Construction and evaluation method of liver injury organ-like model

Abstract

The invention discloses a construction and evaluation method of a liver injury organoid model, which comprises the following steps: (1) selecting mouse liver cells, perfusing a mouse with pancreatin through a sterile operation technology, removing blood in the liver, collecting liver tissues, and carrying out fine enzymolysis digestion treatment to obtain a single-cell suspension; (2) inoculating the single-cell suspension into a three-dimensional culture system, and carrying out preliminary stimulation by adopting a culture medium combined with growth factors and inhibition small molecules, so as to promote the growth, proliferation and differentiation of cells in a three-dimensional space; according to the cell growth condition, subculture operation is carried out in good time, so that continuous culture and amplification of liver organs are ensured; and (3) exposing the maturely cultured liver organoid in a stimulating factor for simulating a liver injury pathological environment, and constructing different types of liver injury organoid models. According to the liver injury organ-like model construction method, model construction can be completed in a short time, the research period is greatly shortened, the efficiency is high, and the cost is low.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a method for constructing and evaluating a liver injury organoid model. Background Art

[0002] In the field of biomedical research, the construction of liver injury models is of great significance for drug screening, toxicity assessment, exploration of disease mechanisms, and research on treatment methods. In the prior art, the construction of liver injury models mainly relies on animal experiments and two-dimensional cell culture models.

[0003] Although animal experiments can simulate the pathological process of liver injury to a certain extent, they have many limitations. First, the cost of animal experiments is relatively high, including the purchase and breeding of experimental animals and the investment in experimental equipment, which limits large-scale experimental research. Second, the cycle of animal experiments is relatively long. From the preparation of experimental animals to the acquisition of experimental results, it often takes several months or even years, which is extremely unfavorable for rapidly promoting the process of drug research and development and disease research. In addition, animal experiments also face ethical controversies. With the increasing attention to animal welfare, the conduct of animal experiments is restricted more and more, which also affects the progress of related research to a certain extent.

[0004] The two-dimensional cell culture model is another commonly used method for constructing liver injury models. However, this model has obvious defects, namely the lack of three-dimensional structure and cell-cell interactions. The liver is a complex organ, and its physiological and pathological processes involve the mutual cooperation between multiple cell types and the close connection between cells and the extracellular matrix. The two-dimensional cell culture model cannot accurately reflect the complexity of the liver, so when simulating the pathological process of liver injury, it often cannot provide accurate and reliable results, which limits its application value in drug screening and disease research.

[0005] In summary, the methods for constructing liver injury models in the prior art have problems such as high cost, long cycle, ethical controversies, and difficulty in accurately reflecting the complex physiological and pathological processes of the liver. There is an urgent need for a method for constructing and evaluating a liver injury model with high efficiency and low cost to meet the growing needs of biomedical research. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for constructing and evaluating a liver injury organoid model with high efficiency and low cost.

[0007] To solve the problems of the prior art, the present invention provides the following technical solutions: In the first aspect, the present application provides a method for constructing a liver injury organoid model.

[0008] In a second aspect, the present application provides a liver injury organoid model constructed by the method for constructing a liver injury organoid model described in the first aspect.

[0009] In a third aspect, the present application provides an application of a liver injury organoid model constructed based on the method for constructing a liver injury organoid model in drug screening, toxicity assessment, disease mechanism exploration, and treatment method research.

[0010] In a fourth aspect, the present application provides an application of an evaluation method based on a liver injury organoid model in evaluating the pathological characteristics, cellular responses, gene expression changes, and degree of functional impairment of a liver injury model.

[0011] In the first aspect of the present application, a method for constructing a liver injury organoid model is provided, including the following steps: (1) Induction, culture, and passage of liver organoids: Select mouse liver cells, perfuse the mouse with trypsin through aseptic surgical techniques, remove the blood in the liver, collect the liver tissue, and perform fine enzymatic digestion to obtain a single-cell suspension; (2) Inoculate the single-cell suspension into a three-dimensional culture system, and perform preliminary stimulation using a culture medium combined with growth factors and inhibitory small molecules to promote the growth, proliferation, and differentiation of cells in three-dimensional space; According to the cell growth situation, perform passage operations in a timely manner to ensure the continuous culture and expansion of liver organoids; (3) Construction of a liver injury organoid model: Expose the cultured mature liver organoids to stimulatory factors that simulate the pathological environment of liver injury, and construct different types of liver injury organoid models by adjusting the types, concentrations, and action times of the stimulatory factors.

