Method for constructing fat-rich hepatocellular carcinoma mouse model based on combination of high-pressure hydrodynamic injection and ketogenic diet and application of fat-rich hepatocellular carcinoma mouse model

Abstract

The invention discloses a method for constructing a fat-rich hepatocellular carcinoma mouse model based on combination of high-pressure hydrodynamic injection and ketogenic diet and application, and belongs to the technical field of biomedicine. Expression plasmids containing Nras oncogene, Myc oncogene and sleeping beauty transposase are introduced into the body of a mouse in a high-pressure hydrodynamic tail vein injection mode, the ratio of the volume of a plasmid solution to the body weight of the mouse is 0.1 mL / g, injection is conducted within 5-8 seconds, and the plasmids enter hepatocytes; after injection is completed, ketogenic diet is given to the mice every 7 days, the mice are continuously fed for 6-8 weeks, and modeling is conducted; and verifying the characteristics of the fat-rich hepatocellular carcinoma. The method disclosed by the invention is short in construction period, simple and convenient to operate, low in death rate, high in modeling rate and clear in tumor specific lipid droplet deposition. The model truly simulates the pathological characteristics and metabolic state of SH-HCC, and can be used for SH-HCC generation mechanism research, targeted drug screening and immunotherapy evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method and application for constructing a lipid-rich hepatocellular carcinoma mouse model based on high-pressure hydrodynamic injection and a ketogenic diet. Background Technology

[0002] Primary liver cancer is a common malignant tumor and a leading cause of cancer death, with hepatocellular carcinoma (HCC) accounting for 75%–85% of cases. Despite advancements in traditional therapies such as surgery, interventional procedures, and radiotherapy, as well as innovative therapies like targeted therapy and immunotherapy, the overall survival rate for liver cancer patients remains unsatisfactory. This is primarily attributed to the high heterogeneity of liver cancer, with significant differences in response to the same treatment modality among different patients. Given this high heterogeneity, the traditional research model that does not differentiate between liver cancer subtypes is showing its limitations, and its conclusions are often difficult to generalize and apply. Therefore, focusing on research into liver cancer subtypes, deeply elucidating the unique mechanisms of occurrence and development of different subtypes, and identifying subtype-specific treatments are essential for achieving precision medicine for liver cancer patients.

[0003] Steatohepatitic hepatocellular carcinoma (SH-HCC, also known as fatty liver hepatocellular carcinoma) is a special type of hepatocellular carcinoma characterized histologically by the accumulation of numerous lipid droplets in the liver cancer tissue. First proposed in 2010, studies have found that SH-HCC accounts for approximately 5-20% of hepatocellular carcinomas, making it the most prevalent subtype among special types of hepatocellular carcinoma, and its proportion is increasing annually. Despite this, SH-HCC has not yet received sufficient attention; fewer than 50 related articles are indexed on PubMed, and most are case reports or clinically relevant studies. Our previous research found that SH-HCC is insensitive to targeted therapy and immunotherapy. Current research provides little understanding of the pathogenesis and development of SH-HCC, and lacks subtype-specific treatments. The lack of suitable SH-HCC models is a major limiting factor for basic research in this area.

[0004] Mouse liver cancer models, as a core tool in liver cancer research, have made significant progress in recent years. These models mainly include genetically engineered mouse models (GEMMs), chemically induced models, transplantation models, and humanized mouse models. GEMMs, through precise regulation of gene mutations (such as Cre-Lox recombination and CRISPR-Cas9 technology), can simulate the genetic heterogeneity and multi-step carcinogenesis process of human liver cancer, making them particularly suitable for studying the relationship between the immune microenvironment and tumor progression. However, their construction cycle is long, the technology is complex, and they are often limited to single gene alterations. Chemically induced models (such as DEN and CCl4) are simple to operate and can reproduce the cirrhosis-liver cancer transformation process, but tumor formation time is long, the degree of lesion varies, and it is difficult to control the immune response. Transplantation models (including homogeneous or xenograft transplantation) are rapid and efficient, suitable for drug screening, but ectopic transplantation lacks the authenticity of the liver microenvironment. While humanized models can partially reconstruct the immune system to evaluate immunotherapy, they face challenges due to high technical difficulty and cost.

