Hepatocyte injury model as well as construction method and application thereof

Abstract

The invention relates to the technical field of cell models, in particular to a hepatocyte injury model as well as a construction method and application thereof. The invention provides a model for over-expressing a phosphoethanolamine phosphatase gene ETNPPL to damage liver cell functions, after the ETNPPL is over-expressed in liver cells, reduced glutathione is reduced, and active oxygen, glutamic oxalacetic transaminase and glutamic-pyruvic transaminase are increased; mitochondria vacuolation and structure damage are caused, the number of mitochondria is reduced, and the cell proliferation ability is reduced; the protein expression quantity of Akt phosphorylation sites p-Akt-Thr308 and p-Akt-Ser473 of a signal path PI3K / AKT is reduced, and the PI3K / AKT can be used as a liver cell metabolic function impaired model, is used for analyzing a liver cell injury molecular mechanism and is used as a material for evaluating targeted therapy measures.

Description

Technical Field

[0001] The present invention relates to the technical field of cell models, and in particular to a liver cell injury model and a construction method and application thereof. Background Art

[0002] As the largest solid organ in the body, the liver has multiple functions such as detoxification, digestion, immunity and metabolism. Dysfunction directly leads to the occurrence of many diseases. Metabolic disorder-related fatty liver disease caused by lipid metabolism disorders is becoming increasingly serious. Metabolic disorder-related fatty liver disease significantly increases the incidence and mortality of type 2 diabetes and cardiovascular diseases, but there are no approved drugs for its treatment so far. Metabolic disorder-related fatty liver disease develops from simple hepatic fatty degeneration to the more harmful metabolic disorder-related fatty hepatitis, liver fibrosis and cirrhosis. It is the result of "multiple hits" such as genetic susceptibility, insulin resistance, oxidative stress and intestinal flora disorders. Among them, liver cell damage caused by lipid metabolism disorders is a key link in the cascade reaction of the pathological process.

[0003] The phosphoethanolamine phosphatase ETNPPL gene, also known as alanine-glyoxylate aminotransferase 2 analog 1AGXT2L1, encodes the phosphoethanolamine phosphatase that can irreversibly decompose phosphoethanolamine PEtN into inorganic phosphate, ammonia, and acetaldehyde, and ETNPPL is highly specific for the hydrolysis of PEtN. PEtN is a precursor for the synthesis of phosphatidylethanolamine in the CDP-ethanolamine (Kennedy) pathway. In addition, PEtN is also considered a potential inhibitor of mitochondrial respiration. Acetaldehyde is mainly metabolized in the liver and is cytotoxic, which can lead to hepatic steatosis, inflammation, oxidative stress, and other injuries.

[0004] ETNPPL is abundantly expressed in the brain and liver, and its abnormal expression is associated with nervous system and liver diseases. A recent study showed that the expression of ETNPPL increased dramatically during early mouse brain development, and the loss of ETNPPL altered the phospholipid profile of the mouse brain, indicating that ETNPPL can regulate brain lipid metabolism. In IDH1-mutated gliomas, the expression level of ETNPPL was negatively correlated with the grade of gliomas, and overexpression of ETNPPL reduced the growth of glioma stem cells, suggesting that ETNPPL could inhibit the occurrence of gliomas. ETNPPL was significantly upregulated in the brains of patients with schizophrenia and mania, which may be a compensatory response to neurochemical imbalance. In liver cancer tissues and cells, low expression of ETNPPL was associated with poor prognosis, while overexpression of ETNPPL inhibited the migration and invasion of hepatocellular carcinoma (HCC), suggesting that ETNPPL could be a therapeutic target for HCC. ETNPPL plays an important role in lipid metabolism in HCC tissues. Silencing the expression of ETNPPL with siRNA can promote lipogenesis in 97H and LM3 liver cancer cells. In insulin-resistant HepG2 cells, ETNPPL expression is upregulated, and silencing ETNPPL can improve the insulin resistance of HepG2 cells. In addition, overexpression of ETNPPL can induce insulin resistance, reactive oxygen species (ROS) generation, and inactivation of the PI3K / AKT pathway in HepG2 cells, thereby causing insulin resistance in HepG2 cells. Other studies have shown that overexpression of ETNPPL can also promote the activation of arginase (ARG2), revealing that ETNPPL can inhibit autophagy by regulating the ARG2-ROS signaling axis in HepG2 cells.