[0012] Further, in step (1), the passage operation uses enzymatic digestion or mechanical dissociation methods to disperse the cells with a stable three-dimensional structure into single cells or small cell clusters, and re-inoculate them into a new three-dimensional culture system for continuous culture.

[0013] Further, in step (2), the three-dimensional culture system is an AIgiMatrix tm 3D cell culture system or other similar systems, and the culture medium is DMEM / F12, STEMDIFF APEL2.

[0014] Further, in step (3), the stimulatory factors include inflammatory mediators, drugs, or immune complexes, and the drugs include, but are not limited to, hepatotoxic drugs such as cisplatin. By observing the morphological changes of the organoids under a microscope, recording the cell survival rate and morphological characteristics, and combining molecular biology, immunology, histology, and functional evaluation methods, the effectiveness of the liver injury organoid model is verified.

[0015] The liver injury organoid model constructed by the method for constructing a liver injury organoid model provided in the second aspect of the present application includes the following steps: Pathological feature evaluation: By observing the microscopic structure, cell morphology, and arrangement pattern of the liver injury organoid model, evaluate whether the pathological features of the liver injury model meet the expectations; Or drug screening evaluation: Treat the liver injury organoid model with a positive drug, and evaluate whether the liver injury organoid model meets the expectations in drug screening; Or cell response evaluation: By detecting cell viability, proliferation ability, and apoptosis, evaluate the response degree of the liver injury organoid model to stimulatory factors; Or evaluation of changes in cell protein expression: Use immunofluorescence labeling technology to detect the expression changes of cell injury markers in the liver injury organoid model to reveal its injury conditions; Or evaluation of the degree of functional impairment: Detect the injury indicators of the liver injury organoid model by enzyme-linked immunosorbent assay to evaluate the functional health status and degree of injury of the liver injury organoid model.

[0016] Furthermore, the pathological feature evaluation also includes recording the changes in indicators such as cell morphology, arrangement pattern, and cell gap of the organoids through HE-stained sections.

[0017] Furthermore, in the cell response evaluation, a cell viability detection method is used to measure the cell viability at different time points.

[0018] Even further, the evaluation of the degree of functional impairment also includes immunofluorescence detection of the physiological index proteins such as cell function and detoxification function of the liver organoids, and using specific biochemical detection methods to measure the activities of related enzymes or the contents of metabolites.

[0019] The third aspect of the present application provides an application of a liver injury organoid model constructed based on the method for constructing a liver injury organoid model in drug screening, toxicity assessment, exploration of disease mechanisms, and research on treatment methods.

[0020] The fourth aspect of the present application provides an application of an evaluation method based on a liver injury organoid model in evaluating the pathological features, cell response, gene expression changes, and degree of functional impairment of a liver injury model.

[0021] Beneficial effects: The method for constructing a liver injury organoid model of the present invention can complete the construction of the model in a relatively short time. Compared with traditional animal experiments and two-dimensional cell culture models, the research cycle is greatly shortened, with high efficiency and low cost.

[0022] Compared with the prior art, the present invention has the following advantages: (1) Compared with animal experiments, this method does not require a large number of experimental animals, reducing the breeding cost and the investment in experimental equipment; compared with two-dimensional cell culture models, although the use of a three-dimensional culture system is increased, the constructed model is more representative, which can reduce the repeated experimental cost caused by inaccurate models in the follow-up, and generally reduces the research cost. This enables work such as drug screening, toxicity assessment, exploration of disease mechanisms, and research on treatment methods to be carried out more quickly.

