Human retina radiation injury model based on retina organoid and construction method

Abstract

The invention discloses a human retina radiation injury model based on retina organoid and a construction method, relates to the technical field of retina radiation injury detection, and aims to solve the problem of lack of a human-derived retina injury model. According to the method, cell apoptosis is detected through gamma-ray gradient dose irradiation in combination with flow cytometry, TUNEL dyeing and the like, oxidative stress and mitochondrial functions are analyzed through DCFH-DA and a JC-1 probe, a high-fitting-degree'irradiation dose-damage index 'curve is constructed, accurate evaluation from molecules to the functional level is achieved, the dose-time-effect rule of radiation damage is explored, and the method has the advantages of being high in accuracy, high in sensitivity and high in reliability. A standard intervention group and a control group are set, protection indexes, ROS and mitochondrial membrane potential changes serve as threshold values, a human retina organ radioactive injury model is successfully constructed, the drug curative effect is judged in a graded mode, and standardized evaluation of the protection effect of candidate drugs is achieved. Establishment of the model is expected to rapidly promote human retina radiation injury mechanism excavation and intervention measures of novel drugs.

Description

Technical Field

[0001] This invention relates to the field of retinal radiation injury detection technology, specifically to a human retinal radiation injury model based on retinal organoids and its construction method. Background Technology

[0002] Current technologies for studying radiation-induced retinopathy rely on animal models or two-dimensional cell lines, but these methods have significant limitations. Animal models, due to species differences, exhibit deviations in retinal structure, cellular composition, and radiation damage responses compared to humans, making it difficult to accurately simulate human pathological processes. Furthermore, they present ethical controversies and high costs. Two-dimensional cell lines cannot reproduce the complex multilayered structure of the retina and its intercellular interactions, failing to reflect the overall effects of radiation damage. Simultaneously, existing models lack a quantitative assessment system for the degree of radiation damage, making it difficult to accurately analyze the dose-time-damage correlation, thus hindering mechanistic research and drug development for radiation-induced retinopathy. Summary of the Invention

[0003] The purpose of this invention is to provide a human retinal radiation injury model and construction method based on retinal organoids. Through gamma-ray gradient dose irradiation, combined with flow cytometry and TUNEL staining to detect cell apoptosis, and DCFH-DA and JC-1 probe analysis to analyze oxidative stress and mitochondrial function, a highly fitted "irradiation dose-damage index" curve is constructed to achieve precise assessment from the molecular to the functional level. The dose-time-effect relationship of radiation injury is explored. Standardized intervention and control groups are set up, and the efficacy of drugs is graded and determined using the protection index, ROS, and mitochondrial membrane potential changes as thresholds. This achieves standardized evaluation of the protective effect of candidate drugs and can solve the problems in existing technologies.

[0004] To achieve the above objectives, the present invention provides the following technical solution: Human retinal radiation injury models based on retinal organoids include: human retinal organoid models, human retinal radiation injury models, and drug screening and evaluation models; The human retinal organoid model is generated by induction from human embryonic stem cells, mimicking the multilayered structure and cellular composition of the human retina. The human retinal radiation injury model is constructed by gamma-ray irradiation and quantitatively characterized based on a standard curve of irradiation dose and damage index. The drug screening and evaluation model is based on a human retinal radiation injury model, as well as protection indices and molecular indicator thresholds, to evaluate the efficacy of candidate drugs.

[0005] Methods for constructing human retinal radiation injury based on retinal organoids include: First, human retinal organoids were generated by directed induction from human embryonic stem cells using a human retinal organoid model. These organoids had multi-layered structures and various retinal cell types. Then, the retinal organoids were used to simulate specific maturation stages of the adult retinal structure and function. Then, using the human retinal radiation injury model, retinal organoids were subjected to radiation treatment and grouping, apoptosis detection, oxidative stress and mitochondrial function detection, and damage quantification model construction. Among them, radiation processing and grouping are used to: first set radiation parameters and control the radiation source to irradiate retinal organoids with quantitative gamma rays, and establish radiation damage groups with different doses, then simulate the retinal damage environment under different intensity radiation exposure, and finally collect phenotypic data of irradiated mature retinal organs. Radiation damage detection is used to: quantitatively assess the degree and mechanism of radiation damage from the molecular and functional levels using collected phenotypic data; and to detect the level of oxidative stress in retinal organoids and mitochondrial dysfunction caused by radiation damage. The damage quantification model was constructed to establish a correlation model between radiation dose and damage indicators based on data from radiation treatment and grouping, apoptosis detection, and oxidative stress and mitochondrial function detection. Finally, a drug screening and evaluation model was used to assess the protective effect of candidate drugs against radiation damage and to establish efficacy evaluation criteria.

[0006] Preferably, retinal organoids with multilayered structures and multiple retinal cell types are generated through directed induction from human embryonic stem cells, and the retinal organoids are modeled to simulate specific maturation stages of adult retinal structure and function, including: Human embryonic stem cells were cultured using a serum-free and feeder-free three-dimensional suspension culture system. Small molecule inhibitors and growth factors were added during the culture process, and after the addition was completed, the human embryonic stem cells aggregated to form three-dimensional embryonic bodies. The resulting three-dimensional embryos were transferred to a culture medium containing retinal-specific inducing factors of retinal acid and taurine. Three-dimensional embryos spontaneously form retinal organoids with optic vesicle-like structures in culture medium; The resulting retinal organoids were cultured in a dynamic suspension culture system for 90 days, with the culture medium containing retinal-specific inducing factors being changed periodically during the culture process. The cultured retinal organoids underwent molecular identification of retinal characteristics, including histological analysis and gene expression analysis. Histological analysis involved preparing paraffin or frozen sections of the cultured retinal organoids and observing their overall layered structure. Immunofluorescence techniques were then used to identify cell types, distribution, and proportions using marker antibodies for specific cell types. Gene expression analysis involved using quantitative polymerase chain reaction (PCR) to detect the expression levels of key genes related to retinal development and function in the cultured retinal organoids, and comparing these expression levels with data from mature retina in vivo. Finally, the results of histological and gene expression analysis were used to determine whether the retinal organoids fell within the scope of retinal organoids.

[0007] Preferably, radiation parameters are set, and the radiation source is controlled to quantitatively irradiate retinal organoids with gamma rays. Different doses of radiation damage are grouped, and retinal damage environments under different intensities of radiation exposure are simulated. Finally, phenotypic data of irradiated mature retinal organs are collected, including: The radiation parameters include the radiation source, dose rate, and irradiation method. The radiation source is cobalt-60 as the gamma-ray radiation source; the dose rate is 1.0 Gy / min, which is monitored and confirmed in real time by a radiation dosimeter; and the irradiation method is a single uniform irradiation mode. After the radiation parameters are set, the radiation source is controlled to irradiate the retinal organoids with quantitative gamma rays. The quantitative gamma ray irradiation process is as follows: the retinal organoids cultured for 90 days are transferred from the culture system to the well plate, the well plate is placed in the center of the cobalt-60 source irradiation platform, the center of the well plate is confirmed to be aligned with the focal point of the radiation source by laser positioning, the irradiation time is confirmed according to the set irradiation dose and fixed dose rate, and the irradiation start and stop are automatically controlled by the built-in timer of the radiation source. During quantitative gamma-ray irradiation, a multi-dose gradient setting was used to establish a basis for the correlation between dose and damage. In this study, retinal organoids were randomly divided into 6 groups: control group, 2Gy group, 5Gy group, 10Gy group, and 20Gy group, with 3 replicates in each group. The control group did not receive gamma-ray irradiation; the 2Gy, 5Gy, 10Gy, and 20Gy groups received the corresponding doses of gamma-ray irradiation. Simultaneously, it simulates retinal damage environments under different intensities of radiation exposure, including low-intensity radiation exposure simulation, medium-intensity radiation exposure simulation, and high-intensity radiation exposure simulation; The simulation included: low-intensity radiation exposure (2 Gy group) corresponding to clinical low-dose radiation, expected to cause mild cell damage; medium-intensity radiation exposure (5 Gy and 10 Gy groups) corresponding to conventional radiotherapy target dose, expected to cause moderate to severe apoptosis and structural damage, with the apoptosis rate within a preset range; and high-intensity radiation exposure (20 Gy group) corresponding to accidental high-dose exposure, with an apoptosis rate of 70%. Finally, the time-dependent changes in radiation damage to retinal organoids after irradiation were dynamically recorded, and phenotypic data were generated. The phenotypic data included data points and data contents, including morphological observation data, preliminary assessment data of survival status, and sample preservation data.