[0005] Currently, there are few reports on constructing cell models of hepatocellular injury through gene editing. The development of hepatocellular injury models is of great significance for analyzing the molecular mechanisms of hepatocellular injury and evaluating targeted therapeutic measures. Summary of the invention

[0006] The invention provides a liver cell injury model and a construction method and application thereof.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides a method for constructing a hepatocyte injury model, the method comprising: performing gene editing on hepatocytes so as to increase the content and / or activity of ETNPPL protein in the hepatocytes.

[0009] Although it is currently known that multiple signaling pathways and genes are involved in liver cell metabolism and function, there are still few research reports on obtaining liver cell injury models by simple gene editing. The present invention unexpectedly discovered during the research and development process that increasing the content and / or activity of ETNPPL protein in liver cells can lead to liver cell function damage, and at the same time, physiological and biochemical indicators such as reduced glutathione (GSH), reactive oxygen species (ROS), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), mitochondrial structure and quantity, cell proliferation ability, and liver cell proliferation and survival, energy metabolism, glucose uptake and insulin signaling key signaling pathways all show abnormalities, which are particularly suitable for the construction of liver cell injury models. Compared with the previous single gene editing that only leads to abnormalities in individual physiological and biochemical indicators, cell structure, etc., ETNPPL single gene editing can achieve changes in multiple key physiological and biochemical indicators and abnormalities in cell structure and signal pathways, and can achieve efficient construction of liver cell injury models.

[0010] In the present invention, the increase in the content of the ETNPPL protein is achieved by the following 1) and / or 2): 1) Increase the copy number of ETNPPL gene; 2) Enhance the activity of transcriptional and / or translational regulatory elements of the ETNPPL gene.

[0011] In the present invention, ETNPPL is phosphoethanolamine phosphatase, and the amino acid sequence of the protein is shown in SEQ ID NO. 1. The nucleotide sequence of the ETNPPL gene is shown in SEQ ID NO. 2.

[0012] In some embodiments of the present invention, the increased content of the ETNPPL protein is achieved by increasing the copy number of the ETNPPL gene.

[0013] In some specific embodiments of the present invention, the increased content of the ETNPPL protein is achieved by inserting an expression cassette of the ETNPPL gene into the genome of the hepatocytes.

[0014] Preferably, the expression cassette of the ETNPPL gene comprises a KOZAK sequence and the ETNPPL gene. The KOZAK sequence is gccacc.

[0015] Preferably, the insertion of the expression cassette of the ETNPPL gene into the genome of hepatocytes can be achieved using an integrative plasmid (eg, a PiggyBac overexpression vector and a 4PB helper vector).

[0016] In the present invention, the hepatocytes are normal human hepatocytes.

[0017] Preferably, the hepatocytes are L02 hepatocytes.

[0018] L02 cells are a type of human hepatocyte derived from the liver tissue of a healthy human body. They have a high proliferation capacity and can continue to grow and reproduce in a culture medium, maintaining multiple functions of hepatocytes such as protein synthesis and decomposition of toxic substances. The present invention constructs L02 cells stably transfected with the ETNPPL gene, in which the expression level of the ETNPPL gene is significantly increased; experimental verification has found that overexpression of the ETNPPL gene in L02 cells leads to impaired metabolic function of hepatocytes, and can be used to construct a hepatocyte injury model.

[0019] In a second aspect, the present invention provides a hepatocyte injury model constructed by the method described above.

[0020] Preferably, the hepatocyte injury model is L02 hepatocytes overexpressing the ETNPPL gene.

[0021] In a third aspect, the present invention provides use of the above-mentioned hepatocyte injury model in screening drugs for preventing and / or treating hepatocyte injury.