[0005] SH-HCC is a significant subtype of liver cancer, but currently there is no animal model to mimic its pathogenesis. To meet clinical and research needs, a novel method for constructing a mouse model of SH-HCC is urgently required. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to address the shortcomings of existing technologies by providing a method and application for constructing a lipid-rich hepatocellular carcinoma mouse model based on a combination of high-pressure hydrodynamic injection and a ketogenic diet.

[0007] This invention provides a mouse modeling method combining high-pressure hydrodynamic injection gene transfection and a ketogenic diet for the efficient and stable construction of an SH-HCC model. The invention involves injecting the Nras / Myc oncogene and the Sleeping Beauty transposon into immunocompetent mice via high-pressure hydrodynamic tail vein injection. One week after plasmid injection, a ketogenic diet is then followed to construct an SH-HCC mouse hepatocellular carcinoma model (Nras / Myc-KD). This model provides a powerful tool for studying the mechanisms of lipid-rich hepatocellular carcinoma.

[0008] Technical solution: The objective of this invention is achieved through the following technical solution: This invention provides a method for constructing a lipid-rich hepatocellular carcinoma mouse model based on a combination of high-pressure hydrodynamic injection and a ketogenic diet, comprising the following steps: (1) SPF grade C57BL / 6J mice were acclimatized for 7 days before modeling began; (2) Preparation of plasmid solution: Construct an expression plasmid containing Nras oncogene, Myc oncogene and Sleeping Beauty transposase, dissolve it in physiological saline, and adjust the concentration to be suitable for high-pressure hydrodynamic injection; (3) High-pressure hydrodynamic tail vein injection: Inject the plasmid solution obtained in step (2) into the tail vein of the mouse. The ratio of the volume of the plasmid solution to the mouse body weight is 0.1 mL / g (e.g., for a mouse weighing 20g, the volume of the injected plasmid solution is 2 mL). Inject rapidly within 5-8 seconds to allow the plasmid to enter the hepatocytes. (4) Dietary intervention: After plasmid injection, mice were fed a ketogenic diet for 7 days and fed for 6-8 weeks to establish the model. (5) Verify the characteristics of lipid-rich hepatocellular carcinoma.

[0009] This invention utilizes the Sleeping Beauty (SB) transposon system to load a specific combination of oncogenes and a transposase gene into plasmid DNA. A large volume of plasmid solution is then rapidly injected into immunocompetent mice via high-pressure hydrodynamic tail vein injection (HTVi). The instantaneous high pressure generated by the injection forces the exogenous DNA into hepatocytes efficiently. Within the hepatocytes, the SB transposase stably integrates the carried oncogene fragments into the host genome, driving genetic mutations in the hepatocytes and gradually developing into liver cancer.

[0010] This invention is based on high-pressure hydrodynamic injection combined with dietary induction (ketogenic diet) to simulate human metabolic risk factors to promote the occurrence of liver cancer and accelerate tumor development.

[0011] Preferably, in step (1), the temperature of the SPF grade C57BL / 6J mice is controlled at 22±2℃, the humidity is 55%±10%, the day and night cycle is 12 hours, and the mice have free access to ordinary drinking water and feed.

[0012] Preferably, in step (2), the expression plasmid containing the Nras oncogene is the purified pT / Caggs-NRASV12 plasmid; the expression plasmid containing the Myc oncogene is the purified c-myc-PT3EF1a plasmid; and the expression plasmid containing the Sleeping Beauty transposase is the purified pCMV / SB11 plasmid.

[0013] More preferably, the purified pT / Caggs-NRASV12 plasmid, c-myc-PT3EF1a plasmid and pCMV / SB11 plasmid are mixed at a mass ratio of 19:1:2.

[0014] Preferably, in step (2), the purified pT / Caggs-NRASV12 plasmid, c-myc-PT3EF1a plasmid, and pCMV / SB11 plasmid are dissolved in 0.9% physiological saline, and the volume of physiological saline is 0.1 mL / g relative to the mouse body weight.

[0015] Preferably, in step (4), the feeding method is free access to ketogenic feed.

[0016] Preferably, in step (5), the lipid-rich hepatocellular carcinoma characteristics of the model are verified by gross observation of the liver, H&E staining, lipid staining, detection of triglyceride and ketone body content, and transcriptomic and lipidomics analysis.

[0017] Preferably, the characteristics of the lipid-rich hepatocellular carcinoma include abundant lipid droplet deposition in the tumor tissue, absence of fatty degeneration in the adjacent tissue, abnormal lipid metabolism, and tolerance to immunotherapy.

