Cell model for high-throughput medicine screening of fatty liver model as well as construction method and application of cell model

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a cell model for high-flux medicine screening of a fatty liver model and a construction method and application of the cell model. According to the cell model for high-throughput drug screening of the fatty liver model, a HepaRG cell line is taken as a host and is transfected into a lentiviral vector of an overexpressed PLIN2 gene; the lentiviral vector contains a reporter gene. The invention also discloses a construction method of the cell model. According to the cell model provided by the invention, the formation, fusion, distribution and dynamic change of the lipid droplets can be intuitively observed in real time, the quantity and size change of the lipid droplets can be more accurately evaluated, the pathophysiological process of the fatty liver can be simulated, the abnormal accumulation and metabolic disorder of the lipid droplets in the occurrence process of the fatty liver can be helped to be researched, and the development of the fatty liver model is facilitated. The method is of great significance to research on influence of different factors on fatty liver and screening of potential drugs for fatty liver.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to cell models for high-throughput drug screening in fatty liver models, their construction methods, and applications. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease, with a global prevalence of approximately 25% of the adult population. It is a leading cause of cirrhosis and hepatocellular carcinoma. Due to its high prevalence, NAFLD is now the fastest-growing cause of liver-related deaths worldwide and is becoming a significant cause of end-stage liver disease. A substantial increase in NAFLD-related advanced liver disease, its mortality rate, and its overall disease burden is projected.

[0003] This disease involves the accumulation of intracellular lipid droplets in the liver through a process not fully understood. Lipid droplets consist of a neutral lipid core surrounded by a lipid monolayer and decorated with a series of specific proteins. Because the accumulation of lipid droplets in hepatocytes is a major characteristic of NAFLD, considerable research has been devoted to understanding their formation, regulation, and contribution to NAFLD progression. Lipid droplets are coated with lipid droplet-associated proteins, including the perilipins family (PLIN), which are involved in droplet formation, stability, and transport. Among these, PLIN2 is a cytoplasmic protein primarily expressed in the liver and is particularly important in the pathogenesis and development of NAFLD, being associated with the development of steatosis and insulin resistance. PLIN2 coats the lipid droplet membrane and reduces the entry of triglyceride lipase (ATGL) into the droplets, thereby preventing lipolysis and exacerbating the progression of fatty liver disease. Studies have shown a strong correlation between PLIN2 levels and the degree of steatosis. Therefore, exploring the mechanisms underlying the PLIN2 protein is of paramount importance.

[0004] Currently, in vitro cell models for studying NAFLD are mainly constructed through exposure to free fatty acids such as oleic acid and palmitic acid. However, existing models have significant limitations and cannot meet the needs of mechanistic research and applications. For example, existing models rely on traditional methods such as Oil Red O staining and BODIPY labeling to observe lipid droplets, which requires cell fixation and cannot be tracked in real time, making it difficult to capture the dynamic processes of lipid droplet formation, fusion, and decomposition. At the same time, traditional labeling lacks specificity and cannot accurately anchor the spatial association between lipid droplets and functional proteins such as PLIN2, making it difficult to intuitively analyze the role of PLIN2 in abnormal lipid droplet accumulation. Existing models can only induce the terminal phenotype of lipid droplet accumulation and cannot simulate the core pathological chain of "PLIN2 upregulation - lipid droplet stabilization - lipolysis disorder" in NAFLD. They have a weak correlation with the pathological process of excessive lipid droplet accumulation driven by high PLIN2 expression in clinical practice and are difficult to reveal the molecular mechanism of abnormal lipid droplet accumulation. Moreover, traditional models require complex staining steps, which are time-consuming and prone to introducing experimental errors. In drug screening, they can only assess efficacy through the endpoint lipid droplet state, and cannot reflect the drug's regulatory effect on lipid droplet dynamics and PLIN2 function in real time, thus restricting the discovery and validation of NAFLD treatment targets.

[0005] For the reasons mentioned above, constructing a cell model that can be prepared on a large scale and whose pathological mechanism of NAFLD can be analyzed in depth is of great scientific significance for screening drugs for the treatment of fatty liver disease and understanding the pathogenic mechanism of its related proteins. This is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a cell model for high-throughput drug screening in fatty liver models, a method for constructing the model, and its application, so as to solve the problems in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] This invention provides a cell model for high-throughput drug screening in a fatty liver model, wherein the cell model uses the HepaRG cell line as the host and is transfected with a lentiviral vector that overexpresses the PLIN2 gene.

