing a solution containing EPO, sublimation drying, and desorption drying, and finally obtaining a stable lyophilized product.
[0009] When the drug is a solution preparation, the solvent of the solution includes, but is not limited to, physiological saline, phosphate buffer, and sodium hyaluronate solution;
[0010] If the drug is administered via gene therapy, the gene encoding EPO is introduced into eye cells through a vector. The vector can be a viral vector or a non-viral vector. The viral vector can be one or more of adenovirus vectors, adeno-associated virus vectors, and lentivirus vectors, while the non-viral vector can be one or more of liposomes and polymer nanoparticles.
[0011] Preferably, the corneal injury caused by mechanical trauma includes corneal abrasions, cuts, and contusions, and when treating corneal abrasions, the EPO is administered via eye drops at a concentration of 500 U / mL to 1000 U / mL, with an eye drop frequency of 6 to 10 times per day. This significantly promotes the repair of corneal epithelial cells within 48 to 72 hours after the trauma, reducing the corneal epithelial defect area to less than 30% of the initial area.
[0012] Preferably, the chemical damage includes corneal burns caused by acids or alkalis. In cases of corneal burns caused by alkalis, when using subconjunctival injection, the drug injection dose is 200U to 500U per injection, with an injection interval of 24 hours. This can inhibit corneal inflammation, reduce the number of inflammatory cells infiltrating the corneal stroma to less than 50% of the normal level, and promote the proliferation of corneal stromal cells. Within 7 to 14 days after treatment, the arrangement of corneal stromal collagen fibers tends to become more regular.
[0013] Preferably, the corneal damage caused by the infectious factors is caused by bacterial, viral or fungal infection. In the treatment of corneal damage caused by viral infection, if topical eye drops are chosen, the concentration of EPO in the eye drop solution is 300 U / mL to 800 U / 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.
[0014] Preferably, for diabetic corneal injury, the solution formulation of the drug further includes sodium hyaluronate and an antioxidant. The amount of sodium hyaluronate added accounts for 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 accounts for 0.5%-2% of the total mass of the solution. When treating diabetic corneal injury, this solution formulation can be administered by eye drops at a concentration of 600U / mL to 1200U / mL, with an eye drop frequency of 5 to 9 times per day. This alleviates oxidative stress damage to corneal cells under high glucose conditions. After continuous treatment for 14 to 28 days, it increases the survival rate of corneal epithelial cells to over 70%, enhances the migration ability of corneal cells, and increases the corneal injury repair rate by 30% compared to when no antioxidant is added.
[0015] Preferably, when the drug is a self-lyophilized formulation, 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 desorption drying temperature range is 0°C to 20°C, and the vacuum degree is 1Pa to 10Pa. The lyophilized formulation prepared in this way can achieve a storage stability of 1 to 2 years at room temperature, and the activity retention rate of EPO after reconstitution is more than 80%.
[0016] Preferably, if the drug is administered via 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 corneal epithelial cell proliferation, increasing the proliferation rate by 25% compared to when the gene sequence is not optimized.
[0017] Compared with the prior art, the present invention provides an application of EPO in the preparation of drugs for preventing and treating corneal damage, which has the following beneficial effects:
[0018] Extensive experimental verification has shown that EPO can effectively improve the reduced proliferation and migration capabilities of corneal stromal and corneal epithelial cells induced by glucose. In cell experiments, EPO significantly promoted cell viability, proliferation, and migration in both normally cultured HCSCs (corneal stromal cells) and HCECs (corneal epithelial cells) after high glucose treatment. For example, after specific treatments, healthy HCSCs and HCECs were cultured with different concentrations of EPO. Cell viability was detected using CCK8 reagent, cell proliferation and migration were analyzed using Edu staining and ImageJ software, and RNA expression was analyzed using RNA-seq technology. The results showed that EPO significantly enhanced the relevant physiological activities of the cells.
