Methods for assessing myopia susceptibility and screening candidate interventions based on the Opn1mw gene knockout mouse model

Abstract

This invention relates to a method for assessing myopia susceptibility and screening candidate interventions based on the Opn1mw gene knockout mouse model, belonging to the field of biomedical technology. The method uses the Opn1mw gene knockout mouse model as the experimental subject, and comprehensively evaluates high myopia susceptibility by applying defocus stimulation and jointly detecting refractive status, ocular biological parameters, retinal dopamine levels, and the expression levels of TFAM and SMN1 molecular markers. Furthermore, based on the model, candidate interventions can be screened and their effects evaluated. The preferred candidate interventions are dopamine receptor modulators, more preferably D1 receptor agonists. The evaluation system established by this invention can stably characterize the high sensitivity to defocus stimulation in the Opn1mw deletion background and can be used for myopia susceptibility assessment, myopia-related mechanism research, and candidate intervention strategy development.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for assessing myopia susceptibility and screening candidate interventions based on an Opn1mw gene knockout mouse model. In particular, it relates to constructing a myopia-highly-susceptible mouse model using an Opn1mw gene deletion background and using it for myopia-related mechanism research and candidate intervention strategy evaluation. Background Technology

[0002] Myopia, especially high myopia, has become a significant public health problem affecting visual health. Current research indicates that the development of myopia is influenced by both genetic and environmental factors, with visual defocusing stimulation, retinal neurotransmitter imbalance, and abnormal photosensitivity signals all considered closely related to refractive developmental abnormalities. In particular, cone cell-mediated light perception and color vision-related signals are thought to potentially participate in the regulation of eye growth. Constructing stable and reproducible animal models of myopia is crucial for studying the pathogenesis of myopia and developing intervention strategies. By simulating the formation process of human myopia, animal models can provide an experimental platform for studying the mechanisms of myopia, assessing myopia susceptibility, screening candidate interventions, and evaluating their effectiveness.

[0003] Currently, commonly used animal models of myopia are mainly established through negative lens induction or form deprivation. While these methods can simulate the myopia formation process to some extent, they are insufficient for assessing differences in myopia susceptibility across different genetic backgrounds. Furthermore, existing technologies primarily focus on phenotypic indicators such as refractive error, axial length, and vitreous cavity depth, lacking a comprehensive system that integrates retinal neurotransmitters and molecular markers for evaluation. This limits the application of such models in candidate intervention screening and mechanism validation.

[0004] Existing research has attempted to construct animal models related to myopia or visual abnormalities based on gene editing. For example, CN121022847A discloses a method for knocking out the sgRNA of zebrafish pde6a and its application in visual diseases. By targeting and knocking down zebrafish pde6a using the CRISPR / Cas9 system, a zebrafish model with impaired visual function but essentially normal retinal development was obtained. This model also exhibited a trend of shortened body length, smaller eyeballs, shortened interocular distance, and increased axial length. CN121022848A discloses the application of WNT7B in constructing animal models of myopia. By knocking out or knocking down the homologous genes wnt7ba and wnt7bb of WNT7B in zebrafish, an animal model with significantly increased axial length, decreased eye movement frequency, and weakened light response was obtained, suggesting that WNT7B is associated with high myopia. The above technologies demonstrate the feasibility of constructing myopia-related animal models based on gene regulation and can provide tools for research on the pathogenesis of myopia and drug screening.

[0005] However, current technology still lacks a mouse model based on a genetic background of abnormal cone cell photoreceptor signals. This model would not only demonstrate higher susceptibility to myopia under defocus stimulation but also provide a comprehensive evaluation of high susceptibility to myopia by combining refractive status, ocular biological parameters, and retinal molecular indices, and could be further used for screening candidate interventions and validating related mechanisms. In particular, a mouse model based on an Opn1mw gene deletion background, suitable for assessing high susceptibility to myopia and screening candidate interventions under defocus stimulation conditions, is still lacking.

[0006] Therefore, developing a myopia-susceptible mouse model based on the deletion of the Opn1mw gene and establishing corresponding methods for myopia susceptibility assessment and candidate intervention screening are of great significance for myopia-related mechanism research and candidate intervention strategy development.