[0023] (2) Liver organoids have a three-dimensional structure and can better simulate the physiological and pathological processes of the liver, including cell-cell interactions and the connection between cells and the extracellular matrix. This structure makes the model more accurate in reflecting the pathological characteristics of liver injury, cell responses, changes in gene expression, and the degree of functional impairment, providing a more reliable basis for drug screening and disease research.

[0024] (3) The present invention reduces the dependence on experimental animals, avoids the ethical controversies that may arise in animal experiments, conforms to the trend of modern society's concern for animal welfare, and enables related research to be carried out within a wider range of ethical acceptance.

[0025] (4) The model construction method of the present invention has good scalability. Different types of liver injury organoid models can be constructed by adjusting the type, concentration, and action time of the stimulating factors to meet different research needs. At the same time, the diversity of evaluation methods also guarantees the comprehensive evaluation of the model. Appropriate evaluation means can be selected according to specific research purposes, further expanding the application scope of the model. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0027] Figure 1 It is a morphological comparison diagram after 3D culture of the normal liver organoid control group, liver injury organoid group, and liver injury organoid treatment group of the present invention.

[0028] Figure 2 It is a morphological comparison diagram of HE staining after 3D culture of the normal liver organoid control group, liver injury organoid group, and liver injury organoid treatment group of the present invention.

[0029] Figure 3 It is a diagram showing the change in the survival rate of liver cells in the liver organoid model of the present invention under induction with different concentrations of liver injury drugs for 24 hours.

[0030] Figure 4 This is a graph showing the changes in the survival rate of liver cells in the liver injury organoid model of the present invention under treatment with positive drugs at different concentrations.

[0031] Figure 5 This is a graph of the immunofluorescence staining results of the liver injury organoid model of the present invention.

[0032] Figure 6 This is a graph of the results of detecting liver injury marker proteins in the supernatant of liver injury organoids by enzyme-linked immunosorbent assay in the present invention. Detailed implementation manners

[0033] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application clearer, the following further details this application in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0034] In this application, the term "and / or" describes the association relationship of associated objects and indicates that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B may be singular or plural. The character " / " generally indicates that the associated objects before and after are an "or" relationship.

[0035] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single item (s) or plural item (s). For example, "at least one of a, b, or c", or "at least one of a, b, and c" can both represent: a, b, c, a - b (that is, a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0036] It should be understood that in various embodiments of this application, the sequence numbers of the above processes do not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0037] The terms used in the embodiments of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The singular forms of "a", "the" and "said" used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0038] The first aspect of the embodiments of the present application provides a method for constructing a liver injury organoid model, including the following steps: (1) Induction, culture, and passage of liver organoids: Select mouse liver cells, perfuse the mouse with trypsin through aseptic surgical techniques, remove the blood in the liver, collect liver tissue, and perform fine enzymatic digestion to obtain a single-cell suspension. (2) Inoculate the single-cell suspension into a three-dimensional culture system, and perform preliminary stimulation using a culture medium combined with growth factors and inhibitory small molecules to promote the growth, proliferation, and differentiation of cells in three-dimensional space; according to the cell growth conditions, perform passage operations in a timely manner to ensure the continuous culture and expansion of liver organoids. (3) Construction of the liver injury organoid model: Expose the maturely cultured liver organoids to stimulators that simulate the pathological environment of liver injury, and construct different types of liver injury organoid models by adjusting the type, concentration, and action time of the stimulators.

[0039] In some embodiments, in step (1), the passage operation uses enzymatic digestion or mechanical dissociation to disperse the cells with a stable three-dimensional structure into single cells or small cell clusters, and re-inoculate them into a new three-dimensional culture system for continued culture.

[0040] In some embodiments, in step (2), the three-dimensional culture system is an AIgiMatrix 3D Culture System or other similar systems, and the culture medium is DMEM / F12 and STEMDIFF APEL2.