[0008] Preferably, the collected phenotypic data are used to quantitatively assess the degree and mechanism of radiation damage at both the molecular and functional levels. Simultaneously, the levels of oxidative stress in retinal organoids and mitochondrial dysfunction caused by radiation damage are detected, including: The quantitative assessment of the degree and mechanism of radiation damage at the molecular level is as follows: using molecular marker technology, the proportion of apoptotic cells is quantified and the damaged cells are located in the retinal layer. At the same time, the molecular mechanism of radiation damage is confirmed. First, the apoptosis rate is quantified by flow cytometry, and the process includes sample digestion, staining, detection and analysis. Then, TUNEL immunofluorescence staining is used to locate the apoptotic cell layer, and the process includes sample fixation, sectioning, staining, observation and analysis. The molecular level detection of oxidative stress and mitochondrial function indicators involves identifying the molecular mechanisms of oxidative and metabolic imbalances in radiation damage by detecting oxidative stress products and mitochondrial function indicators. The process includes sample processing, detection, and analysis, firstly using the DCFH-DA probe to detect reactive oxygen species levels, and then using the JC-1 probe to measure changes in mitochondrial membrane potential.

[0009] Preferably, the collected phenotypic data are used to quantitatively assess the degree and mechanism of radiation damage at both the molecular and functional levels. Simultaneously, the levels of oxidative stress in retinal organoids and mitochondrial dysfunction caused by radiation damage are detected. The method also includes: The quantitative assessment of the degree and mechanism of radiation damage at the functional level is as follows: by dynamically changing molecular indicators, the degree of damage to retinal cell function is correlated, and the pattern of dose, time and functional damage is confirmed. In this process, the degree of damage is first quantitatively graded, including combining flow cytometry to quantify the apoptosis rate, reactive oxygen species level and mitochondrial membrane potential changes of cells, and the degree of damage is graded as mild, moderate and moderate. Functional analysis of the damage mechanism based on the graded degree of damage, including the functional correlation of cell layer and metabolic function correlation; By comparing the changes in molecular indicators at different time points, time-damage curves were constructed. Finally, radiation damage detection of mature retinal organs was completed.

[0010] Preferably, the degree of damage is graded by combining flow cytometry to quantify cell apoptosis rate, reactive oxygen species levels, and changes in mitochondrial membrane potential, including: The fluorescence intensity corresponding to the change in mitochondrial membrane potential before and after modulation was measured. The fluorescence intensity before the change in mitochondrial membrane potential is taken as the first potential fluorescence intensity, and the fluorescence intensity after the change in mitochondrial membrane potential is taken as the second potential fluorescence intensity. The ratio of the fluorescence intensity at the first potential to the fluorescence intensity at the second potential is processed to obtain the first fluorescence intensity ratio; Obtain the fluorescence intensity corresponding to the changes in reactive oxygen species levels before and after the changes; The fluorescence intensity before the change in reactive oxygen species level is taken as the first reactive fluorescence intensity, and the fluorescence intensity after the change in reactive oxygen species level is taken as the second reactive fluorescence intensity. The ratio of the first active fluorescence intensity to the second active fluorescence intensity is processed to obtain the second fluorescence intensity ratio. Compare the first fluorescence intensity ratio and the second fluorescence intensity ratio; The degree of damage is graded based on the quantitative relationship between the first fluorescence intensity ratio and the second fluorescence intensity ratio.

[0011] Preferably, the degree of damage is graded according to the quantitative relationship between the first fluorescence intensity ratio and the second fluorescence intensity ratio, including: When the quantitative relationship between the first fluorescence intensity ratio and the second fluorescence intensity ratio indicates that the first fluorescence intensity ratio is greater than the second fluorescence intensity ratio, flow cytometry is used to quantify the apoptosis rate of cells. The apoptosis rate of the cells quantified by flow cytometry and the ratio of the second fluorescence intensity are processed to obtain the absolute difference between the apoptosis rate of the cells quantified by flow cytometry and the ratio of the second fluorescence intensity, which is taken as the first absolute difference. The damage degree coefficient is obtained by combining the first absolute difference with the first fluorescence intensity ratio. When the quantitative relationship between the first fluorescence intensity ratio and the second fluorescence intensity ratio indicates that the first fluorescence intensity ratio is not greater than the second fluorescence intensity ratio, then flow cytometry is used to quantify the apoptosis rate of cells. The apoptosis rate of the cells quantified by flow cytometry and the ratio of the first fluorescence intensity are processed to obtain the absolute difference between the apoptosis rate of the cells quantified by flow cytometry and the ratio of the first fluorescence intensity, which is used as the second absolute difference. The damage degree coefficient is obtained by combining the second absolute difference with the second fluorescence intensity ratio. The damage degree coefficient is compared with a preset first coefficient threshold and a second coefficient threshold; If the damage severity coefficient is lower than the first coefficient threshold, the current damage severity is determined to be minor. When the damage severity coefficient is not lower than the first coefficient threshold and the damage severity coefficient is lower than the second coefficient threshold, the current damage severity is determined to be moderate. If the damage severity coefficient is not lower than the second coefficient threshold, the current damage severity is determined to be severe.

[0012] Preferably, a correlation model between radiation dose and damage indicators is established based on data from radiation treatment and grouping, apoptosis detection, and oxidative stress and mitochondrial function detection, including: First, the data were organized, including the detection data of different irradiation dose groups and control groups at various time points, including cell apoptosis data, oxidative stress data and mitochondrial function data. The damage index data in the detection data is then confirmed and used as the core parameters of the model. The damage index data are the index data that are significantly correlated with the radiation dose. At each time point, each irradiation dose and corresponding damage index were paired and analyzed. A scatter plot was drawn with the irradiation dose as the x-axis and the quantitative value of each damage index as the y-axis, and the damage curve was finally obtained. The fitting effect of the damage curve is evaluated by calculating the coefficient of determination. When the coefficient of determination is >0.95, the fit is acceptable. If the coefficient of determination is <0.95, the data should be re-examined or the fitting method should be adjusted until the standard is met. The fitted curve data of apoptosis data, oxidative stress data and mitochondrial function data were integrated, and then a correlation model was established using a model correlation tool. The process of establishing the association model is as follows: First, the apoptosis data, oxidative stress data and mitochondrial function data are standardized, including unit unification and time anchor alignment. Machine learning tools were used to model the standardized data. The machine learning tools used the irradiation dose as the independent variable and the standardized parameters of apoptosis data, oxidative stress data and mitochondrial function data as the dependent variable to establish a coupling equation between dose and multiple indicators. Meanwhile, the coupling equations of dose and multiple indicators were cross-validated, and after successful validation, a correlation model between irradiation dose and damage indicators was obtained.

