Use of pdgf-c in corneal injury repair

Abstract

The application discloses application of PDGF-C in corneal injury repair. The application research finds that in a corneal injury model mouse in which Pdgf-c is knocked out, Pdgf-c knockout reduces the area of corneal healing in the corneal injury mouse model, and the number of corneal limbal stem cells with proliferative activity is significantly less than that of wild type mice. Through AAV-mediated PDGF-C overexpression, the proliferation of corneal limbal stem cells can be significantly promoted, and the area of corneal healing in the corneal injury mouse model is significantly increased. These results show that overexpression of PDGF-C significantly improves the ability of corneal injury healing. Therefore, the application provides a new method for treating corneal injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of PDGF-C in corneal injury repair. Background Technology

[0002] Corneal diseases are a leading cause of vision impairment and blindness. Corneal defects and vision impairment can be caused by a variety of factors, including eye trauma and infection, with the extent depending on the severity of the injury. Corneal injuries can be broadly categorized into trauma-related injuries such as corneal perforation and foreign bodies; exposure-related injuries such as chemical burns and radiation burns; and genetically related injuries such as Fuchs' endothelial corneal dystrophy (FECD). Notably, the loss of limbal stem cells (LSCs) has a severe impact on vision. Events such as chemical burns or severe infections can deplete the LSC reserve, leading to limbal stem cell deficiency (LSCD). In LSCD, persistent epithelial defects are not only difficult to treat but can also cause scarring and vision loss. The corneal healing process mainly depends on cell migration, proliferation, differentiation, and extracellular matrix remodeling. Corneal epithelial regeneration relies on the replenishment of LSCs and the reconstruction of the basement membrane structure. Therefore, limbal stem cells play a crucial role in corneal injury repair and are important cells that promote corneal repair. However, there are currently problems such as insufficient donor sources and immune rejection. How to restore the function of limbal stem cells and repair corneal damage is an urgent ophthalmic problem to be solved.

[0003] Platelet-derived growth factor C (PDGF-C) belongs to the PDGF ligand family, which regulates cellular life processes by activating tyrosine kinase receptors (PDGFR-α / β). Within PDGF-C, homodimers, PDGF-CC, are formed through interchain disulfide bonds. PDGF-CC can bind to PDGFR-αα and PDGFR-αβ receptors, thereby regulating biological processes such as cell proliferation, differentiation, migration, and phenotypic transformation. PDGF-C is expressed in tissues with active angiogenesis and promotes angiogenesis in mouse cornea.

[0004] PDGF-C plays a crucial role in vascular-related fields. It is not only expressed in actively angiogenic tissues but is also a key angiogenic protective factor required for maintaining vascular viability. Under ischemic conditions, PDGF-C mobilizes endothelial progenitor cells, induces bone marrow cell differentiation into endothelial cells, and stimulates endothelial cell migration, enhancing revascularization after ischemia in the heart and limbs. Furthermore, studies investigating mouse embryonic development have shown that knocking out the PDGF-C gene results in severe hemorrhage and edema in embryos, ultimately leading to perinatal death, demonstrating the critical role of PDGF-C in embryonic development. However, to date, no studies have reported on the functional role of PDGF-C in limbal stem cells, leaving its specific function unclear. Summary of the Invention

[0005] The purpose of this invention is to explore a novel strategy for effective repair of corneal damage. This invention discovers that AAV-mediated PDGF-C overexpression can promote the proliferation of limbal stem cells, significantly enhancing the corneal healing capacity.

[0006] According to one aspect of the present invention, the use of PDGF-C in the preparation of a medicament that promotes the repair of corneal damage is provided.

[0007] Preferably, in the above applications, the PDGF-C promotes the repair of corneal damage by promoting the proliferation of limbal stem cells.

[0008] According to a second aspect of the present invention, a medicament for promoting corneal damage repair is provided, comprising a vector overexpressing the PDGF-C gene and pharmaceutically acceptable excipients.

[0009] Preferably, the vector overexpressing the PDGF-C gene is an adeno-associated virus vector overexpressing the PDGF-C gene.

[0010] Preferably, the dosage form of the drug is an injection.

[0011] Preferably, the injectable includes an injection solution and a lyophilized powder for injection.

[0012] Preferably, the excipient is physiological saline.

[0013] According to a third aspect of the invention, the use of PDGF-C in the preparation of products that promote the proliferation / stem maintenance of limbal stem cells is provided.

[0014] Preferably, in the above-described applications, the step of increasing the expression level of the PDGF-C gene in cells is included.

