An alcoholic liver injury cell model and a construction method and application thereof

Abstract

The application discloses an alcoholic liver injury cell model and a construction method and application thereof, and belongs to the technical field of cell models. The technical problem to be solved is to improve the stability and accuracy of the constructed alcoholic liver injury cell model. The technical solution is a construction method of an alcoholic liver injury cell model, which comprises the following steps: after HepG2 cells are digested, the cells are resuspended and diluted by using DMEM complete culture medium, the cell liquid obtained after dilution is inoculated into a hole plate, alcohol solution is added after a certain time, and a sealing plate film is added at the same time, and then incubation is continued, and the alcoholic liver injury cell model is obtained.

Description

Technical Field

[0001] This invention relates to the field of cell model technology, specifically to a cell model of alcoholic liver injury, its construction method, and its application. Background Technology

[0002] Cell models are a cost-effective and convenient method for screening specific responsive substances, and are an important tool in food and drug research. Compared with animal models, cell models have the advantages of simpler and more convenient data acquisition at a lower cost.

[0003] Hepatocytes, comprising 80% of liver cells, are the most important parenchymal cells for liver function. They play crucial metabolic and detoxification roles, including the metabolism of fats, carbohydrates, and proteins; the synthesis and secretion of most plasma proteins (such as albumin and prothrombin); the conversion of drugs, toxins, and metabolic waste products into water-soluble substances for excretion; and the formation and excretion of bile, bile pigments, and bile acids. Under stimulation by toxins or chemicals, hepatocyte structure is damaged, leading to a series of functional disorders, such as decreased protein synthesis and secretion, lipid peroxidation, biomembrane disruption, and the leakage of intracellular enzymes (LDH, ALT, AST, etc.). Therefore, a damaged hepatocyte model can be obtained by directly stimulating cultured normal hepatocytes with toxins or chemicals in vitro to observe the effects of drugs on this model cell and understand the anti-hepatotoxic effects and mechanisms of action of drugs.

[0004] Cellular models of alcoholic liver injury are important tools for studying the mechanisms of alcoholic liver disease and for drug screening. They mainly simulate the pathological process by culturing hepatocytes in vitro and exposing them to ethanol or its metabolites.

[0005] Relevant patent documents retrieved:

[0006] The document, published in China (CN111733137A) on July 13, 2020, discloses a cell model of alcoholic liver injury. The preparation method is as follows: human colon cancer cells Caco-2 are first cultured in cell culture medium until a complete and dense Caco-2 monolayer is formed. Then, the Caco-2 monolayer is incubated in cell culture medium with 3-7% alcohol by volume at a temperature of 36-38℃ for 23-25 ​​hours.

[0007] Relevant non-patent literature retrieved: The book, titled "Experimental Methodology of Traditional Chinese Medicine Pharmacology," was published in October 2006. This document discloses an in vitro induced model of hepatocyte damage. First, hepatocytes were isolated and cultured from normal animals. Then, ethanol or acetaldehyde was directly added to the culture medium at a concentration of 0.1-1 mol / L for 20-140 min. With the extension of the treatment time, the hepatocyte damage aggravated, and the levels of LDH, AST, ALT, etc. in the culture supernatant increased.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The stability of the obtained cell model of alcoholic liver injury was not investigated.

[0009] Therefore, it is essential to develop a cell model of alcoholic liver injury that can solve the above-mentioned technical problems and its construction method. Summary of the Invention

[0010] The purpose of this invention is to provide: A method for constructing a cell model of alcoholic liver injury, and related technologies, to address technical issues such as improving the stability and accuracy of the cell model, or a combination thereof.

[0011] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] The definition of standard chemical terms can be found in the reference "Li Yikui. Experimental Methodology of Traditional Chinese Medicine Pharmacology [M]. Shanghai: Shanghai Science and Technology Press, 2nd edition, October 2006".

[0014] Unless otherwise stated, conventional methods within the scope of the art, such as methods for detecting cell viability and methods for detecting aspartate aminotransferase (AST), shall be used.