[0009] The NCBI accession number for the PLIN2 gene is NM_001122.4; the lentiviral vector contains a reporter gene.

[0010] Gene editing technology can achieve stable gene modification, avoiding concentration fluctuations or cell state differences that may occur during free fatty acid induction. This stability makes experimental results more reproducible and suitable for long-term studies. The PLIN2 gene-editing model can maintain a stable gene expression state over a longer period, while free fatty acid-induced models may be affected by changes in cell metabolism. The PLIN2 gene-editing model can more accurately simulate the pathological process of fatty liver, especially metabolic disorders related to the PLIN2 gene. The PLIN2 gene-editing model can be used to screen drugs targeting the PLIN2 gene and verify their potential role in the treatment of fatty liver. Therefore, fatty liver models constructed using PLIN2 gene editing technology have significant advantages in terms of specificity, stability, accuracy of disease simulation, and research and application scope, providing a more powerful tool for basic research and clinical applications of fatty liver.

[0011] In some embodiments of the present invention, the reporter gene is the EGFP gene.

[0012] Compared to other commonly used hepatocyte cell lines (such as G2), the HepaRG cell line exhibits higher metabolic enzyme activity and expression levels in constructing stable cell lines. It stably expresses multiple drug-metabolizing enzymes (such as CYP450 family enzymes), with expression levels and activities more closely resembling those of primary human hepatocytes. During culture, the HepaRG cell line forms polar hepatocyte structures, exhibiting histological characteristics similar to in vivo hepatocytes. This cell polarity helps to better mimic the physiological functions of the liver in vivo, including drug uptake, metabolism, and secretion. The gene expression profile of the HepaRG cell line is closer to that of primary human hepatocytes and liver tissue, while HepG2 cells show significant differences in gene expression and metabolic function compared to primary hepatocytes. Therefore, the HepaRG cell line is more representative in simulating liver physiological and pathological processes. Higher drug metabolism and transport capabilities: The HepaRG cell line exhibits higher activity in drug uptake and transport, with expression levels of key uptake and efflux transport proteins very similar to those of primary human hepatocytes. This makes the HepaRG cell line more valuable in studying drug interactions and drug transport mechanisms. The HepaRG cell line maintains stable metabolic enzyme activity and cell function during long-term culture. In contrast, other hepatocyte cell lines (such as G2 cells) may experience a decline in metabolic function during long-term culture. The HepaRG cell line has significant advantages over other hepatocyte cell lines in terms of metabolic enzyme activity, cell polarity, gene expression profile, drug metabolism, and transport capabilities, making it more valuable for drug development and the construction of liver disease models.

[0013] This invention also provides a method for constructing a cell model for high-throughput drug screening in a fatty liver model, comprising the following steps:

[0014] The PLIN2 gene described in NCBI accession number NM_001122.4 was inserted into a lentiviral vector to obtain a lentiviral vector plasmid overexpressing the PLIN2 gene.

[0015] The PLIN2 gene overexpression lentiviral vector, packaging helper plasmid, and envelope plasmid were transfected into packaging cells to obtain lentiviral particles.

[0016] The HepaRG cell line was infected with the lentiviral particles described above, and a cell model for high-throughput drug screening in a fatty liver model was obtained by culturing the cells.

[0017] In some embodiments of the present invention, the lentiviral vector plasmid contains a reporter gene. The reporter gene facilitates observation of vector transfection and expression using a fluorescence microscope. Specifically, the reporter gene in this invention is the EGFP gene.

[0018] In some embodiments of the present invention, the promoter selected is Ubc (ubiquitin C promoter), which has the advantages of high starting efficiency, strong transcriptional stability and wide species applicability, and can ensure the efficient co-expression of PLIN2 gene and EGFP gene.

[0019] In some specific embodiments of the present invention, the lentivirus vector backbone is GV218.

[0020] In some embodiments of the present invention, the packaging aid plasmid is pHelper 1.0; the coating plasmid is pHelper 2.0.

[0021] In some embodiments of the present invention, the mass ratio of the PLIN2 gene overexpression lentiviral vector, packaging helper plasmid, and envelope plasmid is 4:3:2.