[0018] This invention establishes an SH-HCC model in immunocompetent mice by combining high-pressure hydrodynamic injection gene transfection with a ketogenic diet. This model is suitable for experimental research and drug screening, and it is also characterized by its simple and easy production process, low mouse mortality rate due to operation, high success rate, and ease of promotion.

[0019] The present invention also provides the application of the mouse model established by the above method in animal experiments for studying and developing the occurrence and development mechanism of lipid-rich hepatocellular carcinoma, as well as in targeted drug screening and immunotherapy evaluation. Beneficial effects

[0020] This invention introduces a Sleeping Beauty transposon system plasmid carrying the Nras and Myc oncogenes into immune-healthy mice via high-pressure hydrodynamic tail vein injection, inducing hepatocytes to undergo tumor transformation. One week after injection, the mice are given a ketogenic diet to induce the specific accumulation of lipid droplets in the tumor tissue, thereby successfully constructing a lipid-rich hepatocellular carcinoma mouse model that conforms to the histological characteristics of SH-HCC.

[0021] The model construction method of this invention has advantages such as short construction cycle, simple operation, low mortality rate, high model success rate, and clear tumor-specific lipid droplet deposition. The model constructed by this invention not only realistically simulates the pathological characteristics and metabolic state of SH-HCC, but can also be used for the study of the pathogenesis of SH-HCC, targeted drug screening, and immunotherapy evaluation, and has broad prospects for scientific research and clinical translational applications. Attached Figure Description

[0022] Figure 1 For modeling flowchart ( Figure 1 A) Gross specimen images of ordinary hepatocellular carcinoma (Nras / Myc-ND) and fatty liver carcinoma (Nras / Myc-KD) based on Nras / Myc. Figure 1 B).

[0023] Figure 2 To investigate the effects of a ketogenic diet on the development and progression of hepatocellular carcinoma in Nras / Myc mice, a control group (Nras / Myc-ND) and a model group (Nras / Myc-KD) were selected. Figure 2 A represents the number of tumor nodules on the liver surface in the two groups; Figure 2 B represents the difference in liver weight between the two groups; Figure 2 C represents the weight difference between the two groups; Figure 2 D represents the difference between liver weight and body weight; Figure 2 E represents the difference in peripheral blood ketone body concentration between the two groups.

[0024] Figure 3 Typical photographs of H&E staining results for the control group (Nras / Myc-ND) and the model group (Nras / Myc-KD).

[0025] Figure 4 The results of H&E staining are shown for all model mice.

[0026] Figure 5 The results of triglyceride (TAG) and β-hydroxybutyrate (BHB, ketone bodies) content detection in tumor tissue (T) and adjacent normal tissue (N) of Nras / Myc-ND and Nras / Myc-KD models were presented. Figure 5 A represents the triglyceride (TAG) content test result; Figure 5 B represents the result of the β-hydroxybutyric acid (BHB) content detection.

[0027] Figure 6 This presents the results of a differential analysis of omics characteristics and immune infiltration between conventional and lipid-rich hepatocellular carcinoma models. Figure 6 A represents principal component analysis (PCA) of the transcriptome data. Figure 6 B represents the difference in expression of genes related to fatty acid metabolism; Figure 6 C represents the changes in free fatty acid content in cancer and adjacent tissues of the model group and control group detected by lipidomics analysis; Figure 6 D represents the degree of immune cell infiltration inferred from the transcriptome; Figure 6 E and Figure 6 F represents the results of immunohistochemical staining.

[0028] Figure 7 The results of H&E staining in a MASH-related hepatocellular carcinoma model. Figure 7 A represents MASH-HCC induced by STZ combined with a high-fat diet; Figure 7 B represents the WD-induced MASH-HCC model.

[0029] Figure 8 This analysis presents the differences in metabolic characteristics between conventional and lipid-rich hepatocellular carcinoma tissues and their adjacent tissues. Figure 8 A shows a comparison of the expression levels of enzymes related to fatty acid and ketone body metabolism; Figure 8 B represents the difference in the level of β-hydroxybutyrylation (Kbhb) modification and the expression of its regulatory enzymes (Writer and Eraser).