[0015] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0016] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0017] As used herein, the term "treatment" means: to achieve the desired pharmacological and / or physiological effect. An effect may be preventative, but not necessarily preventative, in relation to the prevention or partial prevention of a disease, symptom, or condition (such as dermatitis). An effect may be therapeutic in relation to the partial or complete cure of a disease, condition, symptom, or adverse reaction attributable to that disease, condition, or condition. Specifically, the term "treatment" can include any treatment of a subject (especially a person) for a relevant symptom or disease, and may include any one or more of the following: (a) prevention of the disease in subjects who may be susceptible to the disease but have not yet been diagnosed with it; (b) suppression of the disease, i.e., prevention of its development; and (c) relief of the disease, i.e., reduction or improvement of the disease and / or its symptoms or condition. The term "treatment" can mean therapeutic treatment alone, preventative treatment alone, or both therapeutic and preventative treatment. A person requiring treatment (a subject requiring treatment) can include a person who already has a symptom and / or a person who needs to prevent a symptom.

[0018] As used herein, the term "prevention" refers to the act of administering a drug as an active ingredient to a healthy individual who is not suffering from disease or is in good health at the time of administration. The administration of a "preventive agent," as used herein, to such a healthy individual aims to prevent the development of disease, i.e., it is expected to be suitable for individuals who have prior symptoms of the disease or are considered to have an increased risk of developing the disease.

[0019] The term "cell viability" used in this article has the same meaning as "cell survival rate," and is calculated using the following formula:

[0020] The indefinite articles “a” and “an” preceding an element or component of this invention do not impose any limitation on the quantity (i.e., number of occurrences) of the element or component. Therefore, “an” or “a” should be interpreted as including one or at least one, and the singular form of an element or component also includes the plural form, unless the quantity clearly refers only to the singular form.

[0021] In this invention, features specified as "first" and "second" may explicitly or implicitly include one or more of those features, used to distinguish and describe features, without any order or emphasis. In the description of this invention, unless otherwise stated, "multiple" means two or more.

[0022] In a first aspect, the present invention provides a method for constructing a cell model of alcoholic liver injury, comprising the following steps: digesting HepG2 cells, resuspending and diluting them in DMEM complete culture medium, seeding the diluted cell solution into well plates, adding alcohol solution after a certain period of time and simultaneously covering the plate with a sealing film, and continuing incubation to obtain the cell model.

[0023] This includes: HepG2 cells, digestion, DMEM complete culture medium, resuspension, dilution, inoculation, well plates, alcohol solution, sealing film, incubation, etc.

[0024] Among them, digestion is selected from: digestion using enzymes.

[0025] The preferred digestion method is to use trypsin.

[0026] The digestion process is further optimized as follows: after discarding the cell supernatant from HepG2 cells in the logarithmic growth phase, wash them 2-3 times with PBS buffer, add trypsin to degrade intercellular proteins, and then discard the trypsin.

[0027] The specific parameters for adding trypsin are: 2 mL.

[0028] The HepG2 cells in the logarithmic growth phase are further preferably obtained by culturing HepG2 cells in DMEM complete medium at a temperature of 35-38℃ and a CO2 concentration of 3-8%.

[0029] The DMEM complete medium is selected from a medium containing DMEM basal medium and fetal bovine serum.

[0030] The preferred DMEM complete culture medium is composed of 80-90% DMEM basal culture medium and 10-20% fetal bovine serum by volume percentage.

[0031] The preferred basic DMEM culture medium is one containing penicillin and streptomycin.

[0032] The basic DMEM culture medium is further preferably composed of 90-100 U / mL penicillin and 90-100 μg / mL streptomycin.

[0033] The dilution factor is selected from 50-80 times.

[0034] The preferred dilution is a cell density of 50,000-80,000 cells / mL after dilution.

[0035] Inoculation is performed in any well of the well plate.

[0036] The preferred method for inoculation is to avoid inoculation within the outermost ring of the well plate.

[0037] The inoculation process is further preferably carried out outside the first and second rings of the well plate, calculated from the outermost edge inwards.