[0013] Preferably, the protective effect of candidate drugs against radiation damage is evaluated, and efficacy evaluation criteria are established, including: First, the drug intervention group and the control group were set up. The drug to be tested was added to the retinal organoids 1 hour before radiation treatment or immediately after irradiation. At the same time, a radiation damage control group without drug and a normal control group were set up. After the intervention and control groups were set up, the damage indicators after drug intervention were detected, and the detection method was the same as that for radiation damage detection. Based on the damage index data of the control group and the drug intervention group, the protection index was calculated, and the overall protection index of the drug was obtained after the protection index was calculated. Effective drugs are determined based on an overall protection index threshold. The criteria for determining an effective drug are: overall protection index ≥ 30%; reactive oxygen species level ≥ 25%; and mitochondrial membrane potential recovery ≥ 20%. Based on the threshold range for determining effective drugs, effective drugs are divided into Level 1 effective and Level 2 effective drugs; Finally, the efficacy evaluation criteria for effective drugs corresponding to retinal organoids were completed.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The human retinal radiation injury model and construction method based on retinal organoids provided by this invention adopts a serum-free, feeder-free three-dimensional suspension culture system, and sequentially adds signal pathway modulators to induce the generation of mature organoids containing eight major types of retinal cells in 90 days. Histological and gene expression analysis verified that it faithfully simulates the structure and function of the adult retina, overcomes the species differences of animal models and the defects of two-dimensional cell line structure, and provides an ideal in vitro model for radiation injury research.

[0015] 2. The human retinal radiation injury model and construction method based on retinal organoids provided by this invention, through gradient dose irradiation with gamma rays, combined with flow cytometry, TUNEL staining and other methods to detect cell apoptosis, DCFH-DA and JC-1 probe analysis of oxidative stress and mitochondrial function, constructs a high-fit "irradiation dose-damage index" curve, realizes accurate assessment from the molecular to the functional level, and explores the dose-time-effect law of radiation injury.

[0016] 3. The human retinal radiation injury model and construction method based on retinal organoids provided by this invention sets up a standardized intervention group and control group, and uses the protection index, ROS, and mitochondrial membrane potential changes as thresholds to grade and determine the efficacy of drugs, realizing the standardized evaluation of the protective effect of candidate drugs, providing an efficient screening tool for the development of new drugs for radiation-induced retinopathy, and solving the problem of the lack of unified standards for intervention evaluation of existing models. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the steps of the method for constructing a human retinal radiation injury model according to the present invention. Detailed Implementation

[0018] 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.

[0019] To address the shortcomings of existing technologies in studying radiation-induced retinopathy, such as the lack of ideal models, species-specific differences in animal models, and the inability of two-dimensional cell lines to simulate retinal structure, please refer to [link to relevant documentation]. Figure 1 This embodiment provides the following technical solution: Human retinal radiation injury models based on retinal organoids include: human retinal organoid models, human retinal radiation injury models, and drug screening and evaluation models; The human retinal organoid model is generated by induction from human embryonic stem cells, mimicking the multilayered structure and cellular composition of the human retina. The human retinal radiation injury model is constructed by gamma-ray irradiation and quantitatively characterized based on a standard curve of irradiation dose and damage index. The drug screening and evaluation model is based on a human retinal radiation injury model, as well as protection indices and molecular indicator thresholds, to evaluate the efficacy of candidate drugs.

[0020] Methods for constructing human retinal radiation injury based on retinal organoids include: First, human retinal organoids were generated by directed induction from human embryonic stem cells using a human retinal organoid model. These organoids had multi-layered structures and various retinal cell types. Then, the retinal organoids were used to simulate specific maturation stages of the adult retinal structure and function. Then, using the human retinal radiation injury model, retinal organoids were subjected to radiation treatment and grouping, apoptosis detection, oxidative stress and mitochondrial function detection, and damage quantification model construction. Among them, radiation processing and grouping are used to: first set radiation parameters and control the radiation source to irradiate retinal organoids with quantitative gamma rays, and establish radiation damage groups with different doses, then simulate the retinal damage environment under different intensity radiation exposure, and finally collect phenotypic data of irradiated mature retinal organs. Radiation damage detection is used to: quantitatively assess the degree and mechanism of radiation damage from the molecular and functional levels using collected phenotypic data; and to detect the level of oxidative stress in retinal organoids and mitochondrial dysfunction caused by radiation damage. The damage quantification model was constructed to establish a correlation model between radiation dose and damage indicators based on data from radiation treatment and grouping, apoptosis detection, and oxidative stress and mitochondrial function detection. Finally, a drug screening and evaluation model was used to assess the protective effect of candidate drugs against radiation damage and to establish efficacy evaluation criteria. The models and their corresponding construction methods are shown in the table below:

[0021] Specifically, a serum-free and feeder-free three-dimensional suspension culture was employed to eliminate fluctuations in serum components and interference from feeder cells, reducing exogenous variables. By adding small molecule inhibitors and growth factors, the stem cell differentiation pathway was precisely regulated, improving the uniformity and efficiency of three-dimensional embryonic body formation and laying a stable foundation for subsequent differentiation. Using a culture medium containing retinal-specific inducing factors such as retinal acid and taurine, the embryonic bodies were directionally induced to form vesicle-like structures, significantly improving the specificity of retinal organoid generation, reducing interference from irrelevant cell types, and ensuring the retinal properties of the organoids. The dynamic suspension culture system simulated the mechanical stimulation and material exchange state of the in vivo physiological microenvironment. The 90-day culture cycle matched the retinal development sequence, and regular medium changes ensured stable concentrations of inducing factors, promoting the structural maturation and functional perfection of the organoids. Histological analysis confirmed cell types through section observation and immunofluorescence. Gene expression analysis used qPCR to compare key genes with in vivo data, providing dual verification at the structural and molecular levels to ensure that the organoids reached the specific stage standards of mature retina, improving the realism and reliability of the model.

[0022] Directional induction of human embryonic stem cells to generate retinal organoids with multilayered structures and multiple retinal cell types, and simulation of specific maturation stages of adult retinal structure and function of the retinal organoids, including: Human embryonic stem cells were cultured using a serum-free and feeder-free three-dimensional suspension culture system. Small molecule inhibitors and growth factors were added during the culture process, and after the addition was completed, the human embryonic stem cells aggregated to form three-dimensional embryonic bodies. The resulting three-dimensional embryos were transferred to a culture medium containing retinal-specific inducing factors of retinal acid and taurine. Three-dimensional embryos spontaneously form retinal organoids with optic vesicle-like structures in culture medium; The resulting retinal organoids were cultured in a dynamic suspension culture system for 90 days, with the culture medium containing retinal-specific inducing factors being changed periodically during the culture process. The cultured retinal organoids underwent molecular identification of retinal characteristics, including histological analysis and gene expression analysis. Histological analysis involved preparing paraffin or frozen sections of the cultured retinal organoids and observing their overall layered structure. Immunofluorescence techniques were then used to identify cell types, distribution, and proportions using marker antibodies for specific cell types. Gene expression analysis involved using quantitative polymerase chain reaction (PCR) to detect the expression levels of key genes related to retinal development and function in the cultured retinal organoids, and comparing these expression levels with data from mature retina in vivo. Finally, the results of histological and gene expression analysis were used to determine whether the retinal organoids fell within the scope of retinal organoids.

[0023] Specifically, a serum-free and feeder-free three-dimensional suspension culture was employed to eliminate fluctuations in serum components and interference from feeder cells, reducing exogenous variables. By adding small molecule inhibitors and growth factors, the stem cell differentiation pathway was precisely regulated, improving the uniformity and efficiency of three-dimensional embryonic body formation and laying a stable foundation for subsequent differentiation. Using a culture medium containing retinal-specific inducing factors such as retinal acid and taurine, the embryonic bodies were directionally induced to form vesicle-like structures, significantly improving the specificity of retinal organoid generation, reducing interference from irrelevant cell types, and ensuring the retinal properties of the organoids. The dynamic suspension culture system simulated the mechanical stimulation and material exchange state of the in vivo physiological microenvironment. The 90-day culture cycle matched the retinal development sequence, and regular medium changes ensured stable concentrations of inducing factors, promoting the structural maturation and functional perfection of the organoids. Histological analysis confirmed cell types through section observation and immunofluorescence. Gene expression analysis used qPCR to compare key genes with in vivo data, providing dual verification at the structural and molecular levels to ensure that the organoids reached the specific stage standards of mature retina, improving the realism and reliability of the model.