[0007] Purpose of the invention The purpose of this invention is to provide a method for assessing myopia susceptibility and screening candidate interventions based on the Opn1mw gene knockout mouse model, in order to solve the problem that the existing technology lacks an experimental system that can stably characterize the high susceptibility to myopia under a specific genetic background and further be used for screening candidate interventions and verifying mechanisms.

[0008] Another objective of this invention is to provide a method for assessing myopia susceptibility based on the Opn1mw gene knockout mouse model, which combines refractive status, ocular biological parameters, and retinal molecular indicators to comprehensively evaluate high susceptibility to myopia.

[0009] A further objective of the present invention is to provide a candidate intervention screening method based on the aforementioned model, for screening candidate interventions that can alleviate myopia phenotype and / or improve related molecular abnormalities.

[0010] Another objective of this invention is to provide a mechanism verification method based on the aforementioned model for verifying the role of dopamine-related pathways and their related molecules in the formation of myopia susceptibility in the context of Opn1mw deficiency. Summary of the Invention

[0011] To address the lack of an experimental system in existing technologies that can stably characterize myopia susceptibility under specific genetic backgrounds and can be used for candidate intervention screening and mechanism verification, this invention provides a method for assessing myopia susceptibility and screening candidate interventions based on the Opn1mw gene knockout mouse model, aiming to provide an experimental basis and evaluation tool for myopia-related mechanism research and candidate intervention strategy development.

[0012] To achieve the above objectives, the present invention adopts the following technical solution: First aspect This invention provides a method for constructing a myopia animal model, the method comprising: Experimental animals with Opn1mw gene deletion were selected, and negative lens defocusing stimulation was applied to their experimental eyes to obtain a myopia animal model.

[0013] Optionally, the experimental animal is a mouse.

[0014] Optionally, the mouse is a C57BL / 6J genetic background mouse.

[0015] Optionally, the mouse is a 4-week-old mouse.

[0016] Optionally, the experimental eye is the right eye, and the left eye serves as an untreated control eye.

[0017] Optionally, the negative lens defocusing stimulation is implemented using a negative diopter lens.

[0018] Optionally, the refractive power of the negative refractive lens is -10D to -30D.

[0019] Optionally, the refractive power of the negative refractive lens is -20D to -25D.

[0020] Optionally, the negative diopter lens has a diopter of -25D.

[0021] Optionally, the duration of the negative lens defocus stimulation is 1 to 6 weeks.

[0022] Optionally, after applying negative lens defocus stimulation, the method further includes detecting at least one of refractive state, ocular biological parameters, retinal dopamine level, TFAM expression level, or SMN1 expression level.

[0023] Optionally, the ocular biological parameters include axial length and / or vitreous cavity depth.

[0024] Optionally, the method for detecting retinal dopamine levels includes any one of high performance liquid chromatography, liquid chromatography-mass spectrometry, or enzyme-linked immunosorbent assay.

[0025] Optionally, the detection methods for TFAM expression level and / or SMN1 expression level include any one of qPCR, Western blot, immunofluorescence, or proteomics analysis.

[0026] This invention reveals that animals lacking the Opn1mw gene are more sensitive to defocus stimulation. Compared to the wild-type defocus stimulation group, Opn1mw gene-deficient mice exhibit more pronounced myopic refractive changes and more significant changes in axial length-related structures after defocus stimulation, thus serving as a potential animal model for high susceptibility to myopia.

[0027] Second aspect This invention provides a method for screening candidate interventions, the method comprising: The candidate intervention was applied to the myopia animal model constructed in the first aspect, and the changes in myopia phenotype and / or molecular indicators in the candidate intervention treatment group and the control group were compared; when the candidate intervention reduced the myopia phenotype and / or restored the molecular indicators to the normal direction, the candidate intervention was determined to have myopia intervention potential.

[0028] Optionally, the candidate intervention includes at least one of dopamine receptor modulators, metabolic regulators, mitochondrial function regulators, epigenetic regulators, or spectral intervention protocols.

[0029] Optionally, the dopamine receptor modulator includes a D1 receptor agonist.

[0030] Optionally, the D1 receptor agonist includes SKF38393 or a pharmaceutically acceptable salt thereof.

[0031] Optionally, the myopia phenotype includes refractive status and / or ocular biological parameters.