[0019] In animal experiments, a mouse model of corneal injury was established. After mechanical wound treatment of the mouse cornea, EPO eye drops of 1500 U / mL were administered every 8 hours. After 48 hours of treatment, corneal injury was marked with sodium fluorescein and observed under a slit lamp. It was found that corneal injury in the EPO treatment group was significantly improved, which strongly demonstrated the efficacy of EPO in treating corneal injury in vivo.
[0020] In conclusion, EPO holds promise as a novel and effective drug for treating corneal damage and related eye diseases, providing more options for clinical ophthalmology treatment, alleviating patient suffering, and reducing the risk of corneal blindness. Attached Figure Description
[0021] Figure 1 shows the effect of different concentrations of EPO on the cell viability of HCSCs and HCECs. The experimental procedure is as follows: HCSCs and HCECs in good condition with a density of approximately 80%-90% were digested with 0.25% trypsin, and 1×10⁴ cells were seeded into 96-well plates. After cell attachment, the cells were starved for 12 hours in DMEM / F12 containing 1% FBS. Then, different concentrations of EPO were added and the cells were cultured for another 24 hours. The CCK8 reagent was diluted with DMEM / F12 to prepare the detection working solution (10 μL of CCK8 reagent per 100 μL of culture medium). The culture medium was removed, and the detection working solution was added. After incubation in an incubator for 2 hours, the absorbance at 450 nm was measured using a microplate reader. The absorbance data visually reflects the regulatory effect of different concentrations of EPO on cell viability.
[0022] Figure 2 shows the effect of EPO on the proliferation of HCSCs and HCECs after high glucose treatment. Specific procedures: HCSCs and HCECs at a healthy density of approximately 80%-90% were digested with 0.25% trypsin, and 2 × 10⁵ cells were seeded into 12-well plates. After cell attachment, the cells were starved for 12 hours in DMEM / F12 containing 1% FBS. Then, 800 UI of EPO or 50 mM glucose was added, and the cells were cultured for another 24 hours. EPO was diluted with complete culture medium to a final concentration of approximately 10 μM and added to the wells for further incubation for 4 hours. The cell culture medium was then removed, and the cells were fixed with 4% paraformaldehyde at room temperature for approximately 15 minutes. The fixative was removed, and the cells were washed three times with PBS for 5 minutes each time. Permeation buffer (0.3% Triton X-100 in PBS) was added, and the cells were incubated at room temperature for 10 minutes. Remove the permeabilization solution, wash 1-2 times with PBS for 5 minutes each time, and incubate with Edu reaction solution at room temperature in the dark for 30 minutes. Remove the reaction solution, wash 3 times with PBS for 5 minutes each time. Stain nuclei with DAPI for 5 minutes, then wash 3 times with PBS for 5 minutes each time. Photograph under a fluorescence microscope, and then calculate the proliferation capacity using the formula: Proliferation capacity = Number of Edu-stained cells / Number of DAPI-stained cells, to quantify the promoting effect of EPO on cell proliferation after high glucose treatment.
[0023] Figure 3 shows the effect of EPO on the migration ability of HCSCs and HCECs after high glucose treatment. The experimental procedure was as follows: HCSCs and HCECs at a good density of approximately 80%-90% were digested with 0.25% trypsin, and 2 × 10⁵ cells were seeded into 12-well plates. After cell adhesion, the cells were starved for 12 hours in DMEM / F12 containing 1% FBS. When the cell confluence reached approximately 70%-80%, a suitable sterile pipette tip was used. Holding the tip perpendicular to the 12-well plate, the pipette tip was gently pressed to draw a straight line smoothly on the cell layer. After washing with PBS, 800 UI of EPO or 50 mM glucose dissolved in 1% FBS DMEM / F12 was added. After microscopic photography, the cells were incubated for another 24 hours. Microscopic photography was then performed again. Finally, the scratch area was statistically analyzed using ImageJ software. Cell migration rate is calculated using the formula: Migration rate = (Initial scratch area - Scratch area after 24 hours) / Initial scratch area × 100%, providing intuitive migration rate data to demonstrate the effect of EPO on improving cell migration ability.