[0022] In some embodiments of the present invention, when the lentiviral particles are used to infect the HepaRG cell line, the MOI of the lentiviral particles is 1 to 100.

[0023] In some embodiments of the present invention, the HepaRG cell line is infected with the lentivirus particles and then incubated for 16-24 hours.

[0024] In some embodiments of the present invention, after infecting the HepaRG cell line with the lentiviral particles, a cell resistance screening reagent is added to finally obtain a positive cell line, which is the cell model used for high-throughput drug screening in a fatty liver model.

[0025] In some embodiments of the present invention, the cell resistance screening reagent is Puromycin.

[0026] In some embodiments of the present invention, the HepaRG cell line is cultured in a culture system containing FBS, and then lentiviral particles and infection enhancement solution are added and incubated for 16-24 hours to infect the HepaRG cell line. After that, Puromycin is added for selection for 7-14 days, and finally positive clones are obtained, which are cell models for high-throughput drug screening in fatty liver models.

[0027] In some embodiments of the present invention, the infection enhancement solution is HiTransGP. The volume percentage of HiTransGP in the culture system is 4%.

[0028] In some embodiments of the present invention, the final concentration of Puromycin in the culture system is 2 μg / mL.

[0029] This invention also provides the application of cell models for high-throughput drug screening in fatty liver models in evaluating or developing the efficacy of fatty liver drugs.

[0030] Based on the technical solution of the present invention, the present invention has the following beneficial effects compared with the prior art:

[0031] The cell model provided by this invention for high-throughput drug screening in a fatty liver model:

[0032] (1) Precise positioning and visualization: intuitive observation of lipid droplet dynamics. PLIN2 is mainly located on the surface of lipid droplets. After fusion with EGFP, the formation, fusion, distribution and dynamic changes of lipid droplets can be observed in real time and intuitively through a fluorescence microscope.

[0033] (2) More accurate quantitative analysis: The intensity of the fluorescence signal is positively correlated with the expression level of the PLIN2-EGFP fusion protein. Quantitative analysis of fluorescence intensity can more accurately assess the changes in the number and size of lipid droplets. This is of great significance for studying the effects of different factors on fatty liver.

[0034] (3) Simulating pathophysiological processes: In the fatty liver model induced by a high-fat diet, the increased expression of PLIN2 is closely related to the excessive accumulation of lipid droplets. This model can simulate the pathophysiological process of fatty liver and help study the abnormal accumulation of lipid droplets and metabolic disorders during the development of fatty liver.

[0035] (4) Easy to construct and detect: The construction of the PLIN2-EGFP fusion protein vector is relatively simple, and the fluorescence signal of EGFP is easy to detect without complicated staining steps. This makes the experimental operation more convenient and saves time and costs.

[0036] (5) Drug screening and efficacy evaluation: Using the PLIN2-EGFP fusion protein model, potential drugs for fatty liver can be screened, and the effect of drugs on lipid droplet metabolism and fatty liver improvement can be evaluated by changes in fluorescence signals.

[0037] The cell model construction method for high-throughput drug screening in fatty liver model provided by this invention uses the Ubc promoter to drive the expression of PLIN2-EGFP fusion gene, which has higher stability and lower silencing rate than the traditional CMV promoter. Attached Figure Description

[0038] Figure 1 This is a structural diagram of the carrier skeleton.

[0039] Figure 2 This is a schematic diagram of the structure of the PLIN2 recombinant vector.

[0040] Figure 3 The image shows the sequencing verification results of the positive clone.

[0041] Figure 4 Bright-field, green-fluorescence, and red-fluorescence images of lentiviruses packaged and transfected into 293T cells.

[0042] Figure 5 The figure shows the statistical results of the identification of stable PLIN2 cells; where RG represents the negative control group, i.e., untransfected HepaRG cells; GFP represents the empty vector control group, i.e., cell lines transfected with an empty vector containing only GFP fluorescent protein; and PLIN2 represents the target gene overexpression group, i.e., stable cell lines transfected with the PLIN2 gene expression vector.

[0043] Figure 6 The figure shows the results of Western blotting validation of the stable PLIN2 strain.

[0044] Figure 7 Visual diagram of lipid droplets in Plin2-GFP transfected HepaRG cell line.

[0045] Figure 8 This is a graph showing the results of drug screening suitability validation.