[0038] More preferably, the inoculation process involves inoculating each well of the plate with 0.1-1 mL of diluted cell solution.

[0039] The well plates are selected from cell culture plates with small wells, and there are no special restrictions on the shape or material of the wells.

[0040] The perforated plate is preferably composed of 4 or more rows and columns, and can be any integer such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, etc. It is further preferred to be more than 4, and even more preferred to be more than 6.

[0041] The orifice plate is further preferably selected from: 48-well plate, 96-well plate, 384-well plate, 1536-well plate, etc.

[0042] The specified time period is selected from 18-24h, with a choice of 18h, 19h, 20h, 21h, 22h, 23h, 24h, etc.

[0043] The alcohol solution is selected from alcohol solutions with a volume concentration of 3-10%.

[0044] The preferred alcohol solution is one with a volume concentration of 4-6%.

[0045] The preferred alcohol solution is prepared from anhydrous ethanol and DMEM complete culture medium.

[0046] The sealing film is selected from rayon sealing film.

[0047] The incubation conditions are selected from the following: temperature of 35-38℃, CO2 concentration of 3-8%, and incubation time of 18-24h.

[0048] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred embodiment is a method for constructing a cell model of alcoholic liver injury, characterized by the following steps: HepG2 cells are digested, resuspended and diluted in DMEM complete medium, and the diluted cell solution is seeded into well plates. After a certain period of time, an alcohol solution is added and the plates are sealed with a sealing film, and incubation continues. The seeding is not performed within the first and second rings of the well plate counted from the outermost edge. This technical solution, based on solving the technical problem of "improving the stability of the cell model," further solves the technical problem of "improving the stability of the cell model."

[0049] The second preferred embodiment: A method for constructing a cell model of alcoholic liver injury, characterized by the following steps: HepG2 cells are digested, resuspended and diluted in DMEM complete culture medium, the diluted cell solution is seeded into well plates, an alcohol solution is added after a certain period of time, and the plates are sealed with a sealing film and incubated for a further period of time; the volume concentration of the alcohol solution is 3-10%. This technical solution, while solving the technical problem of "improving the stability of the cell model", further solves the technical problem of "improving the accuracy of the cell model".

[0050] The third preferred embodiment: A method for constructing a cell model of alcoholic liver injury, characterized by the following steps: HepG2 cells are digested, resuspended and diluted in DMEM complete culture medium, and the diluted cell solution is seeded into well plates. After a certain period of time, an alcohol solution is added and the plates are sealed with a sealing film, and incubation continues until the desired cell model is obtained. The digestion is performed using trypsin. This technical solution, while addressing the technical problem of "improving the stability of the cell model," further addresses the technical problem of "improving the stability and accuracy of the cell model."

[0051] The fourth preferred embodiment: A method for constructing a cell model of alcoholic liver injury, characterized by the following steps: HepG2 cells are digested, resuspended and diluted in DMEM complete medium, and the diluted cell solution is seeded into well plates. After a certain period of time, an alcohol solution is added and the plates are sealed with a sealing film, and incubation continues to yield the desired cell model. The DMEM complete medium, by volume percentage, consists of 80-90% DMEM basal medium and 10-20% fetal bovine serum, and the basal DMEM medium contains 90-100 U / mL penicillin and 90-100 μg / mL streptomycin. This technical solution, in addition to solving the technical problem of "improving the stability of the cell model," further solves the technical problem of "improving the stability and accuracy of the cell model."

[0052] The fifth preferred embodiment: A method for constructing a cell model of alcoholic liver injury, characterized by the following steps: HepG2 cells are digested, resuspended and diluted in DMEM complete culture medium, and the diluted cell solution is seeded into well plates. After a certain period of time, an alcohol solution is added and the plates are sealed with a sealing film, and incubation continues. The dilution factor is 50-80 times, and the cell density after dilution is 50,000-80,000 cells / mL, more preferably 67,000 cells / mL. This technical solution, while solving the technical problem of "improving the stability of the cell model," further solves the technical problem of "improving the stability and accuracy of the cell model."