[0022] In a fourth aspect, the present invention provides application of the above-mentioned hepatocyte injury model in analyzing the molecular mechanism of hepatocyte injury.

[0023] In a fifth aspect, the present invention provides the use of ETNPPL protein or its encoding gene in constructing a hepatocyte injury model.

[0024] In a sixth aspect, the present invention provides the use of ETNPPL protein or its encoding gene or an activator of ETNPPL protein or its encoding gene in the preparation of a product for reducing the antioxidant capacity of hepatocytes, reducing the number of hepatocyte mitochondria, destroying the mitochondrial structure of hepatocytes and / or inhibiting the activation of the hepatocyte signaling pathway PI3K / AKT.

[0025] In the above application, the activator of ETNPPL protein or its encoding gene is a substance that can increase the expression and / or activity of ETNPPL protein or its encoding gene, including but not limited to compounds, compositions, proteins or nucleic acids.

[0026] The beneficial effects of the present invention include at least: the present invention provides a model of liver cell function damage by overexpression of phosphoethanolamine phosphatase gene ETNPPL. After ETNPPL is overexpressed in liver cells, reduced glutathione (GSH) decreases, reactive oxygen species (ROS), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increase; mitochondrial vacuolization and structural damage, mitochondrial number decreases, cell proliferation capacity decreases; Akt phosphorylation site p-Akt-Thr308 and p-Akt-Ser473 protein expression levels of PI3K / AKT, a key signaling pathway for liver cell proliferation and survival, energy metabolism, glucose uptake and insulin signaling, are reduced. The liver cells overexpressing ETNPPL can be used as a model of liver cell metabolic function damage, and can be used for analyzing the molecular mechanism of liver cell damage and as a material for evaluating targeted therapeutic measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 This is an electrophoresis diagram of the PCR amplification product of the CDS region of the phosphoethanolamine phosphatase gene ETNPPL in Example 1 of the present invention, wherein M is a DNA marker, and primers 1, 2, and 3 are the PCR amplification products of the CDS region of ETNPPL.

[0029] Figure 2 This is the sequencing result of the CDS region of the phosphoethanolamine phosphatase gene ETNPPL connected to the PiggyBac overexpression vector in Example 1 of the present invention.

[0030] Figure 3 After the phosphoethanolamine phosphatase gene ETNPPL overexpression vector in Example 1 of the present invention was transfected into L02 cells, the luminescence of the transfected cells was observed under a fluorescence microscope, wherein Light Field represents the light field.

[0031] Figure 4 After the phosphoethanolamine phosphatase gene ETNPPL overexpression vector in Example 1 of the present invention was transfected into L02 cells, qPCR was used to detect the change in the expression level of ETNPPL.

[0032] Figure 5 After the phosphoethanolamine phosphatase gene ETNPPL overexpression vector in Example 1 of the present invention was transfected into L02 cells, the expression change of ETNPPL was detected at the protein level.

[0033] Figure 6 After L02 cells were transfected with the phosphoethanolamine phosphatase gene ETNPPL overexpression vector in Example 2 of the present invention, ELISA was used to detect changes in indicators such as GSH, SOD, MDA, ROS, AST and ALT related to liver cell damage.

[0034] Figure 7 After L02 cells were transfected with the phosphoethanolamine phosphatase gene ETNPPL overexpression vector in Example 2 of the present invention, the characteristics of mitochondrial vacuolization and structural damage were detected by transmission electron microscopy.

[0035] Figure 8 This is the change in the number of mitochondria detected by immunofluorescence after L02 cells were transfected with the phosphoethanolamine phosphatase gene ETNPPL overexpression vector in Example 2 of the present invention.

[0036] Fig. 9 This is the detection of cell proliferation after L02 cells were transfected with the phosphoethanolamine phosphatase gene ETNPPL overexpression vector according to Example 2 of the present invention.