[0024] Radiation parameters were set, and the radiation source was used to quantitatively irradiate retinal organoids with gamma rays. Different doses of radiation damage were grouped, and retinal damage environments under different intensities of radiation exposure were simulated. Finally, phenotypic data were collected from irradiated mature retinal organs, including: The radiation parameters include the radiation source, dose rate, and irradiation method. The radiation source is cobalt-60 as the gamma-ray radiation source; the dose rate is 1.0 Gy / min, which is monitored and confirmed in real time by a radiation dosimeter; and the irradiation method is a single uniform irradiation mode. After the radiation parameters are set, the radiation source is controlled to irradiate the retinal organoids with quantitative gamma rays. The quantitative gamma ray irradiation process is as follows: the retinal organoids cultured for 90 days are transferred from the culture system to the well plate, the well plate is placed in the center of the cobalt-60 source irradiation platform, the center of the well plate is confirmed to be aligned with the focal point of the radiation source by laser positioning, the irradiation time is confirmed according to the set irradiation dose and fixed dose rate, and the irradiation start and stop are automatically controlled by the built-in timer of the radiation source. During quantitative gamma-ray irradiation, a multi-dose gradient setting was used to establish a basis for the correlation between dose and damage. In this study, retinal organoids were randomly divided into 6 groups: control group, 2Gy group, 5Gy group, 10Gy group, and 20Gy group, with 3 replicates in each group. The control group did not receive gamma-ray irradiation; the 2Gy, 5Gy, 10Gy, and 20Gy groups received the corresponding doses of gamma-ray irradiation. Simultaneously, it simulates retinal damage environments under different intensities of radiation exposure, including low-intensity radiation exposure simulation, medium-intensity radiation exposure simulation, and high-intensity radiation exposure simulation; The simulation included: low-intensity radiation exposure (2 Gy group) corresponding to clinical low-dose radiation, expected to cause mild cell damage; medium-intensity radiation exposure (5 Gy and 10 Gy groups) corresponding to conventional radiotherapy target dose, expected to cause moderate to severe apoptosis and structural damage, with the apoptosis rate within a preset range; and high-intensity radiation exposure (20 Gy group) corresponding to accidental high-dose exposure, with an apoptosis rate of 70%. Finally, the time-dependent changes in radiation damage to retinal organoids after irradiation were dynamically recorded, and phenotypic data were generated. The phenotypic data included data points and data contents, including morphological observation data, preliminary assessment data of survival status, and sample preservation data.

[0025] Specifically, Cobalt-60 was selected as the gamma-ray source because its radiation energy is stable and easy to quantify. A fixed dose rate of 1.0 Gy / min, combined with real-time monitoring by a radiation dosimeter, ensures accurate and traceable irradiation intensity. The single-stage uniform irradiation mode avoids the cumulative dose error caused by multiple irradiations, ensuring the consistency of radiation dose from the source. Laser positioning ensures that the center of the well plate is strictly aligned with the focal point of the radiation source, eliminating dose unevenness caused by spatial position deviation. The irradiation time is calculated by back-calculating the irradiation dose and fixed dose rate, and the built-in timer automatically starts and stops, achieving accurate conversion between dose and time. This significantly reduces human error and improves repeatability within and between groups. The six gradients cover a 0 Gy blank control, a control group, and a multi-dose range of 2-20 Gy. The setting of three replicates in each group meets the requirements for statistical repeatability. Multi-dose gradients not only construct complete dose-damage correlation curves, but also effectively distinguish radiation-specific damage from environmental interference through control designs. Low, medium, and high-intensity irradiation correspond to clinical low-dose radiation, conventional radiotherapy target areas, and accidental high-dose exposure, respectively. The expected damage and actual radiation damage characteristics are highly matched, enabling the model to directly serve clinical radiation risk assessment and protection research. Time-dependent recording, combined with data on morphology, survival status, and sample preservation, comprehensively captures the temporal evolution of radiation damage, providing a complete dynamic data chain for analyzing damage mechanisms.

[0026] To address the current limitations of effective assessment methods for radiation-induced retinopathy, and the inability of existing models to quantitatively assess the extent and mechanism of damage at the molecular and functional levels, thus failing to accurately analyze the patterns of radiation damage, please refer to [the relevant documentation / reference]. Figure 1 This embodiment provides the following technical solution: The collected phenotypic data will be used to quantitatively assess the degree and mechanism of radiation damage at both the molecular and functional levels. Simultaneously, the levels of oxidative stress in retinal organoids and mitochondrial dysfunction induced by radiation damage will be detected, including: The quantitative assessment of the degree and mechanism of radiation damage at the molecular level is as follows: using molecular marker technology, the proportion of apoptotic cells is quantified and the damaged cells are located in the retinal layer. At the same time, the molecular mechanism of radiation damage is confirmed. First, the apoptosis rate is quantified by flow cytometry, and the process includes sample digestion, staining, detection and analysis. Then, TUNEL immunofluorescence staining is used to locate the apoptotic cell layer, and the process includes sample fixation, sectioning, staining, observation and analysis. The molecular level detection of oxidative stress and mitochondrial function indicators is as follows: by detecting oxidative stress products and mitochondrial function indicators, the molecular mechanism of oxidative and metabolic imbalance in radiation damage is confirmed. Specifically, the DCFH-DA probe is first used to detect reactive oxygen species levels, and the process involves sample processing, detection and analysis. Then, the JC-1 probe is used to measure changes in mitochondrial membrane potential, and the process involves sample processing, detection and analysis. The specific procedure for quantifying cell apoptosis rate by flow cytometry is as follows: Retinal organoids are collected at various time points after irradiation (24h, 72h, 7d, 14d), gently digested into single-cell suspensions with trypsin, passed through a 200-mesh sieve to remove impurities, and the cell concentration is adjusted to... Annexin V-FITC (labeling early apoptotic cells) and PI (propidium iodide, labeling late apoptotic / necrotic cells) were added and incubated in the dark for 15 minutes. Fluorescence signals were detected by flow cytometry, and the percentage of apoptotic cells in the total cells was calculated to obtain apoptosis rate data at different irradiation doses and time points. The specific procedure for TUNEL immunofluorescence staining to locate apoptotic cell layers is as follows: Irradiated organoids are fixed with 4% paraformaldehyde for 24 hours, dehydrated, embedded in paraffin blocks, cut into 5μm thick continuous sections, mounted on glass slides, dewaxed, and treated with a TUNEL detection kit to fluorescently label the DNA breaks in apoptotic cells; simultaneously, immunofluorescence co-staining is performed with retinal cell layer-specific antibodies to distinguish different cell layers, and the number and proportion of TUNEL cells in each retinal layer are counted by confocal microscopy to identify the cell layers preferentially affected by radiation damage.