[0053] Secondly, the present invention provides: a cell model of alcoholic liver injury constructed by the aforementioned construction method.

[0054] Thirdly, the present invention provides the application of the aforementioned alcoholic liver injury cell model in screening products for the prevention and / or treatment of alcoholic liver injury.

[0055] The products are selected from: pharmaceuticals or health foods.

[0056] In this invention, Example 1 at least supports the protection scope of "a method for constructing a cell model of alcoholic liver injury, characterized by the following steps: digesting HepG2 cells, resuspending and diluting them in DMEM complete culture medium, seeding the diluted cell solution into well plates, adding alcohol solution after a certain time and covering the plate with a sealing film, and continuing incubation to obtain the cell model".

[0057] The beneficial effects of this invention are as follows: Compared with existing technologies, the present invention has better technical effects in terms of stability and accuracy in constructing cell models. Attached Figure Description

[0058] Figure 1 To investigate the effect of different concentrations of alcohol solutions on the viability of HepG2 cells, compared with the control group, P < 0.0001.

[0059] Figure 2 To replicate the effect of 3% alcohol solution on the viability of HepG2 cells in the experiment.

[0060] Figure 3 The effect of applying 6% ethanol solution at different locations in a 96-well plate on the viability of HepG2 cells was compared with that of the control group. P < 0.0001.

[0061] Figure 4 The effect of different columns in a 96-well plate treated with cyclic DMEM complete medium and 6% ethanol solution on the viability of HepG2 cells was investigated, compared with the control group. P < 0.0001.

[0062] Figure 5 To investigate the effect of rayon sealing film on HepG2 cell viability, compared with the control group, P < 0.0001.

[0063] Figure 6 The effect of different cell dilutions of 3% ethanol solution on HepG2 cell viability compared to the control group. P < 0.001.

[0064] Figure 7 The effect of different cell dilutions of 4% ethanol solution on HepG2 cell viability compared to the control group. p < 0.05 p < 0.01, p < 0.001, P < 0.0001.

[0065] Figure 8 The effect of different cell dilutions of 6% ethanol solution on HepG2 cell viability compared to the control group was investigated. P < 0.0001.

[0066] Figure 9 The results show the stability of the cell model constructed using 6% ethanol solution and sealing membrane in the middle 8 columns of a 96-well plate. p < 0.01.

[0067] Figure 10 The AST measurement results are for a cell model constructed using 6% ethanol solution and sealing membrane in the middle 8 columns of a 96-well plate. p < 0.01.

[0068] Figure 11 To investigate the effect of different concentrations of silymarin on the survival rate of HepG2 cells induced in the middle 8 wells of a 96-well plate using 6% ethanol solution and sealing film, compared with the control group, ####P<0.0001; compared with the model group, p < 0.05 p < 0.001, P < 0.0001.

[0069] Figure 12 The effect of silymarin on AST activity of HepG2 cells induced in the middle 8 wells of a 96-well plate using 6% ethanol solution and sealing film was investigated. Compared with the control group, ###P<0.001; compared with the model group, p < 0.01. Detailed Implementation

[0070] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0071] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0072] Example 1 1. Experimental Objective Determine the modeling conditions and establish a cell model of alcoholic liver injury.

[0073] 2 Experimental Design 2.1 Reagents / Equipment 2.1.1 Reagents DMEM complete medium: Prepared by adding 10% fetal bovine serum (PBS) to 90% DMEM basal medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin, purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number 12100) and PAN (Germany), catalog number ST30-3302. CCK8 kit, Western blotting and IP lysis buffer (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number P0013), BCA detection kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number P0010), AST detection kit (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number BC1565), and rayon sealing film (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number FSF033-100pcs).

[0074] 2.1.2 Equipment Low-speed centrifuge, microscope, vortex mixer, enzyme-linked immunosorbent assay (ELISA) reader.