[0041] The DMEM / F12 culture medium is a modified mixture of DMEM culture medium and Ham's F-12 culture medium in a ratio of 1:1. This mixed culture medium combines the high concentrations of glucose, amino acids, and vitamins in DMEM with various trace elements in Ham’s F-12 culture medium, thus providing a more comprehensive nutrient composition. Components: Amino acids and vitamins: Contain rich essential amino acids and vitamins in DMEM and F-12, supporting cell growth and maintaining normal functions. Inorganic salts: Provide a balanced inorganic salt concentration to maintain the osmotic pressure and pH value inside and outside the cells. Glucose: Usually contains 3151 mg / L of D-glucose. L-glutamine: Usually contains 365 mg / L of L-glutamine. Sodium pyruvate: Usually contains 110 mg / L of sodium pyruvate. Phenol red: Usually contains 8.1 mg / L of phenol red. Non-essential amino acids: Contain non-essential amino acids. HEPES: Some formulations do not contain HEPES, but it can be added to enhance the buffering capacity.

[0042] STEMDIFF APEL2 Medium is a completely defined, serum-free, and animal component-free medium used for the differentiation of human embryonic stem cells (ES) and induced pluripotent stem cells (iPS). It is based on the APEL formulation published by Dr. Andrew Elefanty and does not contain undefined components such as protein-free hybridoma medium.

[0043] Serum-free and animal component-free: Ensure that the components of the medium are completely defined, avoiding the uncertainties and potential contamination brought by animal-derived components. Low-insulin (LI) version: STEMDIFF APEL2-LI Medium is a low-insulin version, which is particularly suitable for the differentiation of lineages where insulin is a known inhibitory factor, such as cardiomyocytes.

[0044] In some embodiments, in step (3), the stimulating factors include inflammatory mediators, drugs, immune complexes, etc., including but not limited to hepatotoxic drugs such as cisplatin. By observing the morphological changes of the organoids under a microscope, recording the cell viability and morphological characteristics, and combining molecular biology, immunology, histology, and functional evaluation methods, the effectiveness of the liver injury organoid model is verified.

[0045] The second aspect of the embodiments of the present application provides a method for evaluating a liver injury organoid model, including the following steps: Pathological feature evaluation: By observing the microscopic structure, cell morphology, and arrangement pattern of the liver injury organoid model, evaluate whether the pathological features of the liver injury model meet the expectations; Or drug screening evaluation: Treat the liver injury organoid model with a positive drug and evaluate whether the liver injury organoid model meets the expectations in drug screening; Or cell response evaluation: By detecting cell viability, proliferation ability, and apoptosis, evaluate the response degree of the liver injury organoid model to the stimulating factors; Or cell protein expression change evaluation: Use immunofluorescence labeling technology to detect the expression changes of cell injury markers in the liver injury organoid model to reveal its injury situation; Or functional impairment degree evaluation: Detect the injury indicators of the liver injury organoid model by enzyme-linked immunosorbent assay to evaluate the functional health status and injury degree of the liver injury organoid model.

[0046] In some embodiments, the pathological feature evaluation further includes recording the changes in indicators such as cell morphology, arrangement pattern, and cell gap of the organoids through HE-stained sections.

[0047] In some embodiments, in the cell response evaluation, a cell viability detection method is used to measure the cell viability at different time points.

[0048] In some embodiments, the assessment of the degree of functional impairment further includes immunofluorescence detection of physiological index proteins such as the cell function and detoxification function of liver organoids, as well as the determination of the activity of related enzymes or the content of metabolites using specific biochemical detection methods.

[0049] The third aspect of the embodiments of the present application provides an application of a liver injury organoid model constructed based on a liver injury organoid model construction method in drug screening, toxicity assessment, disease mechanism exploration, and treatment method research.

[0050] The fourth aspect of the embodiments of the present application provides an application of an evaluation method based on a liver injury organoid model in assessing the pathological characteristics, cell responses, gene expression changes, and degree of functional impairment of a liver injury model.

[0051] Example 1

[0052] A method for constructing a liver injury organoid model of the present invention includes the following steps: (1) Induction, culture, and passage of liver organoids: Mouse liver cells are selected, and the mouse is perfused with trypsin through aseptic surgical techniques to remove the blood in the liver and collect liver tissue, and then subjected to fine enzymatic digestion treatment to obtain a single-cell suspension; for the passage operation, enzymatic digestion or mechanical dissociation methods are used to disperse the cells with a stable three-dimensional structure into single cells or small cell clusters, and then re-inoculated into a new three-dimensional culture system for continuous culture.