[0037] Fig.10 After L02 cells were transfected with the phosphoethanolamine phosphatase gene ETNPPL overexpression vector in Example 2 of the present invention, Western Blotting was used to detect changes in the expression levels of ETNPPL, PI3K / AKT signaling pathway-related p-Akt-Thr308, p-Akt-Ser473 and Akt protein. DETAILED DESCRIPTION

[0038] The present invention constructs a CDS overexpression vector of the phosphoethanolamine phosphatase gene ETNPPL by downloading the nucleotide sequence of the human phosphoethanolamine phosphatase gene ETNPPL from the NCBI database, integrates the ETNPPL gene into the hepatocyte genome through L02 cell transfection and screening, obtains a hepatocyte cell line stably transfected with ETNPPL, and evaluates hepatocyte damage.

[0039] In the process of evaluating liver cell injury, the present invention found that overexpression of phosphoethanolamine phosphatase gene ETNPPL has an impact on liver cell metabolic function. After culturing liver cells stably transfected with phosphoethanolamine phosphatase gene ETNPPL for 48 hours, the expression efficiency was evaluated; ELISA kits were used to detect indicators such as GSH, SOD, MDA, ROS, AST and ALT related to liver cell injury; transmission electron microscopy and immunofluorescence were used to evaluate the structural integrity and quantity of mitochondria; and Western Blotting was used to detect the protein expression of ETNPPL, p-Akt-Thr308, p-Akt-Ser473 and Akt. The results showed that in hepatocytes expressing the transphosphoethanolamine phosphatase gene ETNPPL, the expression of ETNPPL was significantly upregulated, the antioxidant capacity was reduced, the oxidative damage was increased, the hepatocyte damage was obvious, the number of mitochondria in the cells was reduced, and vacuolation and structural damage were present. The activation of PI3K / AKT, a key signaling pathway that regulates cell proliferation and survival, energy metabolism, glucose uptake and insulin signaling, was inhibited, indicating that hepatocytes overexpressing the ETNPPL gene were functionally impaired and could be used as a cell model for related research or drug screening.

[0040] In a specific embodiment of the present invention, the method for overexpressing the CDS sequence of the phosphoethanolamine phosphatase gene ETNPPL comprises the following steps: first, by PCR amplification, the KOZAK sequence is connected to the upstream of the CDS sequence of the phosphoethanolamine phosphatase gene ETNPPL, and the amplification primers used are ETNPPL-CDS-KOZAK-F and ETNPPL-CDS-KOZAK-R, and their nucleotide sequences are SEQ ID NO.5 and SEQ ID NO.6; secondly, by PCR amplification, the homology arm sequence of the PiggyBac vector is connected to the CDS sequence of the phosphoethanolamine phosphatase gene ETNPPL carrying the KOZAK sequence, and the amplification primers used are ETNPPL-CDS-KOZAK-TYB-F and ETNPPL-CDS-KOZAK-TYB-R, and their nucleotide sequences are SEQ ID NO.7 and SEQID NO.8; finally, the linearized vector is connected to the ETNPPL gene carrying the KOZAK sequence and the homology arm of the PiggyBac vector, and then transfected into the hepatocyte L02.

[0041] In a specific embodiment of the present invention, an ELISA kit is used to evaluate indicators of liver cell damage, including: decreased GSH and increased ROS, AST and ALT.

[0042] In a specific embodiment of the present invention, transmission electron microscopy and immunofluorescence are used to evaluate indicators of cell damage, including mitochondrial vacuolization and structural damage, reduced mitochondrial number, and decreased cell proliferation ability.

[0043] In a specific embodiment of the present invention, Western Blotting is used to evaluate indicators of inhibition of activation of the key signaling pathway PI3K / AKT that regulates cell proliferation and survival, energy metabolism, glucose uptake and insulin signaling, including decreased expression of p-Akt-Thr308 and p-Akt-Ser473 proteins.

[0044] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Example 1: Construction of hepatocytes stably transfected with ETNPPL gene

[0046] (1) Expansion of PiggyBac empty vector and auxiliary vector 4PB

[0047] Stable transformation of the ETNPPL gene involves two vectors, the PiggyBac overexpression vector and the 4PB auxiliary vector, and the restriction sites of the vectors are KpnI and PmeI, respectively.