[0024] Figure 4 illustrates the therapeutic effect of EPO in a mouse model of corneal injury. The results were obtained by labeling corneal lesions with sodium fluorescein and capturing images under a slit lamp. Specifically, after mechanical incision of the mouse cornea, 1500 U / mL of EPO was administered via eye drops every 8 hours. After 48 hours of treatment, corneal injury in the EPO treatment group was significantly improved. The slit-lamp images clearly demonstrate the therapeutic advantages of EPO, providing strong in vivo experimental evidence for its clinical application.
[0025] Figure 5 reflects how EPO enhances the tolerance of human corneal epithelial cells to high glucose levels. Specifically, the distribution of corneal epithelial cells after EPO treatment, high glucose treatment, and combined high glucose and EPO treatment is shown. Cluster analysis in Figure A shows that the RNA expression profile of the high glucose group treated with EPO is similar to that of the control group and the EPO-treated group. Figure B shows that EPO treatment significantly increases the gene levels of oxidative phosphorylation in cells. Figure C shows the cell biological process analysis of gene function reduction caused by EPO treatment, demonstrating that EPO enhances the ability of corneal epithelial cells to process glucose and protects against glucose-induced corneal epithelial damage.
[0026] Figure 6 reflects how EPO enhances the tolerance of human corneal stromal cells to high glucose levels. Specifically, the distribution of corneal stromal cells after EPO treatment, high glucose treatment, and combined high glucose and EPO treatment is shown. Cluster analysis in Figure A shows that the RNA expression profile of the high glucose group treated with EPO is similar to that of the control group and the EPO-treated group. Figures B and C show the effect of EPO treatment on corneal stromal cell processes, demonstrating that EPO enhances the tolerance of human corneal stromal cells to glucose and protects against glucose-induced damage to the corneal stroma. Detailed Implementation
[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0028] The present invention provides the following as shown in Figures 1-6
[0029] The application of EPO in the preparation of drugs for the prevention and treatment of corneal damage includes:
[0030] Corneal injury includes damage to the corneal tissue structure or impairment of function caused by mechanical trauma, chemical damage, infectious factors, or diabetes.
[0031] 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;
[0032] When the drug is a self-lyophilized formulation, its preparation method includes the steps of pre-freezing a solution containing EPO, sublimation drying, and desorption drying, and finally obtaining a stable lyophilized product.
[0033] When the drug is a solution preparation, the solvent of the solution includes, but is not limited to, physiological saline and phosphate buffer.
[0034] If the drug is administered via gene therapy, the gene encoding EPO is introduced into eye cells through a vector. The vector can be a viral vector or a non-viral vector. The viral vector can be one or more of adenovirus vectors, adeno-associated virus vectors, and lentivirus vectors, while the non-viral vector can be one or more of liposomes and polymer nanoparticles.
[0035] The corneal injury caused by mechanical trauma includes corneal abrasions, cuts, and contusions. When treating corneal abrasions, EPO is administered via eye drops at a concentration of 500 U / mL to 1000 U / mL, with an instillation frequency of 6 to 10 times per day. This treatment can significantly promote the repair of corneal epithelial cells within 48 to 72 hours after the trauma, reducing the corneal epithelial defect area to less than 30% of the initial area.
[0036] The chemical damage includes corneal burns caused by acids and alkalis. For corneal burns caused by alkalis, when using subconjunctival injection, the dosage is 200U to 500U per injection, with an interval of 24 hours. This can inhibit corneal inflammation, reduce the number of inflammatory cells infiltrating the corneal stroma to less than 50% of the normal level, and promote the proliferation of corneal stromal cells. Within 7 to 14 days after treatment, the arrangement of corneal stromal collagen fibers tends to become more regular.