[0015] Preferably, in the above applications, the product includes drugs and culture media.

[0016] This invention reveals that in a Pdgf-c knockout model of corneal injury in mice, Pdgf-c knockout reduces the corneal healing area and the number of proliferating limbal stem cells is significantly lower than in wild-type mice. AAV-mediated PDGF-C overexpression significantly promotes limbal stem cell proliferation and significantly increases the corneal healing area in the corneal injury model. These results indicate that PDGF-C overexpression significantly enhances the ability to heal corneal injuries. Therefore, this invention provides a novel method for the treatment of corneal injuries. Attached Figure Description

[0017] Figure 1 PDGF-C knockout significantly reduced limbal stem cells and inhibited corneal wound healing. A shows the knockout efficiency of PDGF-C in the cornea as indicated by Western blot results. B shows the quantitative data from Western blot analysis, which was normalized to ACTIN (n=3). C shows the immunofluorescence images of P63 (green), Ki67 (red), and DAPI (blue) staining in the damaged cornea, with cell nuclei counterstained with DAPI (blue). Scale bar: 50 μm. D shows P63... + Ki67 + Quantitative results of double-positive cells, n=5 per group. E shows a fluorescein sodium staining image indicating slower corneal wound healing after PDGF-C knockout, scale bar: 2mm. F shows the quantitative analysis results of corneal wound area, n=5 per group.

[0018] Figure 2 PDGF-C can increase the number of limbal stem cells and promote corneal wound healing. A shows a schematic diagram of AAV-induced PDGF-C overexpression in the mouse cornea and a mouse corneal injury model. B shows Western blot results demonstrating PDGF-C overexpression in the cornea via AAV. C shows quantitative data from Western blot analysis, standardized according to TUBULIN, where n=3. D shows immunofluorescence images showing the staining results of P63 (green), Ki67 (red), and DAPI (blue) in the damaged cornea, with cell nuclei counterstained with DAPI (blue). Scale bar: 50 μm. E shows P63... + Ki67 + Quantitative results of double-positive cells, n=5 per group. F is a representative fluorescein sodium staining image showing better corneal wound healing after PDGF-C knockout, scale bar: 2mm. G is the quantitative analysis result of corneal wound area, n=5 per group. Detailed Implementation

[0019] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents and materials used are commercially available unless otherwise specified.

[0021] Example 1

[0022] I. Materials

[0023] AAV2 / 9-PDGF-C and AAV2 / 9-GFP were purchased from Heyuan Biotechnology.

[0024] Information on the antibodies used in the experiment is shown in Table 1:

[0025] Table 1 Antibodies

[0026]

[0027] II. Methods

[0028] 1. Corneal injury healing model

[0029] Mice were first anesthetized with sodium pentobarbital (50 mg / kg), and then the cornea was punctured approximately 1-2 mm above the limbus of the temporal horn using a sterile 30G disposable needle. Through this puncture site, 1 μL of AAV2 / 9-PDGF-C or AAV2 / 9-GFP (10 mg / kg) was injected into the anterior chamber of each eye using a 2.5 μL microsyringe equipped with a 33G small-diameter detachable flat-tipped needle (Hamilton, USA). 10 (vg / eye). Two weeks later, a corneal injury model was established. Mice were anesthetized with sodium pentobarbital (50 mg / kg), and approximately half of the corneal epithelium and adjacent limbal tissue were scraped away using an Algerbrush II corneal rust ring remover (Alger, USA). On day 0 and day 1 post-injury, the corneal wound was marked with sodium fluorescein staining, and images were taken using a slit-lamp microscope. The wound area was quantitatively analyzed using ImageJ software. Corneal tissue was collected 72 hours after injury for Western blot and immunofluorescence staining.

[0030] 2. Pdgf-c systemic knockout mice

[0031] The construction of Pdgf-c whole-body knockout mice was performed using a gene targeting strategy, as detailed in (Ding et al., Nature Genetics, 2004, PMID 15361870), and the mice have been bred for more than six generations on the C57BL / 6J background. The primers used for genotyping Pdgf-c deficient mice were: 5'-CTGATGTTCTCGTGACTCTGA-3'; 5'-TAGCTAGTCGATACCGTCGA-3'; 5'-AGCTGACATTTGATGAGAGAT-3'; and 5'-AGTAGGTGAAATAAGAGGTGAACA-3'. A 200bp electrophoresis band represents a wild-type mouse, and a 350bp band represents a Pdgf-c knockout mouse. The procedure for establishing corneal injury models in Pdgf-c whole-body knockout mice is the same as step 1 above.