[0046] Figure 9 This is a graph showing the statistical results of relative quantitative analysis of lipid droplets. Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. 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 protection scope of the present invention.

[0048] The following description is based on specific embodiments.

[0049] Unless otherwise specified, all reagents used in the following examples are commercially available; and all methods used in the following examples are conventional methods unless otherwise specified.

[0050] Example 1

[0051] A method for constructing a cell model for high-throughput drug screening in a fatty liver model includes: inserting the PLIN2 gene into a lentiviral vector to obtain a PLIN2 gene overexpression lentiviral vector plasmid; then transfecting the PLIN2 gene overexpression lentiviral vector, packaging helper plasmid, and envelope plasmid into packaging cells to package lentiviral particles; and finally infecting the HepaRG cell line with the lentiviral particles to obtain a cell model for high-throughput drug screening in a fatty liver model. Specifically, the method includes the following steps:

[0052] 1. HepaRG cell line culture

[0053] The HepaRG cell line (purchased from Thermo Fisher Scientific (China) Co., Ltd., cell code HPRGC10) was cultured in William Medium E medium at 37°C and 5% CO2. After 48 hours, unattached cells were discarded, and fresh DMEM medium was added. Continue culturing until the cell confluence reaches 80%~90%. Wash the cells with PBS and digest them with a digestion solution (Trypsin-EDTA Sulotion, containing 0.25% trypsin and 0.02% EDTA). When 80% of the cells are observed to be rounded and detached in large areas under a microscope, add 10% FBS to stop the digestion. Collect the cell suspension into a 15mL centrifuge tube and centrifuge at 1200rpm for 5min. Discard the supernatant and resuspend the cells in 1mL of DMEM medium. Count the cells using a cell counter and passage or cryopreserve according to the cell count and subsequent experimental arrangements. (The cryopreservation solution is prepared in 1mL of William Medium E medium: fetal bovine serum (FBS):DMSO in a ratio of 5:4:1. Mix the solution with the cell pellet and place 1mL of cryopreservation solution per cell tube at -80℃.)

[0054] 2. Preparation of PLIN2 gene overexpression lentiviral clones

[0055] (1) Obtaining and amplifying the target gene:

[0056] Using the full-length coding region of the human PLIN2 gene in GenBank NM_001122.4 as a template, specific PCR primers containing BamHI / AgeI restriction sites as shown in Table 1 were designed to amplify a 1360bp PLIN2 gene fragment. Sequencing was used to verify the sequence accuracy, and the fragment was then used for the construction of lentiviral vectors.

[0057] Table 1

[0058] (2) Vector digestion and recombination ligation

[0059] like Figure 1 As shown, the GV218 lentiviral vector from Jikai Gene (core element: Ubc-MCS-EGFP-IRES-Puromycin) was linearized by incubation at 37°C for 3 hours or overnight (ensuring complete digestion) with BamHI and AgeI restriction endonucleases (enzyme digestion system shown in Table 2). 10 μL of the digestion product was subjected to 1% agarose gel electrophoresis with a DNA Marker (DL5000, Lembio, catalog number LM1071). The digestion effect was observed using a gel imaging system to confirm the linearized vector fragment (expected size approximately 11131 bp, referring to the total length of the GV218 vector). The linearized vector fragment was recovered using a gel recovery kit to remove undigested circular vectors and impurities, improving subsequent recombination efficiency. The linearized vector was recovered after verification by agarose gel electrophoresis.

[0060] In the lentiviral vector GV218, the EGFP (enhanced green fluorescent protein) gene is introduced as a reporter gene, facilitating observation of vector transfection and expression using a fluorescence microscope. The Ubc (ubiquitin C) promoter is chosen, offering advantages such as high initiation efficiency, strong transcriptional stability, and broad species applicability, ensuring efficient co-expression of the PLIN2 and EGFP genes. The presence of the IRES (internal ribosome entry site) element enables independent expression of the PLIN2 target gene and the Puromycin resistance gene, avoiding expression interference. The inclusion of the puro resistance gene (Puromycin resistance gene) allows for the selection of cell lines stably expressing the target gene through Puromycin drug screening, simplifying subsequent experimental procedures.