[0075] 2.2 Experimental Methods 2.2.1 Preparation of alcohol solution Using DMEM complete medium, anhydrous ethanol was prepared into alcohol solutions with volume fractions of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% for later use.

[0076] 2.2.2 Effects of different concentrations of alcohol solution on HepG2 cells After HepG2 cells reached the logarithmic growth phase, the culture medium was discarded, and the cells were washed twice with PBS. 2 mL of trypsin was added for digestion for 3 min, followed by the addition of complete culture medium to terminate the digestion. The cells were centrifuged at 1000 rpm for 4 min. After centrifugation, the supernatant was discarded, and the cells were resuspended in 3 mL of DMEM complete culture medium. 1 mL of the cell solution was diluted 50-80 times, and seeded at a density of 50,000-80,000 cells / mL into 96-well plates and cultured in a CO2 incubator. After 24 h of culture, the control group received DMEM complete culture medium, while the model group received DMEM complete culture medium containing different concentrations of ethanol. OD values ​​were measured after 24 h of culture. The blank group contained only complete culture medium. Cell viability was calculated using the following formula:

[0077] 2.2.3 Determination of AST After HepG2 cells reached the logarithmic growth phase, the culture medium was discarded, and the cells were washed twice with PBS. 2 mL of trypsin was added for digestion for 3 min, followed by the addition of complete culture medium to terminate the digestion. The cells were centrifuged at 1000 rpm for 4 min. After centrifugation, the supernatant was discarded, and the cells were resuspended in 3 mL of DMEM complete culture medium. 1 mL of the cell solution was diluted 50-80 times and seeded at a density of 50,000-80,000 cells / mL in the central 4×2 well of a 24-well plate, with 1 mL of diluted cell solution added to each well. After 24 h of culture in a CO2 incubator, the control group received 1 mL of DMEM complete culture medium, the model group received 1 mL of DMEM complete culture medium containing 6% ethanol, and the silymarin group received 1 mL of DMEM complete culture medium containing 6% ethanol and 39 μg / mL silymarin. After another 24 h of culture, the supernatant was discarded, and the cells were washed twice with PBS. 50 μL of Western lysis buffer and IP lysis buffer were added to each well. After lysis, 5 μL of the lysed cell sample was used to determine protein content using a BCA assay kit. Take another 5 μL of cell lysis sample and measure AST using an AST detection kit.

[0078] 3. Experimental Results 3.1 Effects of different concentrations of alcohol solution on the viability of HepG2 cells An alcohol-induced liver injury model was established using alcohol solutions of different concentrations, and the results are as follows: Figure 1 As shown, 3%-10% alcohol solutions significantly reduced cell viability. The cell viability in the 3% alcohol solution group was 68%, therefore 3% is suitable as the modeling concentration for this experiment.

[0079] 3.2 Effect of 3% alcohol solution on HepG2 cell viability The experiment was repeated using a 3% ethanol solution as the modeling concentration. It was found that cell viability did not decrease significantly in the 3% ethanol solution group. Figure 2 The results even showed a tendency to increase cell viability, which is inconsistent with the experimental results in section 3.1. Therefore, it is necessary to verify the possible causes of this result. When using 96-well plates in cell experiments, PBS is generally added to the outermost ring to prevent the evaporation of the culture medium. Therefore, the middle 10×6 area is usually used. In section 3.1, the 3% alcohol group was located in column 3, while in this experiment, the 3% alcohol group was located in column 2. The degree of alcohol evaporation may differ between columns 2 and 3, thus causing the difference in the results between the two experiments.

[0080] 3.3 Effect of alcohol evaporation on HepG2 cell viability The discrepancy between the two experimental results is likely related to the evaporation of the alcohol solution in the 96-well plate. Therefore, a 6% alcohol solution was added to the 60 wells excluding the outermost ring of the plate. Data was normalized with the first column on the left (10 wells per column) as 100%. The results are shown below. Figure 3 The cell viability in the middle 8 columns (columns 2-9 from the left) was between 50% and 60%, with very similar data. However, the data in columns 1 and 10 from the left were significantly higher than those in the middle 8 columns, indicating that columns 1 and 10 should not be used for this experiment.

