A method for constructing a model of retinal degenerative disease and application thereof
Patent Information
- Application Number
- CN202611245437.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-17
- Publication Date
- 2026-09-25
AI Technical Summary
[0004]目前，常用的视网膜退行性病变动物模型主要包括三类：第一类是基因突变模型（如rd1、rd10小鼠），其缺陷在于遗传背景复杂、繁殖周期长、饲养成本高，且针对特定基因突变的模型难以普遍适用于不同机制的研究需求；第二类是光损伤模型，其成模稳定性受光照参数及动物状态影响较大，重复性欠佳；第三类是化学诱导模型（如碘酸钠、N-甲基-N-亚硝基脲等），虽然操作相对简便，但现有化学诱导剂普遍存在毒性剧烈、作用靶点不明确或全身副作用大等问题，且大多通过非特异性细胞毒性机制发挥作用，难以满足靶点特异性药物筛选的需求
本发明首次发现USP21的酶活性对维持视网膜的稳态至关重要。在此基础上，本发明采用玻璃体腔注射给予小鼠USP21选择性酶活抑制剂BAY-805，注射后24小时即可诱导小鼠产生典型的视网膜退行性病变表型，包括外核层明显变薄、外核层细胞核数量降低、感光细胞外节缩短、膜盘损伤以及视网膜电图a波和b波振幅显著降低。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of medical engineering technology, specifically relating to a method for constructing a disease model of retinal degeneration and its application. Background Technology
[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.
[0003] Degenerative retinal diseases such as retinitis pigmentosa and age-related macular degeneration are the main causes of irreversible blindness. The common pathological features of these diseases are progressive photoreceptor cell death and thinning of the outer nuclear layer due to retinal homeostasis imbalance. Therefore, constructing animal models that accurately simulate these pathological changes is of fundamental importance for elucidating disease mechanisms and developing therapeutic drugs.
[0004] Currently, commonly used animal models of retinal degenerative diseases mainly fall into three categories: The first category is gene mutation models (such as rd1 and rd10 mice), which have the drawbacks of complex genetic backgrounds, long breeding cycles, high breeding costs, and models targeting specific gene mutations are difficult to universally apply to the research needs of different mechanisms; The second category is light damage models, whose modeling stability is greatly affected by light parameters and animal conditions, and their reproducibility is poor; The third category is chemically induced models (such as sodium iodate, N-methyl-N-nitrosourea, etc.), which are relatively simple to operate, but existing chemical inducers generally have problems such as severe toxicity, unclear targets, or large systemic side effects, and most of them work through non-specific cytotoxic mechanisms, which are difficult to meet the needs of target-specific drug screening.
[0005] Therefore, there is an urgent need in this field for a method to construct a disease model of retinal degeneration that has a short modeling cycle, clear targets, simple operation and good reproducibility, in order to overcome the shortcomings of existing technologies such as long cycle and high cost of gene models, high toxicity of chemical models and unclear mechanisms. Summary of the Invention
[0006] In view of this, the present invention provides a method for constructing a disease model of retinal degenerative diseases and its application.
[0007] In a first aspect, the present invention provides a method for constructing a disease model of retinal degenerative disease, the method comprising administering BAY-805 into the vitreous cavity of an animal eye.
[0008] Preferably, the intraocular vitreous cavity is administered via intravitreal injection.
[0009] Preferably, the intravitreal injection includes: after anesthetizing the animal, dilating the animal's pupils, and injecting BAY-805 solution into the intravitreal cavity along the posterior edge of the cornea.
[0010] Preferably, the concentration of the BAY-805 solution is 180 μM to 540 μM.
[0011] Preferably, the animal is a mouse.
[0012] Secondly, the present invention provides the application of the retinal degenerative disease model constructed by the above-mentioned method in the study of retinal degenerative diseases, wherein the study is for the purpose of non-disease diagnosis or treatment.
[0013] Thirdly, the present invention provides the application of the method for constructing the retinal degenerative disease model and the retinal degenerative disease model constructed therefrom in the early molecular screening of drugs or in the screening of targeted therapies for retinal degenerative diseases.
[0014] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention is the first to discover that the enzyme activity of USP21 is crucial for maintaining retinal homeostasis. Based on this, this invention uses intravitreal injection to administer the selective USP21 enzyme activity inhibitor BAY-805 to mice. Within 24 hours of injection, mice exhibit typical retinal degenerative phenotypes, including significant thinning of the outer nuclear layer, reduced number of outer nuclear cell nuclei, shortening of photoreceptor outer segments, disc damage, and a significant decrease in the amplitude of a and b waves on electroretinograms.