[0027] The quantitative assessment of the degree and mechanism of radiation damage at the functional level is as follows: by dynamically changing molecular indicators, the degree of damage to retinal cell function is correlated, and the pattern of dose, time and functional damage is confirmed. In this process, the degree of damage is first quantitatively graded, including combining flow cytometry to quantify the apoptosis rate, reactive oxygen species level and mitochondrial membrane potential changes of cells, and the degree of damage is graded as mild, moderate and moderate. The specific procedure for detecting reactive oxygen species (ROS) levels using the DCFH-DA probe is as follows: irradiated organoids are collected, washed three times with PBS, and transferred to serum-free culture medium containing 10 μM DCFH-DA probe (2',7'-dichlorofluorescein diacetate). The culture is then incubated at 37°C for 30 minutes. The fluorescence intensity of the DCF (excitation wavelength 488 nm, emission wavelength 525 nm) is detected using a fluorescence microscope or flow cytometry. The fluorescence intensity is positively correlated with ROS levels, and the relative increase in ROS levels for different dose groups is calculated. The specific procedure for measuring changes in mitochondrial membrane potential using the JC-1 probe is as follows: irradiated organoids are taken and incubated with JC-1 staining solution (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbonyl cyanine iodide) for 20 minutes. The ratio of red to green fluorescence is observed using a fluorescence microscope, or the ratio of red to green fluorescence intensity is detected by flow cytometry. A decrease in the ratio indicates a decrease in mitochondrial membrane potential (impaired function). The relative change rate of mitochondrial membrane potential in different dose groups is calculated. Functional analysis of the damage mechanism based on the graded degree of damage, including the functional correlation of cell layer and metabolic function correlation; By comparing the changes in molecular indicators at different time points, time-damage curves were constructed. Finally, radiation damage detection of mature retinal organs was completed.

[0028] Specifically, by combining flow cytometry to quantify cell apoptosis rate, reactive oxygen species levels, and changes in mitochondrial membrane potential, the degree of damage is graded, including: The fluorescence intensity corresponding to the change in mitochondrial membrane potential before and after modulation was measured. The fluorescence intensity before the change in mitochondrial membrane potential is taken as the first potential fluorescence intensity, and the fluorescence intensity after the change in mitochondrial membrane potential is taken as the second potential fluorescence intensity. The ratio of the fluorescence intensity at the first potential to the fluorescence intensity at the second potential is processed to obtain the first fluorescence intensity ratio; Obtain the fluorescence intensity corresponding to the changes in reactive oxygen species levels before and after the changes; The fluorescence intensity before the change in reactive oxygen species level is taken as the first reactive fluorescence intensity, and the fluorescence intensity after the change in reactive oxygen species level is taken as the second reactive fluorescence intensity. The ratio of the first active fluorescence intensity to the second active fluorescence intensity is processed to obtain the second fluorescence intensity ratio. Compare the first fluorescence intensity ratio and the second fluorescence intensity ratio; The degree of damage is graded based on the quantitative relationship between the first fluorescence intensity ratio and the second fluorescence intensity ratio.

[0029] By converting the fluorescence intensity before and after changes in mitochondrial membrane potential and reactive oxygen species (ROS) levels into "first fluorescence intensity ratio" and "second fluorescence intensity ratio," the dynamic changes in fluorescence signals, which were originally qualitative or semi-quantitative, are transformed into quantitative data that can be accurately calculated and compared horizontally. This avoids the ambiguity of directly relying on absolute fluorescence intensity values ​​for assessment and improves the quantitative accuracy of damage-related indicators. Using the "fluorescence intensity ratio before and after change" as the core assessment parameter eliminates the influence of interference factors such as differences in initial fluorescence intensity among different samples (e.g., differences in initial cell viability and staining efficiency) and fluctuations in the sensitivity of detection equipment. This provides a unified benchmark for comparison of assessment data from different batches and samples, reducing the interference of detection environment and individual sample differences on assessment results and improving the stability and accuracy of the assessment. Focusing on the two core damage dimensions of mitochondrial membrane potential (reflecting cellular energy metabolism damage) and ROS levels (reflecting cellular oxidative stress damage), by comparing the quantitative relationship between the fluorescence intensity ratios of these two, the assessment system incorporates the synergistic damage information of "energy metabolism-oxidative stress," which cannot be covered by a single indicator. Compared with single indicator assessment, this provides a more comprehensive coverage of the molecular mechanisms of radiation damage, making the damage severity grading more consistent with the actual damage state. Using a "ratio-based quantitative relationship" as the grading criterion replaces the assessment method that relies on subjective judgment, clarifies the quantitative standards for grading, and reduces the influence of human experience on the grading results. Simultaneously, the entire process is based on the post-processing of routine flow cytometry data, eliminating the need for additional complex detection methods. This lowers the technical implementation threshold while ensuring assessment accuracy, and improves the scalability and reproducibility of the protocol. Changes in mitochondrial membrane potential and reactive oxygen species (ROS) levels have an intrinsic regulatory relationship (e.g., a decrease in membrane potential exacerbates ROS accumulation, while an increase in ROS further damages the membrane potential). By comparing the quantitative relationship between these two ratios, the synergistic effect of "energy metabolism disorder - intensified oxidative stress" during the injury process can be indirectly reflected. This allows the injury grading results to not only reflect the degree of injury but also implicitly contain information on the molecular mechanisms of injury progression, providing a more targeted reference for subsequent injury intervention.

[0030] Specifically, the degree of damage is graded based on the quantitative relationship between the first fluorescence intensity ratio and the second fluorescence intensity ratio, including: When the quantitative relationship between the first fluorescence intensity ratio and the second fluorescence intensity ratio indicates that the first fluorescence intensity ratio is greater than the second fluorescence intensity ratio, flow cytometry is used to quantify the apoptosis rate of cells. The apoptosis rate of the cells quantified by flow cytometry and the ratio of the second fluorescence intensity are processed to obtain the absolute difference between the apoptosis rate of the cells quantified by flow cytometry and the ratio of the second fluorescence intensity, which is taken as the first absolute difference. The damage degree coefficient is obtained by combining the first absolute difference with the first fluorescence intensity ratio. The damage degree coefficient is obtained by the following formula:

[0031] Where U represents the damage degree coefficient; and These represent the ratio of the first fluorescence intensity and the ratio of the second fluorescence intensity, respectively. Indicates the first absolute difference; P represents the apoptosis rate of cells measured by flow cytometry. When the quantitative relationship between the first fluorescence intensity ratio and the second fluorescence intensity ratio indicates that the first fluorescence intensity ratio is not greater than the second fluorescence intensity ratio, then flow cytometry is used to quantify the apoptosis rate of cells. The apoptosis rate of the cells quantified by flow cytometry and the ratio of the first fluorescence intensity are processed to obtain the absolute difference between the apoptosis rate of the cells quantified by flow cytometry and the ratio of the first fluorescence intensity, which is used as the second absolute difference. The damage degree coefficient is obtained by combining the second absolute difference with the second fluorescence intensity ratio. The damage degree coefficient is obtained by the following formula:

[0032] Where U represents the damage degree coefficient; and These represent the ratio of the first fluorescence intensity and the ratio of the second fluorescence intensity, respectively. Indicates the second absolute difference; P represents the apoptosis rate of cells measured by flow cytometry. The damage degree coefficient is compared with a preset first coefficient threshold and a second coefficient threshold; If the damage severity coefficient is lower than the first coefficient threshold, the current damage severity is determined to be minor. When the damage severity coefficient is not lower than the first coefficient threshold and the damage severity coefficient is lower than the second coefficient threshold, the current damage severity is determined to be moderate. If the damage severity coefficient is not lower than the second coefficient threshold, the current damage severity is determined to be severe.

[0033] This embodiment integrates two different dimensions of indicators—fluorescence intensity ratio and apoptosis rate quantified by flow cytometry—to comprehensively capture damage-related biological information, avoiding the one-sidedness of single-indicator assessment. Based on the quantitative relationship between the first and second fluorescence intensity ratios, it employs targeted damage severity coefficient calculation methods for different scenarios, accurately adapting to different indicator relationship scenarios and significantly improving the accuracy of damage severity quantification. By using preset coefficient thresholds for graded judgment, the damage severity is standardized into mild, moderate, and severe, transforming damage severity assessment from qualitative to quantitative and standardized, significantly reducing human subjective judgment errors, enhancing the objectivity and consistency of assessment results, and providing a more scientific, reliable, and operable quantitative basis for damage-related research, diagnosis, or intervention, thus contributing to the standardization and precision development of assessment systems in related fields.