[0048] The PiggyBac and auxiliary vector 4PB without the target gene sequence were transferred into DH5α competent cells for propagation. The experimental process is as follows: 1) Add PiggyBac, 4PB vector and DH5α competent cells in the corresponding proportions. The transformation system is: 50μL DH5α competent cells, 2μL plasmid (concentration is 1000ng / μL). Competent cells are easy to thaw. Take them out at the end and add plasmid when they are just thawed. Blow gently to mix well and avoid violent shaking.

[0049] 2) Place the above reaction system on ice for 30 min; place in a 42°C water bath for 30 s; and immediately place on ice for 2 min.

[0050] 3) Add 300 μL of liquid LB medium without antibiotics to the system and shake at 37°C and 200 rpm for 60 min.

[0051] 4) Take about 200 μL of bacterial solution and spread it evenly on the plate. Incubate the plate upright at 37°C for 1 hour, then invert it for 12-16 hours.

[0052] 5) The next day, pick a single clone and add it to 300 μL of LB medium containing ampicillin. Vortex and culture until the liquid medium becomes turbid. Take about 200 μL of the bacterial solution for sequencing.

[0053] 6) If the sequencing result is correct, take about 100 μL of bacterial solution and add it to 30 mL of culture medium containing ampicillin antibiotics and culture it for 12-16 h (37°C, 200 rpm shaking culture) for subsequent plasmid extraction.

[0054] (2) Vector linearization

[0055] The piggyBac vector was linearized using KpnI and PmeI restriction endonucleases. The linearization system is shown in Table 1.

[0056] Table 1 PiggyBac vector linearization system

[0057] (3) Amplification of the CDS region of the ETNPPL gene

[0058] According to the ETNPPL sequence, the CSD region of the ETNPPL gene was amplified using KOD Fx Neo high-fidelity enzyme. The total length of the amplification was 1500 bp. Its nucleotide sequence was SEQ ID NO.2. The primer used for amplification was ETNPPL-CDS. The PCR detection results of the amplified fragment were as follows: Figure 1 Using the amplified product as a template, KOZAK sequence and vector homology arms were added to both ends of the primers, and the primers were ETNPPL-CDS-KOZAK and ETNPPL-CDS-KOZAK-TYB, as shown in Table 2, the amplification system was shown in Table 3, and the amplification program was shown in Table 4.

[0059] Table 2 List of primers for amplifying the CDS region of the ETNPPL gene

[0060] Table 3 CDS region amplification system

[0061] Table 4 Amplification procedures of CDS region amplification system

[0062] (4) Linearized vector and target fragment ligation

[0063] According to the principle of homologous recombination, the linearized vector recovered from the gel was connected to the target fragment. The connection system is shown in Table 5. The connection conditions are 50℃, 15min or 42℃, 30min. The connection product was placed in a 4℃ refrigerator for subsequent transformation experiments. The bacterial solution was sent to a sequencing company for sequencing. The sequencing results are shown in Figure 2As shown, the correctly sequenced strains were shaken, plasmids were extracted and preserved for subsequent cell transfection experiments.

[0064] Table 5 Linearized vector and target fragment ligation system

[0065] (5) Overexpression of ETNPPL gene in L02 cells

[0066] L02 cells were revived in 10 cm dishes and used for transfection when the cell density reached about 80%. The medium was changed 8 h after transfection and cultured for 48 h. RPMI medium containing puromycin at a final concentration of 2 μg / mL was used for drug screening to increase the proportion of GFP-positive cells.