[0032] Optionally, the ocular biological parameters include axial length and / or vitreous cavity depth.

[0033] Optionally, the molecular indicators include at least one of retinal dopamine levels, TFAM expression levels, or SMN1 expression levels.

[0034] Third aspect This invention provides a method for verifying the molecular mechanisms related to myopia. It utilizes a myopia animal model constructed in the first aspect to detect changes in retinal dopamine levels and at least one molecular indicator among TFAM and SMN1 before and after defocus stimulation, in order to verify the role of dopamine-related pathways in the formation of myopia susceptibility.

[0035] Optionally, the verification method further includes observing synchronous changes in myopia phenotype and molecular indicators by applying dopamine pathway regulators, mitochondrial function regulators, or epigenetic regulators to verify the functional role of the target pathway.

[0036] Beneficial effects Compared with existing technologies, this invention has at least the following beneficial effects: This invention constructs a method for assessing myopia susceptibility and screening candidate interventions based on the Opn1mw gene knockout mouse model. This method uses Opn1mw gene knockout mice as experimental subjects, applies defocus stimulation, and comprehensively evaluates the high susceptibility to myopia by jointly detecting refractive status, ocular biological parameters, retinal dopamine levels, and at least one molecular indicator among TFAM and SMN1. Furthermore, based on this model, candidate interventions can be screened and their effects evaluated. Preferably, the candidate intervention is a dopamine receptor modulator, more preferably a D1 receptor agonist. The evaluation system established by this invention can stably characterize the high sensitivity to defocus stimulation in the Opn1mw deletion background and can be applied to myopia-related mechanism research and candidate intervention strategy development. Attached Figure Description

[0037] Figure 1 The diagram shows the overall experimental flow chart of this invention. Figure A shows the process of model establishment and molecular mechanism research, and Figure B shows the process of evaluating the effects of candidate interventions.

[0038] Figure 2 Figure A shows the PCR identification results and primer information for Opn1mw gene knockout mice. Figure A shows the PCR identification results of WT mice and Opn1mw gene knockout mice (MKO); Figure B shows the primer information used for Opn1mw genotyping. In Figure A, the left-hand label 1 indicates the PCR identification result obtained using the first set of primers; where KO: 443 bp indicates the theoretical band size of the amplified product corresponding to the Opn1mw gene knockout mouse is 443 bp, and WT: 1144 bp indicates the theoretical band size of the amplified product corresponding to the wild-type mouse is 1144 bp. The right-hand label 2 indicates the PCR identification result obtained using the second set of primers; where WT: 656 bp indicates the theoretical band size of the amplified product corresponding to the wild-type mouse is 656 bp. In Figure A, 61, 62, 63, and 64 represent different individual sample numbers; where lane WT61 is the wild-type mouse sample, and lanes 62, 63, and 64 are the Opn1mw gene knockout mouse samples; lanes with multiple horizontal bands in the figure are DNA molecular weight markers. P represents the PCR positive control lane, B6 represents the control lane using C57BL / 6J mouse genomic DNA as a template, and N represents the blank control without template. In Figure B, numbers 1 and 2 correspond to the two PCR primer combinations labeled 1 and 2 in Figure A, respectively. "Number" indicates the primer combination number, "Primer Name" indicates the name of each PCR primer, and "Sequence (5'→3')" indicates the nucleotide sequence of the corresponding primer. Based on the difference in the size of the PCR amplification product bands of different genotype mice, the genotypes of wild-type mice and Opn1mw gene knockout mice can be determined.

[0039] Figure 3 Figure A shows the validation of M-opsin protein expression in Opn1mw gene knockout mice. Figure A shows the Western blot results, and Figure B shows the statistical results of M-opsin protein expression levels. WT mice are wild-type mice, MKO mice are Opn1mw gene knockout mice, and β-Tubulin is an internal control protein. The results show that compared with WT mice, M-opsin protein expression is significantly reduced or absent in MKO mice, indicating that the constructed Opn1mw gene knockout mice have been validated at the protein level.