[0034] On the other hand, the ratio of the first fluorescence intensity to the second fluorescence intensity reflects the relative changes at the molecular level of fluorescently labeled molecules associated with damage (which can be understood as the signal difference of damage at the molecular labeling level); the apoptosis rate quantified by flow cytometry is a direct quantitative indicator of the apoptosis level at the cell population level (representing the degree of cell fate change caused by damage); absolute difference ( , This reflects the degree of difference between the "apoptosis rate" and the "fluorescence intensity ratio" (reflecting the synergistic / discordant relationship between changes in molecular markers and changes in apoptosis). The formula uses a product ( or The operation logic of the device couples the relative changes of molecular markers with the quantitative information of cell population apoptosis, so that the damage degree coefficient (U) can comprehensively reflect the multi-scale damage effect "from molecular marker abnormality to cell apoptosis", and realize the cross-level biological information integration of the damage process.

[0035] Simultaneously, integrating "labeling changes at the fluorescent molecular level" and "apoptosis ratio at the cell population level" covers the complete process of damage from "molecular expression to changes in cell function," overcoming the limitation of a single indicator "only reflecting local damage characteristics" and making damage assessment more comprehensive. This embodiment allows for a more precise differentiation of damage severity coefficients into different damage levels, such as "mild, moderate, and severe," avoiding the problems of "fuzzy grading and unclear boundaries" in traditional assessments. Combining "quantitative calculation with multi-indicator coupling" with "standardized grading based on preset thresholds" replaces the traditional assessment method relying on subjective human judgment, upgrading damage severity from "fuzzy qualitative" to "precise quantitative + standardized grading," improving the objectivity and reproducibility of assessment results, and providing a more reliable quantitative tool for damage diagnosis, dynamic monitoring, or mechanism research.

[0036] Specifically, molecular-level damage assessment employs a combined approach of flow cytometry and TUNEL immunofluorescence staining: Flow cytometry, through standardized sample processing, staining, and analysis procedures, can accurately quantify the apoptosis rate, providing an objective numerical benchmark for the degree of damage; TUNEL immunofluorescence staining, through sample fixation, sectioning, and specific staining, clearly locates the retinal layers where apoptotic cells are located, achieving spatial characterization of the location and proportion of damage. The combination of these two methods comprehensively presents the molecular-level characteristics of radiation damage. Oxidative stress and mitochondrial function detection utilize specific probe technologies, combining accuracy and specificity: the DCFH-DA probe can sensitively capture changes in reactive oxygen species levels, directly reflecting the intensity of radiation-induced oxidative stress; the JC-1 probe can quantitatively monitor mitochondrial membrane potential fluctuations through fluorescence signal conversion, directly correlated with mitochondrial functional status. Both tests ensured data reliability through standardized operating procedures, providing key molecular evidence for elucidating the mechanism of radiation-induced oxidative-metabolic imbalance. From the quantification and localization of apoptosis and the level of oxidative stress to the functional state of mitochondria, a multi-dimensional and progressive detection system was constructed, which not only achieved quantitative assessment of the degree of damage but also revealed the intrinsic relationship between various pathological processes. At the same time, the standardized operating procedures ensured the reproducibility of results, laying a systematic and reliable experimental foundation for in-depth research on the mechanism of radiation damage.

[0037] To address the lack of effective interventions for radiation-induced retinopathy in existing technologies, the limitations of animal models due to species differences, ethical controversies, and high costs, and the inability of traditional two-dimensional cell lines to reproduce retinal structure and cell interactions, please refer to [the relevant documentation / reference needed]. Figure 1 This embodiment provides the following technical solution: A correlation model between radiation dose and damage indices was established based on data from radiation treatment and grouping, apoptosis detection, and oxidative stress and mitochondrial function testing, including: First, the data were organized, including the detection data of different irradiation dose groups and control groups at various time points, including cell apoptosis data, oxidative stress data and mitochondrial function data. The damage index data in the detection data is then confirmed and used as the core parameters of the model. The damage index data are the index data that are significantly correlated with the radiation dose. At each time point, each irradiation dose and corresponding damage index were paired and analyzed. A scatter plot was drawn with the irradiation dose as the x-axis and the quantitative value of each damage index as the y-axis, and the damage curve was finally obtained. The fitting effect of the damage curve is evaluated by calculating the coefficient of determination. When the coefficient of determination is >0.95, the fit is acceptable. If the coefficient of determination is <0.95, the data should be re-examined or the fitting method should be adjusted until the standard is met. The fitted curve data of apoptosis data, oxidative stress data and mitochondrial function data were integrated, and then a correlation model was established using a model correlation tool. The process of establishing the association model is as follows: First, the apoptosis data, oxidative stress data and mitochondrial function data are standardized, including unit unification and time anchor alignment. Machine learning tools were used to model the standardized data. The machine learning tools used the irradiation dose as the independent variable and the standardized parameters of apoptosis data, oxidative stress data and mitochondrial function data as the dependent variable to establish a coupling equation between dose and multiple indicators. Meanwhile, the coupling equations of dose and multiple indicators were cross-validated, and after successful validation, a correlation model between irradiation dose and damage indicators was obtained.

[0038] Specifically, by retrieving data on apoptosis, oxidative stress, and mitochondrial function at various time points in different dose groups and the control group, multi-dimensional and dynamic data coverage was achieved. This included both dose gradient differences and the capture of time-dependent changes in damage, providing a rich variable base for the model. Focusing on damage indicators significantly correlated with irradiation dose as core parameters, and eliminating irrelevant variables, the model's core variables were ensured to have biological relevance, improving the specificity of the dose-damage correlation. Paired analysis of dose and damage indicators was performed at different time points, and the relationship between the two was visually presented through scatter plots and damage curves, taking into account the time dynamic dimension. A strict standard of a coefficient of determination ≥0.95 was used to verify the model's effectiveness. By recalibrating the data and adjusting the fitting method, high accuracy of curve fitting was ensured and model error was reduced. The fitting curve data of cell apoptosis, oxidative stress and mitochondrial function were integrated, and a comprehensive model was constructed through model correlation tools. This broke through the limitations of single indicators and can simultaneously reflect the multi-pathway mechanism of radiation damage (such as cell death and metabolic imbalance). It reveals the complex relationship between dose and overall damage effect. Through standardized data processing, rigorous fitting verification and multi-dimensional integration, a highly reliable dose-damage correlation model was constructed. This provides an accurate tool for quantitatively assessing the degree of radiation damage and analyzing the dose-effect relationship, and also lays a scientific foundation for setting dose benchmarks for subsequent drug screening.

[0039] To evaluate the protective effect of candidate drugs against radiation damage and establish efficacy evaluation criteria, including: First, the drug intervention group and the control group were set up. The drug to be tested was added to the retinal organoids 1 hour before radiation treatment or immediately after irradiation. At the same time, a radiation damage control group without drug and a normal control group were set up. After the intervention and control groups were set up, the damage indicators after drug intervention were detected, and the detection method was the same as that for radiation damage detection. Based on the damage index data of the control group and the drug intervention group, the protection index was calculated, and the overall protection index of the drug was obtained after the protection index was calculated. Effective drugs are determined based on an overall protection index threshold. The criteria for determining an effective drug are: overall protection index ≥ 30%; reactive oxygen species level ≥ 25%; and mitochondrial membrane potential recovery ≥ 20%. Based on the threshold range for determining effective drugs, effective drugs are divided into Level 1 effective and Level 2 effective drugs; Finally, the efficacy evaluation criteria for effective drugs corresponding to retinal organoids were completed.