[0032] 3. Total protein extraction and BCA protein quantification.

[0033] 1) Remove the cells to be tested from the incubator and use a negative pressure pump to remove the old culture medium.

[0034] 2) Wash the cells with 1×PBS, remove the solution using a negative pressure pump, and transfer to ice.

[0035] 3) On ice, using RIPA as a solvent, add protease inhibitor (100×), phosphatase inhibitor solution A and solution B (100×) to RIPA, and dilute the inhibitors to 1× for use as cell lysis buffer.

[0036] 4) Add the cell lysis buffer prepared in step 3) to a culture dish, incubate on ice for 5-10 minutes to fully lyse the cells, transfer to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 15 minutes at 4°C, and collect the supernatant.

[0037] 5) Prepare reference samples of known concentrations by serially diluting the standard with 1×PBS according to the BCA kit; for protein samples, dilute the supernatant prepared in step 4) 10 times with 1×PBS to prepare the test sample, wherein the final volume of the test sample and the reference sample solution is 20μL.

[0038] 6) Preparation of working solution: Prepare the working solution according to the ratio of solution A: solution B = 50:1.

[0039] 7) Add the working solution prepared in step 6) to the test sample and the reference sample solution, with 200 μL of working solution for each tube, and mix thoroughly.

[0040] 8) Take 200 μL of the sample prepared in step 7) and add it to each well of a 96-well plate. Incubate at 37°C for 30 min.

[0041] 9) After incubation, use an ELISA reader to measure the absorbance of the sample at 562 nm and plot a standard curve (where the R value must be greater than 0.99), and calculate the protein concentration of each sample to be tested.

[0042] 4. Western blot experiment

[0043] 1) Preparing liquids:

[0044] ①10×Running Buffer: Add 144g glycine, 10g SDS powder, and 30.3g Tris powder sequentially, using ddH2O as the solvent, and bring the volume to 1L to prepare a storage solution. Dilute to 1×Running Buffer to prepare the electrophoresis working solution for use.

[0045] ②5×SDS Loading Buffer: Add 4g of SDS powder, 20mg of bromophenol blue, 3.085g of DTT, 10mL of Tris-HCl (1M pH 6.8), and 20mL of glycerol in sequence, and use ddH2O as a solvent to make up to 40mL.

[0046] ③ 10× Transfer Buffer: Add 30.3g Tris powder and 144g glycine sequentially, using ddH2O as the solvent, and bring the volume to 1L to prepare a storage solution. Dilute 10× Transfer Buffer 100mL + methanol 200mL + ddH2O 700mL to prepare 1× Transfer Buffer as the working solution for membrane transfer.

[0047] 2) Preparation of separating gel and stacking gel

[0048] ① Prepare 10% separating gel (10 mL):

[0049] Table 2 shows the preparation of 10% separating gel.

[0050]

[0051] ②Prepare a 5% concentrate (5 mL):

[0052] Table 3 shows the preparation of 5% concentrated gel.

[0053]

[0054] 3) Protein gel electrophoresis: Assemble the prepared gel and add the newly prepared electrophoresis solution to the tank. Check for leakage. Remove the comb and add the marker and sample to the gel wells in sequence. Add electrophoresis solution to the electrophoresis tank and set the program to 80V for 30 minutes of constant voltage electrophoresis. Then switch to 120V for 1 hour of constant voltage electrophoresis.

[0055] 4) Transfer: First, activate the PVDF membrane with methanol, and remove the gel from step 3). Assemble the transfer clamps in a "sandwich" structure, taking care not to leave air bubbles during assembly. Assemble the transfer apparatus, add the transfer working solution, set the program to a constant current of 250 mA for 2 hours, and place it on ice for transfer.

[0056] 5) Blocking: Take out the membrane after the transfer is completed, add 1×TBST and place it on a shaker at room temperature for 5 minutes to clean it. Discard the solution and add 5% skim milk as the blocking solution. Place it on a shaker at room temperature for blocking for 1 hour.

[0057] 6) Primary antibody incubation: Discard the blocking solution, add 1×TBST and place on a shaker at room temperature to wash until the blocking solution is clean. Discard the liquid, add the primary antibody prepared with 5% BSA as antibody diluent, and incubate overnight on a shaker in a 4°C cold storage.

[0058] 7) Wash the membrane: Wash the membrane three times with 1×TBST, 10 min each time.

[0059] 8) Secondary antibody incubation: Discard the solution, add secondary antibody prepared with 5% skim milk at a ratio of 1:5000, and incubate on a shaker at room temperature for 1 hour.