[0061] Table 2 Enzyme digestion system (total volume 50 μL)

[0062] The linearized vector was homologously recombinated with the PLIN2 target gene using the ClonExpress™ II One Step Cloning Kit to construct a recombinant lentiviral expression vector, the structure of which is shown below. Figure 2As shown. The specific steps are as follows:

[0063] Prepare a 10 μL recombinant reaction system (Table 3) under ice bath conditions, ensuring that all reagents are mixed evenly at low temperature to avoid generating bubbles; incubate at 37℃ for 30 min to achieve in vitro circularization of the linearized vector and the target gene fragment; immediately place in an ice-water bath for 5 min after the reaction to terminate the recombinant reaction, prevent non-specific ligation, and obtain the crude product of the recombinant lentiviral expression vector, namely the PLIN2 gene overexpression lentiviral vector.

[0064] Table 3. Homologous recombination reaction system (total system volume 10 μL)

[0065] (3) Screening of positive clones and preparation of plasmids

[0066] The recombinant vector was transformed into TOP10 competent cells, and the cells were screened for ampicillin resistance, identified by colony PCR (positive band 877bp), and verified by sequencing (PLIN2 sequence showed no mutation, correct reading frame, and sequencing assembly results were as follows). Figure 3 As shown in the table, (identical to the target sequence), qualified positive clones were obtained. The primers for colony PCR identification and sequencing are shown in Table 4. Plasmids of higher purity (concentration ≥1 μg / μL) were extracted using a plasmid purification kit and stored at -20℃ / -80℃ for later use.

[0067] Table 4. Primers for colony PCR identification and sequencing

[0068] (4) Lentiviral packaging and quality control

[0069] 1) Cell preparation: HEK293T cells were cultured in DMEM medium containing 10% FBS and seeded into 10cm dishes before transfection, with a density of 70%~80% for later use.

[0070] 2) Transfection system: The Gilead three-plasmid packaging system was used, namely recombinant lentiviral expression vector (20 μg), packaging helper plasmid pHelper 1.0 (15 μg), and envelope plasmid pHelper 2.0 (10 μg), with a mass ratio of 4:3:2; the above plasmids were mixed with an appropriate amount of Gilead transfection reagent, the total volume was adjusted to 1 mL, and incubated at room temperature for 15 min;

[0071] 3) Transfection procedure: 1 hour before transfection, replace the cell culture medium with DMEM medium containing 2% FBS; slowly add the incubated transfection system to the cell culture medium, mix gently (avoid blowing the cells), and incubate at 37°C in a 5% CO2 incubator.

[0072] 4) Culture medium replacement: After culturing for 6-8 hours (specifically, in this example, the culturing time is 6 hours), discard the culture medium containing the transfection system, add 10 mL of PBS (Shanghai Jikai Gene, catalog number GCPBS500S) to wash once, and then slowly add 12 mL of DMEM culture medium containing 2% FBS, and continue culturing for 48 hours.

[0073] 5) Virus collection and concentration: 48 hours after transfection (0 h from the start of transfection), the cell supernatant was collected, centrifuged at 4000g to remove debris, filtered through a 0.45μm filter, concentrated by ultracentrifugation at 25000rpm and 4℃ for 2 h, resuspended and stored at -80℃.

[0074] 6) Quality control standard: By visual inspection, the virus solution is pink and clear, with no obvious viscosity or lag in absorption;

[0075] The titer was determined by q-PCR, and the titer result was ≥1.08×10⁻⁶. 9 TU / mL;

[0076] Sixty-eight hours after infection, cell state and fluorescence expression were observed, and bright-field, green fluorescence, and red fluorescence images were taken. The results are as follows: Figure 4 As shown, the positive rate of fluorescent infection is ≥30%.

[0077] 3. Lentiviral infection of HepaRG cell lines and screening of stable strains

[0078] 1) Cell preparation: Culture the HepaRG cell line to the logarithmic growth phase. When the cell confluence reaches 50%~60%, seed them into 6-well plates. Add 2 mL of complete medium containing 10% FBS to each well (DMEM medium + 10% FBS) and pre-culture at 37℃ in a 5% CO2 incubator for 12 h.

[0079] 2) Viral infection: Add PLIN2 lentivirus concentrate at MOI=50, add HiTransGP infection enhancement solution (4μL for every 100μL system), incubate at 37℃ and 5% CO2 for 16~24h (specifically, in this example, the incubation time is 24h), and then replace with fresh culture medium.