[0081] Simultaneously, 10 wells in the 96-well plate were cycled through DMEM complete medium (columns 1, 3, 5, 7, and 9) and 6% ethanol solution (columns 2, 4, 6, 8, and 10). Data from column 1 were normalized to 100%. Results are shown below. Figure 4 The data in the middle 8 columns were found to be generally reliable, and only the middle 8 columns were used in subsequent plate-laying experiments. However, the cell survival rate in the second column, which was used as the model group with added alcohol solution, was significantly higher than that in the other column model groups. Therefore, using the middle 8 columns for experiments may not completely solve the problem, and other methods to improve the evaporation of alcohol solution need to be further explored.

[0082] After adding complete culture medium containing 6% (v / v) ethanol, a rayon sealing membrane was placed on each 96-well plate. This membrane provides some permeability and does not affect cell growth, while also reducing ethanol evaporation to some extent. Cells were divided into a control group (C), an unsealed group (WFB), and a sealed group (FB). The control group received 1 mL of DMEM complete culture medium, the unsealed group received 1 mL of DMEM complete culture medium containing 6% (v / v) ethanol and did not use a rayon sealing membrane, and the sealed group received 1 mL of DMEM complete culture medium containing 6% (v / v) ethanol and sealed with a rayon sealing membrane. Experimental results are shown below. Figure 5 At the same alcohol solution concentration, the cell survival rate was significantly reduced after covering with a sealing membrane, indicating that the sealing membrane played a role in preventing the alcohol solution from evaporating.

[0083] 3.4 Effects of cell seeding density and alcohol solution concentration on cell viability Aside from the evaporation of the alcohol solution in the 96-well plate, other possible reasons for inconsistencies between the first and second experimental results include cell seeding density and alcohol solution concentration. Therefore, experiments were conducted separately after covering the plates with a rayon sealing film. Figure 6 It can be seen that when the alcohol solution concentration is 3%, the dilution factor at the time of cell seeding has a significant impact on the results. At cell dilutions of 50, 60, and 70 times, there was no significant difference in cell viability between the 3% alcohol solution group and the control group. At a cell dilution of 80 times, the cell viability of the 3% alcohol solution group was significantly lower than that of the control group. When the alcohol solution concentration for modeling was 4% ( Figure 7 In all groups, cell viability was significantly lower than that in the control group. The higher the dilution factor, the lower the cell viability.

[0084] When the concentration of the alcohol solution is 6% ( Figure 8Cell viability in all groups was significantly lower than in the control group, with a cell survival rate of approximately 40%. However, cell dilution factor did not have a significant impact on cell viability. To minimize the influence of the seeded cell amount on subsequent experiments, 6% was selected as the modeling concentration for alcoholic liver injury.

[0085] 3.5 Model Stability Verification Through the above experiments, the experimental modeling conditions were determined: the middle 8 columns of a 96-well plate were used for plating, the alcohol solution concentration was 6%, and the plate was sealed with a sealing film after adding the modeling agent. To verify the stability of the model, three experiments were conducted using the above conditions. In cell experiments, the first and second columns are generally used as the control group and the model group, respectively. Therefore, in all three experiments, the first and second columns of the 8-well plate were used as the control group and the model group, respectively. Each group had 4 parallel wells, and the results are as follows: Figure 9 The cell survival rates of the model group (denoted as M) in the three experiments were 44.3%, 40.2% and 41.2%, respectively, indicating that the method has good stability.

[0086] 3.6 Effect of 6% alcohol solution on AST activity in HepG2 cells To verify the stability of 6% alcohol-induced modeling on the AST index, three repeated experiments were conducted. Figure 10 It can be seen that in the three repeated experiments, the AST activity of the model group (denoted as M) was significantly increased compared with the control group (denoted as C), indicating that the experiment was relatively stable.