[0040] Specifically, a radiation injury control group without medication and a normal control group were set up. The drug administration time points covered both pre-radiation prevention and post-radiation treatment scenarios, allowing for precise differentiation between the drug's specific protective effect and the natural recovery effect. Environmental interference factors were eliminated, ensuring the independence of the efficacy assessment. The detection of damage indicators after drug intervention used the same methods as the radiation injury model, ensuring data traceability and comparability. This established the efficacy assessment on a unified benchmark, reducing errors caused by methodological differences. Quantitative characterization of efficacy was achieved through the calculation of the protection index, with clearly defined thresholds (overall protection index ≥30%, reactive oxygen species level ≥25%). The mitochondrial membrane potential recovery (≥20%) transforms qualitative descriptions into verifiable numerical standards, avoiding subjective judgment bias and improving the reliability of results. Effective drugs are classified into primary and secondary levels, and the efficacy intensity is distinguished based on differences in multi-dimensional indicators, providing a tiered reference for subsequent drug development. This facilitates the rapid screening of candidate drugs with outstanding clinical potential. Through standardized control design, unified detection methods, quantitative evaluation standards, and a grading system, a complete chain from efficacy assessment to result classification is constructed, ensuring the scientific nature of experimental data and enhancing the practicality of clinical translation, laying a reliable foundation for the efficient screening of radiation damage protection drugs.

[0041] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0042] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention.

Claims

1. A human retinal radiation injury model based on retinal organoids, characterized in that, include: Human retinal organoid models, human retinal radiation injury models, and drug screening and evaluation models; The human retinal organoid model is generated by induction from human embryonic stem cells, mimicking the multilayered structure and cellular composition of the human retina. The human retinal radiation injury model is constructed by gamma-ray irradiation, and quantitative analysis is achieved based on the standard curve of irradiation dose and damage index. The drug screening and evaluation model is based on a human retinal radiation injury model, as well as protection indices and molecular indicator thresholds, to conduct high-throughput and rapid efficacy assessment of candidate drugs.

2. A method for constructing a human retinal radiation injury model based on retinal organoids, applied in the human retinal radiation injury model based on retinal organoids as described in claim 1, characterized in that, include: First, human retinal organoids were generated by directed induction from human embryonic stem cells using a human retinal organoid model. These organoids had multi-layered structures and various retinal cell types. Then, the retinal organoids were used to simulate specific maturation stages of the adult retinal structure and function. Then, using the human retinal radiation injury model, retinal organoids were subjected to radiation treatment and grouping, apoptosis detection, oxidative stress and mitochondrial function detection, and damage quantification model construction. Among them, radiation processing and grouping are used to: first set radiation parameters and control the radiation source to irradiate retinal organoids with quantitative gamma rays, and establish radiation damage groups with different doses, then simulate the retinal damage environment under different intensity radiation exposure, and finally collect phenotypic data of irradiated retinal organs. Radiation damage detection is used to: quantitatively assess the degree and mechanism of radiation damage from the molecular and functional levels using collected phenotypic data; and to detect the level of oxidative stress and mitochondrial dysfunction in human retinal organoids caused by radiation damage. The damage quantification model was constructed to establish a correlation model between radiation dose and damage indicators based on data from radiation treatment and grouping, apoptosis detection, and oxidative stress and mitochondrial function detection. Finally, a drug screening and evaluation model was used to assess the protective effect of candidate drugs against radiation damage and to establish efficacy evaluation criteria.

3. The method for constructing human retinal radiation injury based on retinal organoids according to claim 2, characterized in that, Directional induction of human embryonic stem cells to generate retinal organoids with multilayered structures and multiple retinal cell types, and simulation of specific maturation stages of adult retinal structure and function of the retinal organoids, including: Human embryonic stem cells were cultured using a serum-free and feeder-free three-dimensional suspension culture system. Small molecule inhibitors and growth factors were added during the culture process, and after the addition was completed, the human embryonic stem cells aggregated to form three-dimensional embryonic bodies. The resulting three-dimensional embryos were transferred to a culture medium containing retinal-specific inducing factors of retinal acid and taurine. Three-dimensional embryos spontaneously form retinal organoids with optic vesicle-like structures in culture medium; The resulting retinal organoids were cultured in a dynamic suspension culture system for 90 days, with the culture medium containing retinal-specific inducing factors being changed periodically during the culture process. The cultured retinal organoids underwent molecular identification of retinal characteristics, including histological analysis and gene expression analysis. Histological analysis involved preparing paraffin or frozen sections of the cultured retinal organoids and observing their overall layered structure. Immunofluorescence techniques were then used to identify cell types, distribution, and proportions using marker antibodies for specific cell types. Gene expression analysis involved using quantitative polymerase chain reaction (PCR) to detect the expression levels of key genes related to retinal development and function in the cultured retinal organoids, and comparing these expression levels with data from mature retina in vivo. Finally, the results of histological and gene expression analysis were used to determine whether the retinal organoids fell within the scope of retinal organoids.

4. The method for constructing human retinal radiation injury based on retinal organoids according to claim 3, characterized in that, Radiation parameters were set, and the radiation source was used to quantitatively irradiate retinal organoids with gamma rays. Different doses of radiation damage were grouped, and retinal damage environments under different intensities of radiation exposure were simulated. Finally, phenotypic data were collected from irradiated mature retinal organs, including: The radiation parameters include the radiation source, dose rate, and irradiation method. The radiation source is cobalt-60 as the gamma-ray radiation source; the dose rate is 1.0 Gy / min, which is monitored and confirmed in real time by a radiation dosimeter; and the irradiation method is a single uniform irradiation mode. After the radiation parameters are set, the radiation source is controlled to irradiate the retinal organoids with quantitative gamma rays. The quantitative gamma ray irradiation process is as follows: the retinal organoids cultured for 90 days are transferred from the culture system to the well plate, the well plate is placed in the center of the cobalt-60 source irradiation platform, the center of the well plate is confirmed to be aligned with the focal point of the radiation source by laser positioning, the irradiation time is confirmed according to the set irradiation dose and fixed dose rate, and the irradiation start and stop are automatically controlled by the built-in timer of the radiation source. During quantitative gamma-ray irradiation, a multi-dose gradient setting was used to establish a basis for the correlation between dose and damage. In this study, retinal organoids were randomly divided into 6 groups: control group, 2Gy group, 5Gy group, 10Gy group, and 20Gy group, with 3 replicates in each group. The control group did not receive gamma-ray irradiation; the 2Gy, 5Gy, 10Gy, and 20Gy groups received the corresponding doses of gamma-ray irradiation. Simultaneously, it simulates retinal damage environments under different intensities of radiation exposure, including low-intensity radiation exposure simulation, medium-intensity radiation exposure simulation, and high-intensity radiation exposure simulation; The simulation included: low-intensity radiation exposure (2 Gy group) corresponding to clinical low-dose radiation, expected to cause mild cell damage; medium-intensity radiation exposure (5 Gy and 10 Gy groups) corresponding to conventional radiotherapy target dose, expected to cause moderate to severe apoptosis and structural damage, with the apoptosis rate within a preset range; and high-intensity radiation exposure (20 Gy group) corresponding to accidental high-dose exposure, with an apoptosis rate of 70%. Finally, the time-dependent changes in radiation damage to retinal organoids after irradiation were dynamically recorded, and phenotypic data were generated. The phenotypic data included data points and data contents, including morphological observation data, preliminary assessment data of survival status, and sample preservation data.