[0040] Figure 4This figure shows the changes in refractive status, axial length, vitreous depth, and their differences between WT mice and Opn1mw gene knockout mice. Figure A shows the changes in refractive power over time for each group; Figure B shows the changes in axial length over time for each group; Figure C shows the changes in vitreous depth over time for each group; Figure D compares the refractive differences among the groups; Figure E compares the differences in axial length among the groups; and Figure F compares the differences in vitreous depth among the groups. In each figure, MKO-R represents the right eye of an Opn1mw gene knockout mouse, MKO-L represents the left eye of an Opn1mw gene knockout mouse, WT-R represents the right eye of a wild-type mouse, and WT-L represents the left eye of a wild-type mouse; p4w, p6w, and p8w represent different age-related detection time points. This figure illustrates the dynamic differences in refractive status and ocular biological parameters between WT mice and Opn1mw gene knockout mice under defocus stimulation conditions.

[0041] Figure 5 This figure compares retinal dopamine metabolism-related indicators between WT mice and Opn1mw gene knockout mice. Figure A compares DOPAC levels in the retina of each group; Figure B compares dopamine levels in the retina of each group; and Figure C compares the DOPAC / DA ratio in the retina of each group. In the figures, WT-R and WT-L represent the right and left eyes of WT mice, respectively, and MKO-R and MKO-L represent the right and left eyes of MKO mice, respectively. This figure illustrates the differences in retinal dopamine metabolism-related indicators between WT and MKO mice.

[0042] Figure 6 This figure shows the changes in TFAM and SMN1 protein expression in the retinas of WT mice and Opn1mw knockout mice under defocus stimulation conditions. Figure A shows the Western blot results and relative grayscale analysis of TFAM protein expression; Figure B shows the Western blot results and relative grayscale analysis of SMN1 protein expression. In the figures, β-Tubulin is the internal control protein; LIM(R) indicates the right eye received defocus stimulation treatment, and L indicates the left eye was not treated. This figure illustrates the changes in TFAM and SMN1 protein expression in the retinas of WT and MKO mice under defocus stimulation conditions.

[0043] Figure 7Figure 1 shows a comparison of phenotypic changes in the Opn1mw gene knockout mouse defocus stimulation model after SKF38393 intervention. Figure A compares the refractive error of the right eye between the SKF38393 treatment group and the solvent control group at time points P4w and P8w; Figure B compares the difference in refractive error between the two eyes between the SKF38393 treatment group and the solvent control group at time points P4w and P8w; Figure C compares the axial length of the right eye between the SKF38393 treatment group and the solvent control group at time points P4w and P8w; Figure D compares the difference in axial length between the two eyes between the SKF38393 treatment group and the solvent control group at time points P4w and P8w. This figure illustrates that, compared with the solvent control group, SKF38393 intervention can improve the refractive status of the Opn1mw gene knockout mouse defocus stimulation model and alleviate myopic changes related to axial elongation, thereby inhibiting the occurrence and development of myopia and reducing myopia susceptibility. Detailed Implementation

[0044] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following embodiments are merely exemplary embodiments of the present invention and are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0045] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained through legitimate commercial channels.

[0046] This invention provides a method for assessing myopia susceptibility and screening candidate interventions based on the Opn1mw gene knockout mouse model. The overall experimental procedure is as follows: Figure 1 As shown. The method includes the following steps: 1) Provide experimental animals, select Opn1mw gene knockout mice as the experimental group, and select wild-type mice with the same genetic background as the control group; 2) Establish defocus stimulation conditions and apply negative lens defocus stimulation to the experimental eye to induce myopia formation; 3) Perform phenotypic detection, and detect the refractive status and ocular biological parameters of mice at preset time points; 4) Perform molecular detection to detect intraretinal dopamine levels, TFAM expression levels, and / or SMN1 expression levels; 5) Make a comprehensive judgment. If Opn1mw gene knockout mice show more obvious myopic refractive changes, ocular biological abnormalities and / or related molecular abnormalities compared with wild-type mice under defocus stimulation, then the Opn1mw gene knockout background can be judged to represent a high susceptibility to myopia. 6) Conduct candidate intervention screening and apply the candidate intervention to the model; if the candidate intervention can alleviate the myopia phenotype and / or restore the relevant molecular indicators to the normal direction, then the candidate intervention is determined to have the potential to improve the myopia susceptibility state in the context of Opn1mw deficiency.