[0053] (2) Inoculate the single-cell suspension into a three-dimensional culture system, and perform preliminary stimulation using a culture medium combined with growth factors and inhibitory small molecules to promote the growth, proliferation, and differentiation of cells in three-dimensional space; according to the cell growth situation, perform passage operations in a timely manner to ensure the continuous culture and amplification of liver organoids; the three-dimensional culture system is an AIgiMatrix 3D Culture System or other similar systems, and the culture medium is DMEM / F12, STEMDIFF APEL2.

[0054] (3) Construction of the liver injury organoid model: Expose the cultured mature liver organoids to stimulators that simulate the pathological environment of liver injury, and construct different types of liver injury organoid models by adjusting the type, concentration, and action time of the stimulators. The stimulators include inflammatory mediators, drugs, immune complexes, etc., including but not limited to hepatotoxic drugs such as cisplatin. Observe the morphological changes of the organoids through a microscope, record the cell viability and morphological characteristics, and verify the effectiveness of the liver injury organoid model in combination with molecular biology, immunology, histology, and functional evaluation methods.

[0055] Example 2

[0056] An evaluation method for a liver injury organoid model of the present invention includes the following steps: Experimental method: Take a sample of the liver injury organoid model, place it under a microscope for observation, adjust the microscope magnification, and clearly observe the cell morphology, arrangement pattern, and cell gaps.

[0057] Pathological feature evaluation: By observing the microscopic structure, cell morphology, and arrangement pattern of the liver injury organoid model, etc., evaluate whether the pathological features of the liver injury model meet the expectations; the pathological feature evaluation also includes recording the changes in indicators such as cell morphology, arrangement pattern, and cell gaps of the organoids through HE-stained sections.

[0058] Example 3

[0059] An evaluation method for a liver injury organoid model of the present invention includes the following steps: Drug screening evaluation: By selecting known positive drugs, such as liver-protecting drugs, treat the liver injury organoid model to evaluate whether the liver injury organoid model meets the expectations in drug screening.

[0060] Example 4

[0061] An evaluation method for a liver injury organoid model of the present invention includes the following steps: Cell response evaluation: By detecting cell viability, proliferation ability, and apoptosis, evaluate the response degree of the liver injury organoid model to stimulatory factors; for example, after taking a sample of the liver injury organoid model, use a cell viability detection method (such as the CCK-8 method) to measure cell viability.

[0062] Example 5

[0063] An evaluation method for a liver injury organoid model of the present invention includes the following steps: Evaluation of changes in cell protein expression: Adopt immunofluorescence labeling technology, select specific antibodies to detect the expression changes of cell injury markers in the liver injury organoid model, and compare the immunofluorescence expression of the liver injury organoid model with that of normal liver organoids to reveal its injury situation.

[0064] Example 6

[0065] An evaluation method for a liver injury organoid model of the present invention includes the following steps: Evaluation of the degree of functional impairment: Detect the expression levels of injury indicators (such as ALT, AST, TNF-α, etc.) of the liver injury organoid model by enzyme-linked immunosorbent assay to evaluate the functional health status and degree of injury of the liver injury organoid model. The evaluation of the degree of functional impairment also includes immunofluorescence detection of the cell function, detoxification function, and other physiological index proteins of the liver organoids, as well as using specific biochemical detection methods to measure the activities of related enzymes or the contents of metabolites.

[0066] Detection of liver injury marker proteins by enzyme-linked immunosorbent assay Method: Collect the supernatant of liver injury organoids and organoids after positive drug treatment, and use enzyme-linked immunosorbent assay to detect the expression level of specific liver injury marker proteins.

[0067] The results are as Figure 6 shown. The positive drug can significantly protect liver cells, further verifying the degree of functional impairment of the liver injury model and the protective effect of the drug.