[0037] The aforementioned infectious factors causing corneal damage are caused by bacterial, viral, or fungal infections. In the treatment of corneal damage caused by viral infection, if intravenous administration is chosen, the injection dose is 100 U / kg to 300 U / kg body weight per dose, combined with topical eye drops. The concentration of EPO in the eye drop solution is 300 U / mL to 800 U / mL, and the frequency of eye drops is 8 to 12 times per day. This can effectively enhance the antiviral ability of the corneal epithelium and reduce the risk of corneal ulcer formation.
[0038] For diabetic corneal injury, the solution formulation of the drug also contains an antioxidant and sodium hyaluronate, with the sodium hyaluronate accounting for 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 accounts for 0.5%-2% of the total mass of the solution. When treating diabetic corneal injury, this solution formulation can be administered by eye drops at a concentration of 600 U / mL to 1200 U / mL, with an eye drop frequency of 5 to 9 times per day, to alleviate oxidative stress damage to corneal cells under high glucose environment. After continuous treatment for 14 to 28 days, it increases the survival rate of corneal epithelial cells to over 70%, enhances the migration ability of corneal cells, and increases the corneal injury repair rate by 30% compared to when no antioxidant is added.
[0039] When the drug is a self-lyophilized formulation, 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 desorption drying temperature range is 0°C to 20°C, and the vacuum degree is 1Pa to 10Pa. The lyophilized formulation prepared in this way can achieve a storage stability of 1 to 2 years at room temperature, and the activity retention rate of EPO after reconstitution is more than 80%.
[0040] If the drug is administered via 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 corneal epithelial cell proliferation, increasing the proliferation rate by 25% compared to when the gene sequence is not optimized.
[0041] Example 1:
[0042] To verify the effect of EPO on the cell viability of normally cultured HCSCs and HCECs;
[0043] Cell preparation: Select healthy HCSC and HCEC cells with a density of approximately 80%-90%, digest them with 0.25% trypsin, and then take 1×10⁴ cells and evenly seed them in a 96-well plate to ensure uniform cell distribution and provide a reliable cell sample basis for subsequent experiments.
[0044] Pretreatment: After the cells adhered, they were starved for 12 hours in DMEM / F12 medium containing 1% FBS to keep the cells in a relatively synchronized growth state and reduce the interference of different cell growth cycles on the experimental results.
[0045] Drug treatment: EPO at different concentration gradients was added to the well plates and cultured for another 24 hours. Multiple concentration groups were set up to comprehensively explore the dose-response relationship between EPO concentration and cell viability.
[0046] Assay: After culture, the CCK8 reagent was diluted with DMEM / F12 to prepare the working solution according to the established procedure. The culture medium was carefully removed, the working solution was added, and the wells were incubated in an incubator for 2 hours. Finally, the absorbance at 450 nm was measured using a microplate reader. By statistically analyzing the absorbance data of different concentration groups, the curve of EPO concentration versus cell viability was accurately plotted, clarifying the influence of EPO on the viability of normally cultured cells.
[0047] Example 2:
[0048] Investigating the effect of EPO on the proliferation capacity of HCSCs and HCECs after high glucose treatment;
[0049] Cell seeding: Select healthy HCSC and HCEC cells with a density of approximately 80%-90%. After digestion with 0.25% trypsin, seed 2×10⁵ cells into a 12-well plate to ensure sufficient space for cell growth in subsequent proliferation experiments.
[0050] Starvation and treatment: After cell adhesion, cells were starved for 12 hours in DMEM / F12 containing 1% FBS, and then 800 IU of EPO or 50 mM glucose were added for 24 hours. A control group (glucose only) and an experimental group (EPO and glucose) were set up to compare and analyze the intervention effect of EPO on cell proliferation under high glucose environment.
[0051] Edu staining: Dilute Edu to a suitable final concentration (approximately 10 μM) with complete culture medium and add it to the well plate for further incubation for 4 hours, so that Edu can be effectively incorporated into the DNA of proliferating cells and serve as a marker of cell proliferation.
[0052] Subsequent processing: Cell fixation, washing, permeabilization, Edu reaction incubation, DAPI staining of nuclei, and multiple PBS washing were performed strictly according to standard procedures to ensure clear and accurate cell staining results and reduce non-specific staining interference.