[0015] Intravitreal injection of BAY-805 enables localized targeted drug delivery, allowing the active ingredient to act directly on retinal tissue. This significantly shortens the modeling period while effectively avoiding the systemic toxicity associated with traditional chemical inducers administered intraperitoneally or subcutaneously. More importantly, because this invention induces disease phenotypes based on precise regulation of a specific target (USP21), its mechanism of action is clear, overcoming the fundamental shortcomings of traditional chemically induced models, such as unclear targets and singular mechanisms. Compared to gene mutation models, this invention eliminates the need for complex breeding and gene identification processes, significantly reducing construction and time costs. This invention provides a novel non-human animal model for the study of retinal degenerative diseases and the screening of drugs or therapies for treating these diseases, greatly enriching the models available for retinal degenerative disease research. Attached Figure Description
[0016] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.
[0017] Figure 1 This is an observation diagram of the overall structure of the retina using HE staining. Figure 2 This is a statistical analysis chart of the thickness of the outer nuclear layer of the retina; Figure 3 This is a statistical analysis chart of the number of cells in the outer nuclear layer; Figure 4 Visual function analysis of mice using ERG; Figure 5 Statistical analysis chart of visual function; Figure 6 An image showing the observation of the outer segments of photoreceptor cells using immunofluorescence staining; Figure 7 Statistical graph of the outer segment of photoreceptor cells; Figure 8 An image showing the fine structure of photoreceptor cells using an electron microscope; Figure 9 Statistical diagram of abnormal structures in photoreceptor cells. Detailed Implementation
[0018] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0019] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.
[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this application are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods in the art or as per the product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this application. The preferred embodiments and materials described herein are for illustrative purposes only.
[0021] Example 1: Preparation of BAY-805 injection solution The USP21 enzyme activity inhibitor BAY-805 (HY-153045; MedChemExpress) was first dissolved in DMSO to prepare a 1.8 mM stock solution. This stock solution was then diluted tenfold in sterile physiological saline (0.9% NaCl) to obtain a 180 μM BAY-805 solution for subsequent intravitreal injection. At this concentration, after injecting 1 μL into the vitreous humor, it was diluted by vitreous fluid, ultimately achieving an effective intraocular concentration of 30 μM.
[0022] Example 2: Preparation of BAY-805 injection solution The USP21 enzyme activity inhibitor BAY-805 (HY-153045; MedChemExpress) was first dissolved in DMSO to prepare a 5.4 mM stock solution. This stock solution was then diluted tenfold in sterile physiological saline (0.9% NaCl) to obtain a 540 μM BAY-805 solution for subsequent intravitreal injection. At this concentration, after injecting 1 μL into the vitreous humor, it was diluted by vitreous fluid, ultimately achieving an effective intraocular concentration of 90 μM.
[0023] Experimental Example 1 1. Laboratory animals The mice used in this study were obtained from our laboratory. There were 12 8-week-old mice.
[0024] 2. Establishment of an animal model by intravitreal injection of BAY-805 Anesthesia and Mydriasis: Mice were induced and maintained into general anesthesia by inhalation of 2% isoflurane. Simultaneously, local surface anesthesia was performed by instilling 0.5% oxysibucaine hydrochloride eye drops (Santen Pharmaceuticals) onto the corneal surface, and the pupils were fully dilated by instilling 0.5% tropicamide / phenylephrine eye drops (Santen).
[0025] Injection: Under a surgical microscope, a microsyringe (Hamilton Company, Reno, NV) with a 30G needle was inserted at the posterior margin of the cornea of the mouse eyeball, obliquely penetrating the sclera to enter the vitreous cavity. 1 μL of BAY-805 solution prepared according to the methods of Examples 1 and 2 was slowly injected into the vitreous cavity of each experimental group mouse. The injection was performed under a surgical microscope, and the needle was left in place for approximately 10 seconds after injection before being slowly withdrawn to prevent backflow of the drug. Control group: Control group mice were injected with an equal volume of the carrier control (i.e., a dilution of DMSO in physiological saline) in the same manner. Various indicators were measured 24 hours after injection.
[0026] 3. Testing Methods (1) HE staining: Mouse eyeballs were first preserved overnight at 4 °C in 4% paraformaldehyde. After removing the cornea and lens, the retina was fixed again at room temperature for two hours. The tissue was then embedded in paraffin and cut into 4 μm thick sections. For standard hematoxylin-eosin (H&E) staining, only sections containing the optic nerve were selected. To measure retinal thickness, sections with the same eccentricity and eyecups embedded in the same direction were selected. Images were observed and captured using a DM3000 microscope (Leica, Wetzlar, Germany). To assess structural regularity, we analyzed the thickness of the outer nuclear layer (ONL). Specifically, the linear thickness of the ONL and the number of cells in the outer nuclear layer at different centrifugation distances in H&E-stained sections were quantified using ImageJ software.