[0080] 3) Resistance screening: After the cell density recovers to 60%~70%, add Puromycin (Shanghai Jikai Gene, catalog number REVG1001) at a final concentration of 2μg / mL for screening for 7~14 days (change the medium every 2~3 days) until the control group cells die completely.

[0081] 4) Monoclonal purification: The resistant cells were seeded into 96-well plates using the limiting dilution method, and the monoclonal culture was expanded to 24-well plates.

[0082] 4. Identification of stable strains

[0083] (1) q-PCR detection: Total RNA was extracted from cells and reverse transcribed into cDNA. GAPDH was used as an internal reference, and the relative expression level of PLIN2 gene was detected using the PLIN2-specific primers as shown in Table 5.

[0084] Identification system (20 μL): ddH2O 9 μL, 2×Taq Plus Master Mix 10 μL, upstream identification primer (10 μM) 0.5 μL, downstream identification primer (10 μM) 0.5 μL;

[0085] Procedure: In a clean bench, use a sterile pipette tip to pick up a single colony, pipette it into the identification system as a template, mix well, and then place it in a PCR instrument (LongGene, A300) for reaction;

[0086] Reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 44 s (calculated at 1 kb / min, expected positive product length 877 bp), for a total of 22 cycles; 72℃ final extension for 8 min.

[0087] Table 5 Primers for identifying stable strains

[0088] (2) Western Blot verification of PLIN2 protein expression

[0089] 1) Total cell protein extraction: Receive cell samples, wash twice with PBS, add an appropriate amount of pre-chilled 2×LysisBuffer (containing protease inhibitors) to lyse cells; scrape cells off with a cell scraper and transfer them to EP tubes, lyse on ice for 10-15 minutes, then sonicate to disrupt cells (200W for 4 times, 5 seconds each time, with 2 seconds interval); centrifuge at 12000g at 4℃ for 15 minutes, collect the supernatant, determine the protein concentration using the BCA method, adjust the protein concentration of each sample to 2μg / μL, and store at -80℃ for later use.

[0090] 2) SDS-PAGE electrophoresis: Prepare a 10% separating gel and a 5% stacking gel according to the molecular weight of the target protein (PLIN2 protein molecular weight is about 55kDa); after the gel solidifies, remove the comb, clean the sample wells with electrophoresis buffer, mix the protein sample with the adjusted concentration and the sample loading buffer, boil to denature and then load the sample; set the electrophoresis parameters: stacking gel 80mA electrophoresis for 20 minutes, separating gel 120mA electrophoresis for 1 hour.

[0091] 3) Immunoblotting (wet transfer): After electrophoresis, the protein was transferred to a PVDF membrane by electrophoresis at 4°C and 200mA constant current for 120 minutes.

[0092] 4) Antibody hybridization: Block the PVDF membrane with TBST solution containing 5% skim milk at room temperature for 1 hour; dilute the PLIN2 primary antibody (1:1000) with blocking buffer and incubate it with the PVDF membrane at 4°C overnight, then wash the membrane 3 times with TBST for 10 minutes each time; dilute the HRP-labeled secondary antibody (1:5000) with blocking buffer and incubate the PVDF membrane at room temperature for 2 hours, then wash the membrane 3 times with TBST for 10 minutes each time.

[0093] 5) X-ray development: Place the PVDF membrane on plastic wrap, mix ECL luminescent solution A and solution B at a ratio of 1:40, and evenly drop the mixture onto the membrane. React in the dark for 5 minutes. Drain excess reaction solution, place the membrane in a dark box, cover it with plastic wrap (to avoid air bubbles), place the X-ray film on it, close the dark box, and expose for 1-2 minutes. Remove the X-ray film and immerse it in the developing solution for 1 minute, rinse with water, and fix it for 2 minutes. After drying, analyze the protein bands.

[0094] 5. Appraisal Results

[0095] 1) q-PCR detection: Results are as follows Figure 5 As shown in the q-PCR statistical graph, lentivirus-mediated PLIN2 overexpression achieved significant and specific upregulation of transcriptional levels in HepaRG cells, indicating that the lentiviral vector overexpressing PLIN2 was successfully constructed.

[0096] 2) Western Blot validation: Results are as follows Figure 6 As shown, the expression level of PLIN2 protein in the overexpression vector was significantly higher than that in the control group (CTRL) and the GFP control group. The GFP-PLIN2 fusion protein was correctly expressed, and the high expression of PLIN2 protein was successfully detected in the experiment, indicating that the lentiviral vector overexpressing PLIN2 was successfully constructed.