[0087] 3.7 Effects of silymarin on HepG2 cells induced by 6% ethanol solution Following the experimental conditions described in section 3.5, the middle eight wells of a 96-well plate were induced for 24 hours using 6% ethanol solution and a sealing membrane, with different concentrations of silymarin samples added. After culture, the cells were incubated with CCK8 reagent, and the OD value was measured to calculate cell viability. Figure 11 It can be seen that 5-40 μg / mL silymarin significantly improved cell survival rate after induction with alcohol solution, with the 40 μg / mL group showing a 23.4% increase.

[0088] Depend on Figure 12 The results showed that the AST content in the control group of HepG2 cells was 2.03 U / mg prot, while the AST content in the model group after induction with 6% ethanol solution for 24 h was 3.11 U / mg prot, which was significantly higher than that in the control group, indicating that the model was successfully established. When 39 μg / mL silymarin was added simultaneously with induction with 6% ethanol solution, the AST content decreased to 2.41 U / mg prot, which was significantly different from that in the model group, indicating that silymarin has a role in improving alcoholic liver injury.

[0089] Silymarin is a commonly used positive control in the evaluation of alcoholic liver injury. Based on the combined results of cell viability and AST activity, the cell model constructed in this invention can be used to evaluate alcoholic liver injury.

[0090] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for constructing a cell model of alcoholic liver injury, characterized in that, The process includes the following steps: HepG2 cells are digested, resuspended and diluted in DMEM complete medium, and the diluted cell solution is seeded into well plates. After a certain period of time, alcohol solution is added and the plates are covered with a sealing film. The cells are then incubated for a longer period of time.

2. The construction method according to claim 1, characterized in that, The volume concentration of the alcohol solution is 3-10%, preferably 4-6%.

3. The construction method according to claim 2, characterized in that, The alcohol solution was prepared from anhydrous ethanol and DMEM complete culture medium.

4. The construction method according to claim 1, characterized in that, The digestion was performed using trypsin.

5. The construction method according to claim 4, characterized in that, The specific digestion procedure is as follows: after discarding the cell supernatant of HepG2 cells in the logarithmic growth phase, wash them 2-3 times with PBS buffer, add trypsin to degrade intercellular proteins, and then discard the trypsin.

6. The construction method according to claim 5, characterized in that, The HepG2 cells in the logarithmic growth phase were obtained by culturing HepG2 cells in DMEM complete medium at a temperature of 35-38℃ and a CO2 concentration of 3-8%.

7. The construction method according to claim 1, characterized in that, The DMEM complete culture medium, by volume percentage, consists of 80-90% DMEM basal culture medium and 10-20% fetal bovine serum. The basal DMEM culture medium contains 90-100 U / mL penicillin and 90-100 μg / mL streptomycin.

8. The construction method according to claim 1, characterized in that, The dilution factor is 50-80 times, and the cell density after dilution is 50,000-80,000 cells / mL.

9. The construction method according to claim 1, characterized in that, The specified time period is 18-24 hours.

10. The construction method according to claim 1, characterized in that, The number of rows and columns of the perforated plate is 4 or more, preferably more than 4, and even more preferably 6 or more.

11. The construction method according to claim 10, characterized in that, The perforated plate is a 96-hole plate.

12. The construction method according to claim 10, characterized in that, The inoculation is not performed within the outermost ring of the well plate.

13. The construction method according to claim 12, characterized in that, The inoculation is not performed within the first and second rings of the well plate, calculated from the outermost edge inwards.

14. The construction method according to claim 1, characterized in that, The sealing film is a rayon sealing film.

15. The construction method according to claim 1, characterized in that, The incubation conditions are: temperature 35-38℃, CO2 concentration 3-8%, and incubation time 18-24h.

16. The construction method according to claim 1, characterized in that, 0.1-1 mL of diluted cell solution was inoculated into each well of the plate.

17. A cell model of alcoholic liver injury constructed by any one of the construction methods described in claims 1-16.

18. The use of the alcoholic liver injury cell model of claim 17 in screening products for the prevention and / or treatment of alcoholic liver injury.

19. The application according to claim 18, characterized in that, The product in question is a pharmaceutical or health food product.

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