[0053] Results Calculation: High-quality images were taken under a fluorescence microscope. Based on the established formula: Proliferation capacity = Number of cells stained with Edu / Number of cells stained with DAPI, the cells in the images were accurately counted and calculated to present the effect of EPO on improving cell proliferation capacity after high glucose treatment in a quantitative and intuitive way.
[0054] Example 3:
[0055] Investigating the effect of EPO on the migration ability of HCSCs and HCECs after high-sugar treatment;
[0056] Preliminary preparation: Select HCSC and HCEC cells in good condition with a density of approximately 80%-90%. After digestion with 0.25% trypsin, take 2×10⁵ cells and seed them in a 12-well plate. After the cells adhere, starve them for 12 hours with DMEM / F12 containing 1% FBS to bring the cells into a stable growth initiation state.
[0057] Scratch procedure: When the cell confluence reaches about 70%-80%, select a sterile pipette tip of appropriate size after sterilization. Hold the pipette tip perpendicular to the 12-well plate and gently press the tip with the pipette to smoothly draw a straight line of uniform width on the cell layer, simulating the initial trauma environment of cell migration. Then wash with PBS to remove the scratched cell debris to avoid interfering with subsequent experiments.
[0058] Drug administration and culture: Add 800 UI of EPO or 50 mM of glucose to 1% FBS DMEM / F12, respectively. Take initial scratch photos under a microscope as a control. Then place the well plate in an incubator and continue to culture for 24 hours to ensure that the cells have enough time to migrate.
[0059] Results statistics: After the culture was completed, the cells were photographed again under a microscope, and the scratch area was accurately counted using professional ImageJ software. The migration rate was calculated using the formula: migration rate = (initial scratch area - scratch area after 4 hours) / initial scratch area × 100%. The intuitive migration rate data comparison clearly shows the promoting effect of EPO on cell migration ability after high glucose treatment.
[0060] Example 4:
[0061] Verify that EPO, when used as an eye drop solution, treats corneal damage and promotes the healing process of the corneal epithelium;
[0062] Animal model establishment: Healthy mice were selected, and the corneas of the mice were precisely mechanically incised using professional instruments to ensure that the size and depth of the wounds were relatively consistent, thus constructing a standardized mouse model of corneal injury, which provides a reliable animal model basis for the subsequent evaluation of drug treatment effects.
[0063] Administration: Mice with corneal damage were given 1500 U / mL of EPO eye drops every 8 hours, strictly following the set dosing intervals and dosages to ensure the stability and continuity of the drug's effect.
[0064] Efficacy evaluation: Forty-eight hours after treatment, corneal damage was marked by intraocular instillation of sodium fluorescein. Subsequently, detailed observation and image acquisition were performed under a slit lamp. The sodium fluorescein staining of the corneal damage area and changes in corneal morphology were compared between the treated and untreated groups. The improvement effect of EPO eye drops on corneal damage was evaluated intuitively and quantitatively, providing strong in vivo experimental evidence for its clinical application in the treatment of corneal damage.
[0065] Example 5:
[0066] To 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;
[0067] Cell treatment: Human corneal epithelial cells and human corneal stromal cells were starved for 12 hours in DMEM / F12 containing 1% FBS, and then cultured for another 24 hours after adding 800 UI of EPO or 50 mM glucose, respectively. A control group (normal culture medium), a high glucose group (added with 50 mM glucose), an EPO group (added with 800 UI of EPO), and an experimental group (added with EPO (800 UI) and 50 mM glucose) were set up to compare and analyze the effect of EPO on the RNA expression level of cells under high glucose environment.
[0068] Data analysis: RNA from each group of cells after processing was collected and high-throughput RNA sequencing was performed. The sequencing results were then subjected to cluster analysis and differential gene biological cluster analysis.
[0069] Results evaluation: EPO enhances the tolerance of human corneal epithelial cells and human corneal stromal cells to high glucose levels and improves the effect of high glucose on RNA expression in corneal epithelial cells and corneal stromal cells, revealing the mechanism of EPO treatment for diabetic corneal damage.