[0060] 9) Same as step 7).

[0061] 10) Exposure and development: Prepare the developer (A solution: B solution = 1:1 ratio, prepare and use immediately). Place the film on the plate, add developer solution to the film, shake well, expose and develop on the machine, and save the results.

[0062] 5. Immunofluorescence staining

[0063] Mouse muscle tissue was fixed with 4% polyoxymethylene (PFA, Sigma) for 2 hours at room temperature, then embedded overnight in 30% sucrose (Sigma) at 4°C. The tissue was then frozen using OCT (SAKURA) and sectioned into 10 μm sections. Sections were blocked for 1 hour at room temperature with a blocking buffer of 5% BSA and 0.5% Triton X-100 prepared in PBS, followed by incubation overnight with primary antibody at 4°C. Secondary antibody (Life Technologies, Waltham, MA, USA) was incubated at room temperature for 1 hour. The sections were imaged using a fluorescence microscope (Zeiss, Oberkosen, Germany). Primary antibodies included Ki67 (AF7649-SP, R&D System) and P63 (ET1610-44, HUABIO). Secondary antibody included donkey anti-rabbit IgG (Alexa Fluor). TM 555, Life Technologies, A-31572), Goat anti-mouse IgG (Alexa Fluor) TM 488, Life Technologies, A-10680) and goat anti-mouse IgG (Alexa Fluor) TM 488, Life Technologies, A-11006).

[0064] 6. Hematoxylin and eosin staining

[0065] All mice were anesthetized with isoflurane and euthanized. Separated muscles were fixed with 4% paraformaldehyde. Fixed tissues were dehydrated, embedded in paraffin, and sectioned. Frozen tissue blocks for hematoxylin and eosin (H&E) staining were cut into 10 μm thin sections. H&E staining was performed by Wuhan Saiwei Biotechnology Co., Ltd. according to standard protocols.

[0066] III. Results

[0067] All data are presented as mean ± standard deviation (SD). Statistical significance was assessed using a one-sample t-test. *p<0.05, **p<0.01, ***p<0.001.

[0068] 1. Limbal stem cell deficiency is a debilitating disease caused by the loss of limbal stem cells. We conducted a corneal injury model to explore its potential to enhance limbal stem cells and promote corneal repair / regeneration. Figure 1 and Figure 2 We first constructed Pdgf-c gene knockout mice, then established the model, and Western blot analysis confirmed that Pdgf-c was knocked out in the cornea. Figure 1 (A and B in the text). Further analysis using sodium fluorescein staining revealed that Pdgf-c knockout reduced the area of ​​corneal healing in a mouse model of corneal injury (A and B in the text). Figure 1(E and F in the text).

[0069] 2. Immunofluorescence staining showed that in a Pdgf-c knockout mouse model of corneal injury ( Figure 1 C in the middle), limbal stem cells (P63) + Ki67 + The number of double-positive cells was significantly less than that of wild-type mice. Figure 1 (D in the middle).

[0070] 3. To explore the therapeutic effect of PDGF-C, we overexpressed PDGF-C in the cornea of ​​wild-type mice by injecting AAV-PDGF-C. Corneal injury was induced 14 days later, and corneal tissue was collected 3 days later for relevant biomarker detection. Figure 2 (A) Western blot analysis showed that PDGF-C was significantly overexpressed in the cornea. Figure 2 (B and C in the text). Immunofluorescence showed that PDGF-C overexpression significantly promoted the proliferation of limbal stem cells, namely P63. + Ki67 + The proportion of double-positive limbal stem cells has increased. Figure 2 (D and E in the text).

[0071] 4. Fluorescein staining revealed that PDGF-C overexpression significantly increased the corneal healing area in a mouse model of corneal injury. Figure 2 The F and G values ​​in the figure indicate that PDGF-C overexpression significantly enhances the corneal injury healing ability. Therefore, these data fully demonstrate that mice overexpressing PDGF-C exhibit better corneal repair than the control group.

[0072] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. Application of vectors overexpressing the PDGF-C gene in the preparation of drugs that promote corneal damage repair.

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

3. The application according to claim 1, characterized in that, The vector overexpressing the PDGF-C gene is an adeno-associated virus vector overexpressing the PDGF-C gene.

4. The application according to claim 1, characterized in that, The drug is in the form of an injection.

5. The application according to claim 4, characterized in that, The injectables include injection solutions and lyophilized powder injections.

6. The application according to claim 2, characterized in that, The excipient is physiological saline.

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