[0068] Example 7

[0069] Application of a liver injury organoid model constructed by the method for constructing a liver injury organoid model of the present invention in drug screening, toxicity assessment, exploration of disease mechanisms and research of treatment methods: For example, using the liver injury organoid model for new drug screening to evaluate the efficacy and toxicity of drugs; inducing liver injury to study the mechanism of disease occurrence and development; using the model to test the effects of different treatment methods to provide a basis for clinical treatment.

[0070] Example 8

[0071] Application of an evaluation method based on a liver injury organoid model of the present invention in evaluating the pathological characteristics, cellular responses, gene expression changes and degree of functional impairment of a liver injury model; For example, comprehensively using methods such as microscopic structure observation and HE staining section analysis to evaluate the pathological characteristics of the liver injury organoid model; evaluating cellular responses through methods such as cell viability detection, proliferation ability detection, and apoptosis detection; using gene expression detection techniques (such as RT-PCR, qPCR, etc.) to evaluate gene expression changes; combining enzyme-linked immunosorbent assay detection, immunofluorescence detection of physiological index proteins and biochemical detection methods to evaluate the degree of functional impairment.

[0072] Example 9 Induction and culture of liver organoids

[0073] 1. Preparation of starting materials: Select the liver tissue of healthy mice as the starting material to ensure no hepatitis virus or other liver disease infections.

[0074] Collect the liver tissue through aseptic surgical techniques and immediately place it on ice after collection to reduce cell damage.

[0075] Use delicate enzymatic digestion treatment (such as trypsin-EDTA solution) to digest the liver tissue into a single-cell suspension, and remove impurities and cell debris by centrifugation and filtration.

[0076] 2. Three-dimensional culture: Inoculate the single-cell suspension into the AIgiMatrix 3D Culture System or other three-dimensional culture systems to ensure uniform cell distribution.

[0077] Use STEMDIFF APEL2 medium as the basal medium, supplemented with 10% fetal bovine serum (FBS) or human serum albumin (HSA), and 1% penicillin / streptomycin double antibody in the basal medium (Advanced DMEM / F12) as the basic nutritional support. At the same time, add Wnt3a (80 ng / mL) and R-spondin1 (800 ng / mL) to act synergistically to enhance the proliferation and maintenance of hepatic progenitor cell stemness; add Noggin (150 ng / mL) to inhibit BMP signaling and prevent premature differentiation of organoids; add FGF4 (30 ng / mL) and HGF (30 ng / mL) to promote hepatocyte proliferation and functional maturation; for the first time, introduce epidermal growth factor (EGF, 10 ng / mL) and retinoic acid, which can promote cell proliferation and differentiation, contribute to the further growth and complexity of organoids, and act as a morphogen to induce the development of liver organoids into more mature structures; finally, add B27 (1x) to provide comprehensive nutrition, and N-acetylcysteine (NAC, 5 mM) as an antioxidant to protect hepatocytes for stimulation and culture to promote cell growth, proliferation and differentiation in three-dimensional space. During the culture process, regularly observe the cell growth conditions and record the changes in cell morphology, colony formation and three-dimensional structure.

[0078] According to the cell growth rate and density, timely perform medium replacement and subculture operations to ensure the continuous culture and expansion of liver organoids.

[0079] 3. Subculture and amplification: After the cells grow to a certain number (such as forming a stable three-dimensional structure), use enzymatic digestion or mechanical dissociation methods to disperse the cells into single cells or small cell clusters.

[0080] Re-inoculate into a new three-dimensional culture system and continue to culture under the same culture conditions.

[0081] Record the cell growth conditions and changes in three-dimensional structure after subculture to ensure the stability and consistency of cells during subculture.

[0082] Example 10 Construction of liver injury model

[0083] 1. Stimulating factor treatment: Expose the maturely cultured liver organoids to different concentrations of liver injury drugs (such as cisplatin, tetracycline, carbon tetrachloride) (concentration gradients such as 1, 2, 5, 10 μg / ml) to simulate the pathological environment of liver injury.

[0084] Observe the microscopic structural changes of the organoids through a microscope, such as changes in cell morphology and an increase in cell gaps.