[0067] 1) After the revived L02 cells grow to about 80%, they are digested with trypsin containing EDTA, and the cells are collected and counted after centrifugation; 2) According to 10 6 Cells / well (six-well plate) were electroporated. The electroporation kit used was the Lonza nuclear transfection kit. The ratio of Solution to Supplement was 100:2 (volume ratio), and they were fully mixed. 3) Add the ETNPPL overexpression vector and the auxiliary vector 4PB to 100 μL of nuclear transfection reagent and mix thoroughly. The calculated ratio of the two vectors is: if the transferred ETNPPL overexpression vector is 5 μg, the length of the ETNPPL overexpression vector is 10609 bp, and the length of the auxiliary vector 4PB is 8531 bp, then the mass of the transferred auxiliary vector 4PB is: 8531 bp × 5000 ng / 10609 bp, and then calculate the transferred volume according to the concentration of the auxiliary vector; 4) Add 100 µL of nuclear transfection reagent to the centrifuge tube containing L02 cells after centrifugation, and gently blow and resuspend the cells with a pipette; 5) Use a pipette to gently add the mixed cell suspension to the electroporation cup. Try not to create bubbles during the process to avoid affecting the electroporation efficiency. 6) Select T-030 as the electroporation program. After transfection, add complete culture medium and gently disperse the cells. Culture at 37℃ for 6-8 hours, then change the medium and continue to culture for 48 hours for drug screening. Observe the luminescence under a fluorescence microscope. Use DMEM medium containing puromycin (2μg / mL) for drug screening for 12 hours. Observe the luminescence of the transfected cells under a fluorescence microscope. Figure 3 shown.

[0068] 7) When the cell density reaches 80%-90%, collect cell samples for overexpression efficiency detection and subsequent experiments.

[0069] (6) ETNPPL overexpression efficiency detection

[0070] After scraping the L02 cells overexpressing the ETNPPL gene in the 6-well plate with a cell scraper, the RNA and protein of the cells were extracted, and the overexpression efficiency of the ETNPPL gene was detected at the mRNA and protein levels. The primer sequences used are shown in Table 6, the qPCR system is shown in Table 7, and the mRNA expression level of the overexpressed ETNPPL gene is shown in Table 8. Figure 4 As shown in the results, compared with the control group (Ctrl), the expression of ETNPPL in the ETNPPL overexpression group (OA) was significantly upregulated; the protein expression level was detected using ETNPPL antibody (Cat# HPA044546, Sigma-Aldrich, 1:1500), and the results are shown in Figure 5 As shown, it showed that ETNPPL expression was significantly upregulated.

[0071] Table 6 qPCR primer list

[0072] Table 7 Fluorescence quantitative PCR

[0073] Example 2: Evaluation of hepatocyte damage by overexpressing ETNPPL gene

[0074] (1) Determination of liver injury-related indicators

[0075] 1) To detect the antioxidant capacity of hepatocytes overexpressing the ETNPPL gene, antibodies were used to detect the levels of intracellular reduced glutathione (GSH antibody, catalog number BC1175-100T / 96S, Beijing Solebow Technology Co., Ltd.) and superoxide dismutase (SOD antibody, catalog number BC5165-100T / 48S, Beijing Solebow Technology Co., Ltd.). The results showed that the GSH content was reduced, approaching the statistically significant difference level, indicating that the antioxidant capacity of hepatocytes overexpressing the ETNPPL gene was reduced.

[0076] 2) To detect the oxidative stress state of hepatocytes overexpressing the ETNPPL gene, the Cellular ROS AssayKit (Deep Red) detection kit was used to detect ROS in the cells (Cat. No.: Ab186029); the Cell Malondialdehyde (MDA) assay kit was used to detect MDA in the cells (Cat. No.: A003-4-1, Nanjing Jiancheng Bioengineering Institute). The results showed that the ROS content was significantly increased, indicating that the oxidative stress of hepatocytes overexpressing the ETNPPL gene was significantly increased.

[0077] 3) Aspartate aminotransferase (AST antibody, catalog number C010-2-1, Nanjing Jiancheng Bioengineering Institute) and alanine aminotransferase (ALT antibody, catalog number C009-2-1, Nanjing Jiancheng Bioengineering Institute) are mainly synthesized in hepatocytes in the liver, and their increased content indicates hepatocyte damage. The content of ALT and AST in the cell culture medium of the control (Ctrl) group and the overexpression (OE) group after 24 hours of culture was detected. The results showed that the content of AST and ALT in the cell culture medium of the OE group was significantly increased, indicating that the hepatocytes overexpressing the ETNPPL gene were damaged.

[0078] The above results are Figure 6 shown.