[0047] In this invention, the Opn1mw (Opsin 1, Medium Wave Sensitive) gene encodes medium wave sensitive opsin (M-opsin), which is located at the end of the long arm of the X chromosome.

[0048] Example 1 Establishment of an Opn1mw gene knockout mouse model for assessing myopia susceptibility Twelve male, 4-week-old Opn1mw gene knockout mice (MKO) and 12 wild-type mice (WT) with the same genetic background (C57BL / 6J) were selected. The WT mice were purchased from Shanghai Silex Laboratory Animal Co., Ltd. The Opn1mw gene knockout mice were constructed using CRISPR / Cas9 gene editing technology. Specifically, a frameshift mutation was introduced at the target site of the Opn1mw gene via non-homologous end linkage repair to obtain a loss-of-function phenotype; preferably, the frameshift mutation was formed by deleting exon 3 of the Opn1mw gene.

[0049] During the construction process, Cas9 mRNA obtained through in vitro transcription and sgRNA designed targeting the Opn1mw sequence were co-microinjected into C57BL / 6J mouse zygotes to obtain F0 generation pioneer mice. After PCR amplification and sequencing identification of positive pioneer mice, they were mated with C57BL / 6J wild-type mice to obtain F1 generation mice carrying the target mutation. Further genotyping and screening were then conducted to obtain Opn1mw gene knockout mice for subsequent experiments. The PCR identification results and primer information of the Opn1mw gene knockout mice are as follows: Figure 2 As shown.

[0050] Furthermore, Western blot was used to validate M-opsin protein expression, with β-Tubulin as an internal control. The results are as follows: Figure 3 As shown, compared with WT mice, M-opsin protein expression was significantly reduced in MKO mice, indicating that the Opn1mw gene knockout mice were confirmed at the protein level.

[0051] During the establishment of the myopia model, mice wore a -25D negative lens in their right eye, while the left eye remained untreated. The lens was worn continuously for 28 days, with daily checks of its position and cleanliness to ensure a stable and continuous defocusing stimulus. At time points P4w, P6w, and P8w, the refractive status of each group of mice was assessed using an off-center infrared mouse refractometer, and ocular biological parameters such as axial length and vitreous cavity depth were measured using SD-OCT.

[0052] Results of refractive status, axial length, and vitreous depth measurements are as follows: Figure 4 As shown in the figures, Figure A shows the refractive error changes of each eye in wild-type mice and Opn1mw gene knockout mice at different time points; Figure B shows the changes in axial length of each group of mice over time; Figure C shows the changes in vitreous cavity depth of each group of mice over time; Figure D compares the refractive error difference between the left and right eyes of wild-type mice and Opn1mw gene knockout mice; Figure E compares the axial length difference between the left and right eyes of wild-type mice and Opn1mw gene knockout mice; and Figure F compares the vitreous cavity depth difference between the left and right eyes of wild-type mice and Opn1mw gene knockout mice. In the figures, MKO-R represents the right eye of a gene knockout mouse, MKO-L represents the left eye of a gene knockout mouse, WT-R represents the right eye of a wild-type mouse, WT-L represents the left eye of a wild-type mouse, and p4w (4 weeks), p6w (6 weeks), and p8w (8 weeks) represent different age-related detection time points.

[0053] Depend on Figure 4 As can be seen, with continued defocus stimulation, the right eye of the Opn1mw gene knockout mice showed more pronounced myopic refractive shift, along with more significant axial elongation and vitreous cavity deepening. Furthermore, the difference between the left and right eyes was more pronounced compared to wild-type mice. These results indicate that, compared to the wild-type defocus stimulation group, the Opn1mw gene knockout mice exhibited more significant myopic refractive changes and more pronounced changes in axial-related structures after defocus stimulation, suggesting that the constructed model has higher sensitivity to defocus stimulation and can be used for myopia susceptibility assessment.