[0097] Effect verification

[0098] The cell model prepared in Example 1 for high-throughput drug screening in a fatty liver model was validated using lipid droplet visualization and high-throughput screening, as detailed below:

[0099] 1. Verification of lipid droplet visualization function

[0100] After transfection, the HepaRG cell line was stably passaged and cultured at 37°C and 5% CO2. After 48 hours, lipid droplets were observed under an inverted fluorescence microscope. Since EGFP is the reporter gene, we observed that the lipid droplets emitted green fluorescence and were evenly distributed in the cytoplasm as droplets.

[0101] See results Figure 7This demonstrates that the EGFP reporter gene has been successfully and specifically expressed in cells and precisely targets and labels lipid droplets. It also verifies that the constructed cell model exhibits the typical pathological phenotype of lipid droplet accumulation in fatty liver, with good population uniformity and stability. Furthermore, it shows that the model can quickly reflect changes in lipid droplet content through quantitative analysis of fluorescence signals, meeting the core requirements of high-throughput screening for "visualization, quantification, and high efficiency." This directly proves that the model has been successfully constructed and has practical value, and can be used for high-throughput screening and activity evaluation of lipid-lowering drugs or lipid metabolism modulators related to fatty liver.

[0102] 2. Applicability Validation of High-Throughput Screening

[0103] To demonstrate the applicability of high-throughput drug screening and to identify potential drugs for alleviating NAFLD, resmetirom, a drug already in clinical trials, was used as a positive control, and engletin, a flavonoid compound, was used as the investigational drug. The concentration of both drugs was set at 100 μM. No other treatment was given, and no control drug was added as the control group (CTRL). The cells obtained in Example 1 were seeded in 48-well plates, and after adding the drugs, they were cultured at 37°C and 5% CO2 for 48-72 hours. The morphology and number of lipid droplets were observed using an inverted fluorescence microscope.

[0104] The results are as follows Figure 8 and Figure 9 As shown, both resmetirom and engletin reduced lipid droplets to some extent compared with the model group. The results indicate that this model can serve as a fatty liver model, and that the selected engletin (huangyigan) reduced lipid droplet formation at a concentration of 100 μM.

[0105] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0106] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A cell model for high-throughput drug screening in a fatty liver model, characterized in that, The cell model used the HepaRG cell line as the host and was transfected with a lentiviral vector that overexpressed the PLIN2 gene. The NCBI accession number for the PLIN2 gene is NM_001122.4; the lentiviral vector contains a reporter gene.

2. The cell model as described in claim 1, characterized in that, The reporter gene is the EGFP gene.

3. The method for constructing a cell model for high-throughput drug screening in a fatty liver model as described in claim 1 or 2, characterized in that, Includes the following steps: The PLIN2 gene described in NCBI accession number NM_001122.4 was inserted into a lentiviral vector to obtain a lentiviral vector plasmid overexpressing the PLIN2 gene. The PLIN2 gene overexpression lentiviral vector, packaging helper plasmid, and envelope plasmid were transfected into packaging cells to obtain lentiviral particles. The HepaRG cell line was infected with the lentiviral particles described above, and a cell model for high-throughput drug screening in a fatty liver model was obtained by culturing the cells.

4. The construction method as described in claim 3, characterized in that, The lentiviral vector plasmid contains a reporter gene.

5. The construction method as described in claim 3, characterized in that, The promoter used in the construction of the recombinant lentiviral vector is the Ubc promoter.

6. The construction method as described in claim 3, characterized in that, When the lentiviral particles are used to infect the HepaRG cell line, the MOI of the lentiviral particles is 1~100.

7. The construction method as described in claim 3, characterized in that, After infecting the HepaRG cell line with the lentiviral particles, incubate for 16-24 hours.

8. The construction method as described in claim 3, characterized in that, After infecting the HepaRG cell line with the lentiviral particles, the cell resistance screening reagent is added to finally obtain a positive cell line, which is the cell model used for high-throughput drug screening in fatty liver model.

9. The construction method as described in claim 8, characterized in that, The cell resistance screening reagent is HiTransGP.

10. The use of the cell model for high-throughput drug screening in a fatty liver model as described in claim 1 in evaluating or developing the efficacy of drugs for fatty liver.

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