[0027] (2) Electroretinography analysis: In this study, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital to perform electroretinography (ERG). The simplified procedure is as follows: corneal surface anesthesia was performed using 0.5% oxybuprofen hydrochloride eye drops, and mydriasis was achieved using 0.5% tropicamide and phenylephrine eye drops. Signal acquisition was performed using a RetiMINER-C ERG recorder from IRC Technologies in Bangkok, Thailand: reference electrodes were implanted subcutaneously behind both ears, the ground wire was a gold wire electrode fixed to the tail, and the recording electrodes were placed directly on the corneal surface. Mice were administered 3 cd... Stimulation with white light flashes at s / m² was used to record dark-adapted electroretinogram (ERG) responses.
[0028] The amplitude value of wave a is the voltage difference between the resting baseline and the trough of wave a; the amplitude value of wave b is defined as the voltage difference between the trough of wave a and the peak of wave b. (3) Remove the eyeball and fix it overnight in paraformaldehyde fixative at 4 ℃. Carefully remove the lens and cornea, and embed the remaining eye cup tissue in 5% low melting point agarose (Shanghai Sangon Biotech). Use a Leica VT1200S vibrating microtome to cut 80 μm thick sections. Immunofluorescence staining: Cells were permeabilized and blocked for 1 h at room temperature with a permeabilization blocking buffer containing 0.5% Triton X-100 and 7% normal goat serum; then, primary antibody was added and incubated overnight at 4 °C. After thorough washing with PBS, Alexa Fluor 568 or 488 fluorescently labeled secondary antibody was added and incubated at room temperature in the dark for 1 h; after rinsing three times with PBS, cell nuclei were stained with DAPI. All fluorescence images were acquired using an Andor Dragonfly 200 high-speed confocal imaging system.
[0029] (4) Observation by transmission electron microscopy The intact eyeball was first fixed in 0.1 M sodium diarsinate buffer containing 0.25% glutaraldehyde for 4 hours at room temperature. After carefully removing the cornea and lens, the eye cup tissue was fixed overnight at 4°C in the same fixative. Subsequently, it was fixed twice with 1% osmium tetroxide at room temperature for 1 hour each time.
[0030] After being dehydrated by a gradient of ethanol and impregnated with resin, the samples were embedded in Spurr low-viscosity epoxy resin. After polymerization and curing in a 65 ℃ oven for 3 days, 50 nm ultrathin sections were prepared and double-stained with lead citrate and uranium acetate.
[0031] Electron micrographs were taken using a Hitachi HT-7800 transmission electron microscope from Japan at an accelerating voltage of 80 kV.
[0032] 4. Results Twenty-four hours after intravitreal injection of a USP21 enzyme activity inhibitor, retinal homeostasis abnormalities were rapidly induced. Analysis of the overall retinal structure in mice revealed a significant thinning of the outer nuclear layer and a marked decrease in the number of outer nuclear cell nuclei. Figure 1-3 This indicates that the structure of photoreceptor cells was damaged. We then examined the ERG function of the mice, with wave a representing photoreceptor cell function and wave b representing bipolar and Muller cell function. We found that the amplitudes of both waves a and b were significantly reduced. Figure 4-5 This indicates impaired photoreceptor cell function. Immunofluorescence staining analysis of the photoreceptor cell structure revealed a significant shortening of the outer segment of the photoreceptor cells. Figure 6-7 Furthermore, using transmission electron microscopy to conduct detailed observations of the outer segments of photoreceptor cells, it was discovered that the membrane discs of the photoreceptor cells were damaged. Figure 8-9 ).
[0033] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for constructing an animal model of retinal degenerative disease, characterized in that, The construction method includes administering BAY-805 into the vitreous cavity of an animal's eye.
2. The construction method according to claim 1, characterized in that, The intraocular administration is a vitreous injection.
3. The construction method according to claim 2, characterized in that, The intravitreal injection includes: after anesthetizing the animal, dilating the pupil, and injecting BAY-805 solution into the intravitreal cavity along the posterior edge of the cornea.
4. The construction method according to claim 1, characterized in that, The concentration of the BAY-805 solution is 180 μM to 540 μM.
5. The construction method according to claim 1, characterized in that, The animal in question is a mouse.
6. The application of the retinal degenerative disease model constructed by the method of any one of claims 1-5 in the study of retinal degenerative diseases, wherein the study is for the purpose of non-disease diagnosis or treatment.
7. The application of a method for constructing a retinal degenerative disease model as described in any one of claims 1-5 in the early molecular screening of drugs or in screening for targeted therapies for retinal degenerative diseases.