[0054] Example 2 Detection of model-related molecular indices After the assay in Example 1, retinal tissues from each group of mice were collected, and dopamine-related indicators were detected using high-performance liquid chromatography-electrochemical detection (HPLC-ECD). Western blot was used to detect the mRNA and protein expression levels of TFAM and SMN1 genes, respectively. To minimize diurnal rhythm differences, all retinal tissue separations were completed between 08:00 and 10:00. Separated retinal tissues were rapidly collected on ice, flash-frozen in liquid nitrogen, and stored at -80°C. On the day of assay, the tissues were homogenized in perchloric acid solution and centrifuged, and the supernatant was used for neurotransmitter assays. Dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were detected using an Agilent 1200 HPLC system with an electrochemical detector. The chromatographic column was an Acclaim C18 column (2.2 μm, 2.1 × 100 mm), and peak area integration analysis was performed using ChemStation software. The results are expressed as pg content per retina.

[0055] Results of dopamine-related markers in mouse retina as follows Figure 5 As shown in the figure, Figure A shows the comparison results of DOPAC content in the retina of each group, Figure B shows the comparison results of dopamine content in the retina of each group, and Figure C shows the comparison results of DOPAC / dopamine ratio in the retina of each group.

[0056] The results of TFAM and SMN1 protein expression level detection are as follows: Figure 6 As shown, Figure A is the TFAM protein imprint map and its grayscale value statistics, and Figure B is the SMN1 protein imprint map and its grayscale value statistics.

[0057] The results showed that, compared with the wild-type defocus stimulation group, the Opn1mw gene knockout defocus stimulation group had significantly lower retinal dopamine levels, and the expression of TFAM and SMN1 proteins was significantly downregulated. These results suggest that dopamine-related molecular pathways can serve as a molecular basis for determining the high susceptibility to myopia in this model.

[0058] Example 3 Candidate intervention screening methods This embodiment designs a method for screening candidate interventions. Based on the Opn1mw gene knockout mouse defocus stimulation model established in Example 1, the candidate interventions are evaluated. The overall experimental procedure is as follows: Figure 1 As shown. The candidate interventions can be dopamine pathway regulators, metabolic regulators, mitochondrial function regulators, epigenetic regulators, or spectral intervention protocols.

[0059] Preferably, the candidate intervention is a dopamine D1 receptor agonist; more preferably, the D1 receptor agonist is SKF38393 or a pharmaceutically acceptable salt thereof. The candidate intervention can be administered to the Opn1mw gene knockout mouse defocus stimulation model via intraperitoneal injection, ocular drops, oral administration, or other suitable methods, and refractive status, ocular biological parameters, retinal dopamine levels, and TFAM and SMN1 expression levels are measured after treatment.

[0060] If the treatment group shows at least one of the following changes compared to the model control group: reduced myopic refractive changes, reduced axial elongation, increased or restored retinal dopamine levels, or restoration of TFAM and / or SMN1 expression towards normal, then the candidate intervention is deemed to have the potential to improve myopia susceptibility in the context of Opn1mw deficiency.

[0061] Example 4 Mechanism verification method This embodiment designs a mechanism verification method. A lens-induced myopia (LIM) model was established using 4-week-old (P4w) Opn1mw gene knockout mice. Five mice were included in the solvent control group and five in the SKF38393 treatment group.

[0062] The SKF38393 treatment group received intervention with the dopamine D1 receptor agonist SKF38393 hydrochloride (MedChemExpress, HY-12520A, CAS: 62717-42-4). The drug was dissolved in physiological saline and administered intraperitoneally at a dose of 18 nmol / g body weight, at a volume of 10 μL / g body weight, once daily at 9:00 AM, starting from the day of LIM modeling and continuing until the experimental endpoint P8w. The solvent control group received an equal volume of physiological saline.

[0063] Before the intervention began and after the last intervention, the refractive state (RE) and axial length (AL) of the right eye of mice in each group were measured. Results are as follows: Figure 7 As shown in the figures, Figure A compares the refractive error of the right eye before and after intervention in the SKF38393 treatment group and the solvent control group; Figure B compares the difference in refractive values ​​between the two eyes before and after intervention in the SKF38393 treatment group and the solvent control group; Figure C compares the axial length of the right eye before and after intervention in the SKF38393 treatment group and the solvent control group; and Figure D compares the difference in axial length between the two eyes before and after intervention in the SKF38393 treatment group and the solvent control group.