[0070] Finally, it should be noted that the above description 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 foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. The application of EPO in the preparation of a drug for preventing and treating corneal damage, characterized in that, include: Corneal injury includes damage to the corneal tissue structure or impairment of function caused by mechanical trauma, chemical damage, infectious factors, or 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 self-lyophilized formulation, its preparation method includes the steps of pre-freezing a solution containing EPO, sublimation drying, and desorption 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, and sodium hyaluronate solution; If the drug is administered via gene therapy, the gene encoding EPO is introduced into eye cells through a vector. The vector can be a viral vector or a non-viral vector. The viral vector can be one or more of adenovirus vectors, adeno-associated virus vectors, and lentivirus vectors, while the non-viral vector can be one or more of liposomes and polymer nanoparticles.
2. The application of EPO according to claim 1 in the preparation of a drug for preventing and treating corneal damage, characterized in that: The corneal injury caused by mechanical trauma includes corneal abrasions, cuts, and contusions. When treating corneal abrasions, EPO is administered via eye drops at a concentration of 500 U / mL to 1000 U / mL, with an frequency of 3 to 10 times per day. This treatment can significantly promote the repair of corneal epithelial cells within 48 to 72 hours after the trauma, reducing the corneal epithelial defect area to less than 30% of the initial area.
3. The application of EPO according to claim 1 in the preparation of a drug for preventing and treating corneal damage, characterized in that: The chemical damage includes corneal burns caused by acids and alkalis. For corneal burns caused by alkalis, when using subconjunctival injection, the dosage is 200U to 500U per injection, with an interval of 24 hours. This can inhibit corneal inflammation, reduce the number of inflammatory cells infiltrating the corneal stroma to less than 50% of the normal level, and promote the proliferation of corneal stromal cells. Within 7 to 14 days after treatment, the arrangement of corneal stromal collagen fibers tends to become more regular.
4. The application of EPO according to claim 1 in the preparation of a drug for preventing and treating corneal damage, characterized in that: The aforementioned infectious factors causing corneal damage are caused by bacterial, viral, or fungal infections. In the treatment of corneal damage caused by viral infection, if topical eye drops are chosen, the concentration of EPO in the eye drop solution should be 300 U / mL to 800 U / mL, and the frequency of eye drops should be 8 to 12 times a day. This can effectively enhance the antiviral ability of the corneal epithelium and reduce the risk of corneal ulcer formation.
5. The application of EPO according to claim 1 in the preparation of a drug for preventing and treating corneal damage, characterized in that: For diabetic corneal injury, the solution formulation of the drug also contains sodium hyaluronate and an antioxidant. The sodium hyaluronate is added at a concentration of 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 is added at a concentration of 0.5%-2% of the total mass of the solution. When treating diabetic corneal injury, this solution formulation can be administered as eye drops at a concentration of 600 U / mL to 1200 U / mL, 5 to 9 times daily. This alleviates oxidative stress damage to corneal cells under high glucose conditions. After continuous treatment for 14 to 28 days, it increases the survival rate of corneal epithelial cells to over 70%, enhances the migration ability of corneal cells, and increases the corneal injury repair rate by 30% compared to when no antioxidant is added.
6. The application of EPO according to claim 1 in the preparation of a drug for preventing and treating corneal damage, characterized in that: When the drug is a self-lyophilized formulation, 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 desorption drying temperature range is 0°C to 20°C, and the vacuum degree is 1Pa to 10Pa. The lyophilized formulation prepared in this way can achieve a storage stability of 1 to 2 years at room temperature, and the activity retention rate of EPO after reconstitution is more than 80%.
7. The application of EPO according to claim 1 in the preparation of a drug for preventing and treating corneal damage, characterized in that: If the drug is administered via 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 corneal epithelial cell proliferation, increasing the proliferation rate by 25% compared to when the gene sequence is not optimized.