[0079] (2) Overexpression of the ETNPPL gene damages hepatocyte mitochondria and inhibits hepatocyte proliferation

[0080] 1) In order to evaluate the effect of overexpression of ETNPPL gene on mitochondrial damage in hepatocytes, transmission electron microscopy (TEM) was used to observe cells in the Ctrl group and the OE group. The results showed that the mitochondria in the Ctrl group had normal morphology and intact cristae structure. The mitochondria in the OE group showed vacuolization, mitochondrial autophagy, and cristae disappeared ( Figure 7 ), indicating that overexpression of the ETNPPL gene can cause structural damage to mitochondria.

[0081] 2) To evaluate the effect of overexpression of ETNPPL on the number of mitochondria, MitoTracker DeepRed FM dye (catalog number HYD1783) was used to stain mitochondria in living cells, and the fluorescence intensity of cells in the Ctrl and OE groups was counted. 50 μg of MitoTracker Deep Red FM dye was dissolved in 92 μL of DMSO, and a 1 mM dye storage solution was prepared. The solution was divided into equal parts and stored at -80°C. The results showed that the number of mitochondria in the OE group was significantly reduced, indicating that overexpression of ETNPPL gene not only destroyed the mitochondrial structure, but also reduced the number of mitochondria ( Figure 8 ).

[0082] 3) To evaluate the effect of overexpression of ETNPPL gene on cell proliferation, BeyoClick™ EdU-594 Cell Proliferation Assay Kit was used to detect cell proliferation in Ctrl and OE groups (Cat. No.: C0078S, Bio-Tech Co., Ltd.). The results showed that the proportion of EdU positive signals in OE group was significantly reduced, indicating that overexpression of ETNPPL can inhibit L02 cell proliferation ( Fig. 9 ).

[0083] Based on the above research results, it is shown that ETNPPL can cause liver cell damage by destroying mitochondrial structure, reducing the number of mitochondria, and inhibiting cell proliferation pathways.

[0084] (3) Overexpression of the ETNPPL gene inhibits the PI3K / AKT signaling pathway in hepatocytes

[0085] PI3K / AKT is a star pathway that plays an important role in cell proliferation and survival, energy metabolism, glucose uptake and insulin signal response. To evaluate the effect of overexpression of ETNPPL gene on PI3K / AKT signaling pathway, the phosphorylation levels of AKT-T308 (Cat. No.: 07-1398, Sigma-Aldrich) and AKT-S473 (Cat. No.: 05-1003, Sigma-Aldrich) in Ctrl group and OE group were detected at protein level. The expression levels of AKT-T308 and AKT-S473 in OE group were significantly decreased ( Fig.10 ), indicating that overexpression of ETNPPL gene can inhibit the PI3K / AKT signaling pathway.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a hepatocyte injury model, characterized in that: The method comprises: performing gene editing on hepatocytes so as to increase the content and / or activity of ETNPPL protein in the hepatocytes.

2. The method according to claim 1, characterized in that: The increase in the content of the ETNPPL protein is achieved by the following 1) and / or 2): 1) Increase the copy number of ETNPPL gene; 2) Enhance the activity of transcriptional and / or translational regulatory elements of the ETNPPL gene.

3. The method according to claim 1 or 2, characterized in that: The increase in the content of the ETNPPL protein is achieved by inserting an expression cassette of the ETNPPL gene into the genome of the hepatocytes.

4. The method according to claim 3, characterized in that: The expression cassette of the ETNPPL gene comprises a KOZAK sequence and the ETNPPL gene.

5. The method according to claim 4, characterized in that The hepatocytes are normal human hepatocytes.

6. The method according to claim 5, characterized in that The hepatocytes are L02 hepatocytes.

7. The hepatocyte injury model constructed by the method according to any one of claims 1 to 6.

8. Use of the hepatocyte injury model according to claim 7 in screening drugs for preventing and / or treating hepatocyte injury.

9. Use of the hepatocyte injury model according to claim 7 in analyzing the molecular mechanism of hepatocyte injury.

10. Application of ETNPPL protein or its encoding gene in the construction of hepatocyte injury model.

Images