[0064] The results showed that, compared with the solvent control group, the refractive status of the right eye in the SKF38393 treatment group was improved and the axial length-related myopia changes were reduced, indicating that SKF38393 can inhibit the myopia phenotype induced by defocus stimulation in the context of Opn1mw gene knockout, suggesting that the dopamine D1 receptor-related pathway is involved in the formation of myopia susceptibility in the context of Opn1mw deficiency.

[0065] In summary This invention develops a method for assessing myopia susceptibility and screening candidate interventions based on the Opn1mw gene knockout mouse model. Using Opn1mw gene knockout mice as experimental subjects, this method comprehensively evaluates high myopia susceptibility by applying defocus stimulation and simultaneously detecting refractive status, ocular biological parameters, retinal dopamine levels, and TFAM and / or SMN1 expression levels. Furthermore, based on the model, candidate interventions can be screened and their effects evaluated. The preferred candidate interventions are dopamine receptor modulators, and more preferably D1 receptor agonists. The evaluation system established in this invention can stably characterize the high sensitivity to defocus stimulation in the Opn1mw knockout background and can be used for myopia-related mechanism research and candidate intervention strategy development.

[0066] The applicant declares that the above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any equivalent modifications or substitutions made within the scope of the technology disclosed in this invention should fall within the scope of protection of this invention.

Claims

1. A method for constructing a myopia animal model, characterized in that, include: Experimental animals with Opn1mw gene deletion were selected, and negative lens defocusing stimulation was applied to their experimental eyes to obtain a myopia animal model.

2. The method according to claim 1, characterized in that, The experimental animals are mice; preferably C57BL / 6J genetic background mice; preferably 4-week-old mice; the experimental eye is the right eye, and the left eye is used as an untreated control eye.

3. The method according to claim 1 or 2, characterized in that, The negative lens defocus stimulation is performed using a negative diopter lens; the diopter of the negative diopter lens is preferably -10D to -30D, more preferably -20D to -25D, and even more preferably -25D; the duration of the negative lens defocus stimulation is preferably 1 to 6 weeks.

4. The method according to any one of claims 1 to 3, characterized in that, After applying negative lens defocus stimulation, the method further includes detecting refractive state, ocular biological parameters, retinal dopamine level, TFAM expression level, and / or SMN1 expression level; wherein, the ocular biological parameters preferably include axial length and / or vitreous cavity depth; the detection method for retinal dopamine level preferably includes any one of high performance liquid chromatography, liquid chromatography-mass spectrometry, or enzyme-linked immunosorbent assay; the detection method for TFAM expression level and / or SMN1 expression level preferably includes any one of qPCR, Western blot, immunofluorescence, or proteomics analysis.

5. A method for screening candidate interventions, characterized in that, include: The candidate intervention is applied to the animal model constructed by the method of any one of claims 1 to 4, and the changes in myopia phenotype and / or molecular indicators in the candidate intervention treatment group and the control group are compared; when the candidate intervention reduces the myopia phenotype and / or restores the molecular indicators to the normal direction, the candidate intervention is determined to have myopia intervention potential.

6. The method according to claim 5, characterized in that, The candidate interventions include at least one of dopamine receptor modulators, metabolic regulators, mitochondrial function regulators, epigenetic regulators, or spectral intervention protocols; wherein the dopamine receptor modulators preferably include D1 receptor agonists, more preferably SKF38393 or a pharmaceutically acceptable salt thereof.

7. The method according to claim 5 or 6, characterized in that, The myopia phenotype includes refractive status and / or ocular biological parameters; the ocular biological parameters preferably include axial length and / or vitreous cavity depth; the molecular indicators include retinal dopamine levels, TFAM expression levels and / or SMN1 expression levels.

8. A method for verifying the molecular mechanisms related to myopia, characterized in that, Using an animal model constructed by the method described in any one of claims 1 to 4, the levels of retinal dopamine, TFAM expression, and / or SMN1 expression were detected before and after defocus stimulation to verify the role of dopamine-related pathways in the formation of myopia susceptibility.

9. The method according to claim 8, characterized in that, It also includes applying dopamine pathway regulators, mitochondrial function regulators, or epigenetic regulators, and observing synchronous changes in myopia phenotype and molecular indicators to verify the functional role of the target pathway.

10. Application of animal models with Opn1mw gene deletion in myopia susceptibility assessment and candidate intervention screening.

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