5. The method for constructing human retinal radiation injury based on retinal organoids according to claim 4, characterized in that, The collected phenotypic data will be used to quantitatively assess the degree and mechanism of radiation damage at both the molecular and functional levels. Simultaneously, the levels of oxidative stress in retinal organoids and mitochondrial dysfunction induced by radiation damage will be detected, including: The quantitative assessment of the degree and mechanism of radiation damage at the molecular level is as follows: using molecular marker technology, the proportion of apoptotic cells is quantified and the damaged cells are located in the retinal layer. At the same time, the molecular mechanism of radiation damage is confirmed. First, the apoptosis rate is quantified by flow cytometry, and the process includes sample digestion, staining, detection and analysis. Then, TUNEL immunofluorescence staining is used to locate the apoptotic cell layer, and the process includes sample fixation, sectioning, staining, observation and analysis. The molecular level detection of oxidative stress and mitochondrial function indicators involves identifying the molecular mechanisms of oxidative and metabolic imbalances in radiation damage by detecting oxidative stress products and mitochondrial function indicators. The process includes sample processing, detection, and analysis, firstly using the DCFH-DA probe to detect reactive oxygen species levels, and then using the JC-1 probe to measure changes in mitochondrial membrane potential.

6. The method for constructing human retinal radiation injury based on retinal organoids according to claim 5, characterized in that, The collected phenotypic data will be used to quantitatively assess the degree and mechanism of radiation damage at both the molecular and functional levels. Simultaneously, the levels of oxidative stress in retinal organoids and mitochondrial dysfunction caused by radiation damage will be detected. Other aspects include: The quantitative assessment of the degree and mechanism of radiation damage at the functional level is as follows: by dynamically changing molecular indicators, the degree of damage to retinal cell function is correlated, and the pattern of dose, time and functional damage is confirmed. In this process, the degree of damage is first quantitatively graded, including combining flow cytometry to quantify the apoptosis rate, reactive oxygen species level and mitochondrial membrane potential changes of cells, and the degree of damage is graded as mild, moderate and moderate. Functional analysis of the damage mechanism based on the graded degree of damage, including the functional correlation of cell layer and metabolic function correlation; By comparing the changes in molecular indicators at different time points, time-damage curves were constructed. Finally, radiation damage detection of mature retinal organs was completed.

7. The method for constructing human retinal radiation injury based on retinal organoids according to claim 6, characterized in that, By combining flow cytometry to quantify cell apoptosis rate, reactive oxygen species levels, and changes in mitochondrial membrane potential, the degree of damage was graded, including: The fluorescence intensity corresponding to the change in mitochondrial membrane potential before and after modulation was measured. The fluorescence intensity before the change in mitochondrial membrane potential is taken as the first potential fluorescence intensity, and the fluorescence intensity after the change in mitochondrial membrane potential is taken as the second potential fluorescence intensity. The ratio of the fluorescence intensity at the first potential to the fluorescence intensity at the second potential is processed to obtain the first fluorescence intensity ratio; Obtain the fluorescence intensity corresponding to the changes in reactive oxygen species levels before and after the changes; The fluorescence intensity before the change in reactive oxygen species level is taken as the first reactive fluorescence intensity, and the fluorescence intensity after the change in reactive oxygen species level is taken as the second reactive fluorescence intensity. The ratio of the first active fluorescence intensity to the second active fluorescence intensity is processed to obtain the second fluorescence intensity ratio. Compare the first fluorescence intensity ratio and the second fluorescence intensity ratio; The degree of damage is graded based on the quantitative relationship between the first fluorescence intensity ratio and the second fluorescence intensity ratio.

8. The method for constructing human retinal radiation injury based on retinal organoids according to claim 7, characterized in that, The degree of damage is graded based on the quantitative relationship between the first fluorescence intensity ratio and the second fluorescence intensity ratio, including: When the quantitative relationship between the first fluorescence intensity ratio and the second fluorescence intensity ratio indicates that the first fluorescence intensity ratio is greater than the second fluorescence intensity ratio, flow cytometry is used to quantify the apoptosis rate of cells. The apoptosis rate of the cells quantified by flow cytometry and the ratio of the second fluorescence intensity are processed to obtain the absolute difference between the apoptosis rate of the cells quantified by flow cytometry and the ratio of the second fluorescence intensity, which is taken as the first absolute difference. The damage degree coefficient is obtained by combining the first absolute difference with the first fluorescence intensity ratio. When the quantitative relationship between the first fluorescence intensity ratio and the second fluorescence intensity ratio indicates that the first fluorescence intensity ratio is not greater than the second fluorescence intensity ratio, then flow cytometry is used to quantify the apoptosis rate of cells. The apoptosis rate of the cells quantified by flow cytometry and the ratio of the first fluorescence intensity are processed to obtain the absolute difference between the apoptosis rate of the cells quantified by flow cytometry and the ratio of the first fluorescence intensity, which is used as the second absolute difference. The damage degree coefficient is obtained by combining the second absolute difference with the second fluorescence intensity ratio. The damage degree coefficient is compared with a preset first coefficient threshold and a second coefficient threshold; If the damage severity coefficient is lower than the first coefficient threshold, the current damage severity is determined to be minor. When the damage severity coefficient is not lower than the first coefficient threshold and the damage severity coefficient is lower than the second coefficient threshold, the current damage severity is determined to be moderate. If the damage severity coefficient is not lower than the second coefficient threshold, the current damage severity is determined to be severe.

9. The method for constructing human retinal radiation injury based on retinal organoids according to claim 6, characterized in that, A correlation model between radiation dose and damage indices was established based on data from radiation treatment and grouping, apoptosis detection, and oxidative stress and mitochondrial function testing, including: First, the data were organized, including the detection data of different irradiation dose groups and control groups at various time points, including cell apoptosis data, oxidative stress data and mitochondrial function data. The damage index data in the detection data is then confirmed and used as the core parameters of the model. The damage index data are the index data that are significantly correlated with the radiation dose. At each time point, each irradiation dose and corresponding damage index were paired and analyzed. A scatter plot was drawn with the irradiation dose as the x-axis and the quantitative value of each damage index as the y-axis, and the damage curve was finally obtained. The fitting effect of the damage curve is evaluated by calculating the coefficient of determination. When the coefficient of determination is >0.95, the fit is acceptable. If the coefficient of determination is <0.95, the data should be re-examined or the fitting method should be adjusted until the standard is met. The fitted curve data of apoptosis data, oxidative stress data and mitochondrial function data were integrated, and then a correlation model was established using a model correlation tool. The process of establishing the association model is as follows: First, the apoptosis data, oxidative stress data and mitochondrial function data are standardized, including unit unification and time anchor alignment. Machine learning tools were used to model the standardized data. The machine learning tools used the irradiation dose as the independent variable and the standardized parameters of apoptosis data, oxidative stress data and mitochondrial function data as the dependent variable to establish a coupling equation between dose and multiple indicators. Meanwhile, the coupling equations of dose and multiple indicators were cross-validated, and after successful validation, a correlation model between irradiation dose and damage indicators was obtained.

10. The method for constructing human retinal radiation injury based on retinal organoids according to claim 9, characterized in that, To evaluate the protective effect of candidate drugs against radiation damage and establish efficacy evaluation criteria, including: First, the drug intervention group and the control group were set up. The drug to be tested was added to the retinal organoids 1 hour before radiation treatment or immediately after irradiation. At the same time, a radiation damage control group without drug and a normal control group were set up. After the intervention and control groups were set up, the damage indicators after drug intervention were detected, and the detection method was the same as that for radiation damage detection. Based on the damage index data of the control group and the drug intervention group, the protection index was calculated, and the overall protection index of the drug was obtained after the protection index was calculated. Effective drugs are determined based on an overall protection index threshold. The criteria for determining an effective drug are: overall protection index ≥ 30%; reactive oxygen species level ≥ 25%; and mitochondrial membrane potential recovery ≥ 20%. Based on the threshold range for determining effective drugs, effective drugs are divided into Level 1 effective and Level 2 effective drugs; Finally, the efficacy evaluation criteria for effective drugs corresponding to retinal organoids were completed.

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