[0085] Use a cell viability detection method (such as the CCK-8 method) to measure the cell viability after treatment with different concentrations of drugs, and screen out the optimal induction concentration.

[0086] 2. Model evaluation: Verify the liver injury model by combining molecular biology, immunology, histology, and functional evaluation methods.

[0087] Use immunofluorescence staining technology to detect the expression changes of hepatocyte markers, such as the expression of hepatocyte-specific antibodies, albumin, cytokeratin, etc.

[0088] Immunofluorescence staining analysis Method: Perform immunofluorescence staining on the liver injury organoid model and the organoids after treatment with positive drugs to detect the expression changes of specific markers.

[0089] The results are as Figure 5 shown. The fluorescence expression level in the liver injury organoid group decreased significantly. After drug treatment, the expression level of hepatocyte markers increased significantly and approached that of the normal control group, further verifying the effectiveness of the liver injury organoid model.

[0090] Use enzyme-linked immunosorbent assay to detect the expression levels of liver injury marker proteins, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), etc.

[0091] Evaluate the therapeutic effects of various positive drugs on the liver injury organoid model (such as bicyclol, polyene phosphatidylcholine, reduced glutathione) to assess their roles in pharmacodynamic evaluation.

[0092] 3D culture and morphological observation of organoids Method: Perform 3D culture on normal liver cells, liver cells after liver injury induction, and liver injury cells after treatment with different drugs (bicyclol, polyene phosphatidylcholine, reduced glutathione) respectively.

[0093] The results are as Figure 1 shown. The normal organoid control group has a tight structure and a complete morphology; the liver injury group shows obvious cell dispersion and signs of injury; the liver injury organoid treatment group has a relatively tight cell structure. Although there are signs of injury, it has a greater improvement compared to the liver injury group.

[0094] Record the pathological characteristics and cell proliferation of the liver injury organoid model at different time points after positive drugs to evaluate its dynamic change process.

[0095] Example 11 Establishment and Application of Evaluation Method

[0096] 1. Pathological Feature Evaluation: By observing the microscopic structural changes of organoids through a microscope, evaluate whether the pathological features of the hepatitis model meet the expectations.

[0097] Record the changes in indicators such as cell morphology, arrangement pattern, and cell gap of organoids through HE-stained sections.

[0098] HE Staining Morphology Comparison Method: Perform HE staining on the organoids after 3D culture and observe the cell structure and morphology.

[0099] The results are as Figure 2 shown. In the normal organoid control group, the cell structure is clear, the morphology is relatively normal, and the cells are arranged tightly; in the liver injury organoid group, obvious pathological changes can be seen, the cell arrangement is disordered, and obvious inflammatory cell infiltration and cell damage occur; in the liver injury organoid treatment group, the cell structure of different drug treatment groups has been improved to varying degrees. Among them, the cell structure of the polyene phosphatidylcholine treatment group is relatively complete, and the inflammatory cell infiltration and damage are significantly reduced.

[0100] 2. Cell Response Evaluation: Use the cell viability assay method to determine the cell viability at different time points.

[0101] 3. Degree of Functional Impairment Evaluation: Use immunofluorescence to detect the protein levels of physiological indicators such as cell function and detoxification function in liver organoids.

[0102] Analysis of Changes in Liver Cell Viability Method: Induce liver organoids with different concentrations of liver injury drugs (such as cisplatin) for 24 hours and detect the cell viability.

[0103] The results are as Figure 3 shown. At a concentration of 5 μg / ml, cisplatin can cause the liver cell injury rate to reach more than 50%, meeting the requirements of the liver injury model.

[0104] Further experiment: Treat liver injury organoids with different concentrations of positive drugs and detect the cell viability. The results are as Figure 4 shown. The data show that this model can be applied to drug screening.

[0105] Use specific biochemical detection methods to determine the activity of related enzymes or the content of metabolites.

[0106] Record and analyze the dynamic change process of the degree of functional impairment.

[0107] Through the detailed description of the above specific embodiments, the operation steps and experimental conditions of the method for constructing a hepatitis cell injury model based on liver organoids and its evaluation system provided by the present invention can be further clarified. These embodiments not only help to ensure the reliability and repeatability of experimental results, but also provide a solid model basis for subsequent drug screening, toxicity assessment, disease mechanism exploration and treatment method research.

[0108] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements. The scope of protection required by the present invention is defined by the appended claims, the specification and their equivalents.

Claims

1. A method for constructing an organoid model of liver injury, characterized in that The steps include: (1) Select mouse liver cells, perfuse the mice with pancreatic enzymes through sterile surgical techniques, remove the blood in the liver, collect liver tissue, and perform fine enzymatic digestion to obtain a single cell suspension; (2) Inoculate the single-cell suspension into a three-dimensional culture system and use a culture medium combined with growth factors and inhibitory small molecules for initial stimulation to promote cell growth, proliferation, and differentiation in three-dimensional space; perform subculture operations based on cell growth to ensure continuous culture and expansion of liver organoids; (3) Expose the cultured mature liver organoids to stimulating factors that simulate the pathological environment of liver injury, and construct different types of liver injury organoid models by adjusting the type, concentration, and duration of action of the stimulating factors.

2. The method for constructing a liver injury organoid model according to claim 1, characterized in that: In step (1), the subculture operation uses enzymatic or mechanical dissociation to disperse the cells that have formed a stable three-dimensional structure into single cells or small cell clusters, and re-inoculate them into a new three-dimensional culture system for continued culture.

3. The method for constructing a liver injury organoid model according to claim 1, characterized in that: In step (2), the three-dimensional culture system is AIgiMatrix tm 3D cell culture system or other similar systems, the culture medium is DMEM / F12, STEMDIFF APEL2.

4. The method for constructing a liver injury organoid model according to claim 1, characterized in that: In step (3), the stimulation factors include inflammatory mediators, drugs or immune complexes. The morphological changes of organoids are observed under a microscope, and the cell survival rate and morphological characteristics are recorded. The effectiveness of the liver injury organoid model is verified by combining molecular biology, immunology, histology and functional evaluation methods.

5. A liver injury organoid model constructed based on the liver injury organoid model construction method according to claim 1, characterized in that The steps include: Pathological feature evaluation: by observing the microstructure, cell morphology and arrangement of the liver injury organoid model of claim 1, evaluate whether the pathological features of the liver injury model meet expectations; or drug screening evaluation: treating the liver injury organoid model of claim 1 with a positive drug to evaluate whether the liver injury organoid model meets expectations in drug screening; Or cell response evaluation: evaluating the response of the liver injury organoid model of claim 1 to the stimulating factor by detecting cell survival rate, proliferation ability, and apoptosis; Or evaluation of changes in cell protein expression: using immunofluorescence labeling technology to detect changes in the expression of cell injury markers in the liver injury organoid model of claim 1 to reveal its injury status; Or assessment of the degree of functional damage: detecting the damage indicators of the liver injury organoid model of claim 1 by enzyme-linked immunosorbent assay to assess the functional health status and degree of damage of the liver injury organoid model.

6. The method for evaluating the liver injury organoid model according to claim 5, characterized in that: The pathological feature assessment also includes recording changes in the cell morphology, arrangement, and cell gap indicators of the organoids through HE staining sections.

7. The method for evaluating the liver injury organoid model according to claim 5, characterized in that: In the cell response assessment, a cell viability detection method is used to measure the cell viability at different time points.

8. The method for evaluating the liver injury organoid model according to claim 5, characterized in that: The functional impairment degree assessment also includes immunofluorescence detection of physiological indicator proteins such as cellular function and detoxification function of liver organoids, as well as the use of specific biochemical detection methods to determine the activity of related enzymes or the content of metabolites.

9. Use of a liver injury organoid model constructed based on the liver injury organoid model construction method described in any one of claims 1 to 4 in drug screening or toxicity assessment.

10. Use of an evaluation method based on the liver injury organoid model described in any one of claims 5 to 8 in evaluating the pathological characteristics, cellular responses, gene expression changes and degree of functional impairment of the liver injury model.

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