[0030] Figure 9Model building and immunotherapy flowchart ( Figure 9 A) Magnetic resonance imaging and gross tumor images of the two groups of mice after treatment ( Figure 9 B) Survival analysis curves ( Figure 9 C Figure 9 D). Detailed Implementation

[0031] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0032] Unless otherwise specified, the experimental methods used in the examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0033] The ketogenic diet used in this embodiment of the invention is a product of Dyets, catalog number HF89.5.

[0034] The pT / Caggs NRASV12 plasmid (#20205), c-myc-PT3EF1a plasmid (#92046), and pCMV / SB11 plasmid (#26552) used in the embodiments of this invention were purchased from Addgene.

[0035] Example 1: Construction of a lipid-rich hepatocellular carcinoma mouse model (1) Laboratory animals SPF-grade C57BL / 6J mice, male, 6 weeks old, 18-22g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Before the experiment, the mice were acclimatized for 7 days in an SPF-grade environment with a temperature controlled at 22±2℃ and humidity at 55%±10%, with a 12-hour day-night cycle. They had free access to standard drinking water and feed.

[0036] (2) Model building method according to Figure 1 The modeling process shown in A was followed. The Nras / Myc oncogene (N-Ras 19 μg, c-Myc 1 μg) and Sleeping Beauty transposon (SB 2 μg) were injected into 6-week-old C57BL / 6J mice via high-pressure hydrodynamic tail vein injection. One week after plasmid injection, the mice were fed a ketogenic diet for 8 consecutive weeks to establish the SH-HCC mouse liver cancer model (Nras / Myc-KD).

[0037] 1. Preparation of plasmid solution Large-scale plasmid purification was performed using the FastPure Enhanced EndoFree Plasmid Maxi (Plus) Kit (catalog number: DC212-01, Vazyme) (following the product instructions). 19 μg of pT / Caggs NRASV12 plasmid (#20205), 1 μg of cmyc PT3EF1a plasmid (#92046), and 2 μg of pCMV / SB11 plasmid were dissolved in 2 mL of sterile water. The plasmid solution was prepared in 0.9% physiological saline for later use.

[0038] 2. High-pressure hydrodynamic tail vein injection The mice were secured with tape and kept warm with an electric blanket (37°C). After exposing the tail vein, 2 mL of plasmid solution was rapidly injected over 5-8 seconds using a 27G injection needle. The DNA plasmid was then forced into the liver cells by instantaneous high pressure.

[0039] Closely monitor the mice's condition after injection and maintain a constant temperature of 37°C for recovery. During the injection process, ensure skillful operation to avoid stress-induced death. After injection, return the mice to their cages to recover and observe them continuously for 24 hours.

[0040] 3. Dietary intervention One week after plasmid injection, the mice were divided into two groups: Normal diet group (Nras / Myc-ND): Continue to be given normal experimental feed.

[0041] The standard experimental feed was selected from Jiangsu Xietong Biotechnology XTI01WC-009. Its ingredients include Northeast corn, wheat, imported fish meal, etc., with protein providing 21.5% energy, fat providing 11.1% energy, and carbohydrates providing 67.4% energy.

[0042] Ketogenic diet group (Nras / Myc-KD): High-fat, low-carb ketogenic diet (product number HF89.5) provided by Dyets. Both groups of mice were fed freely for 8 weeks to establish a common hepatocellular carcinoma model (control group) and a high-fat ketogenic diet (control group). Liver cancer model (model group).

[0043] Mice were fed freely, and their weight was recorded every three days. Blood ketone and blood glucose levels were monitored regularly to assess ketosis. A constant temperature environment was maintained throughout the experiment, and the mice's mental state and metabolic adaptation were closely observed.

[0044] The concentration of ketone bodies in serum was detected using a commercial ketone body detection kit (catalog number: MAK134, Sigma). The experimental method was performed in accordance with the kit instructions.

[0045] After anesthetizing the mice, the livers were dissected and obtained; the number of nodules on the surface of the livers was counted by the naked eye.

[0046] Figure 2 To investigate the effects of a ketogenic diet on the development and progression of hepatocellular carcinoma in Nras / Myc mice, a control group (Nras / Myc-ND) and a model group (Nras / Myc-KD) were selected. Figure 2 A represents the number of tumor nodules on the liver surface in the two groups; Figure 2 B represents the difference in liver weight between the two groups; Figure 2 C represents the weight difference between the two groups; Figure 2 D represents the difference between liver weight and body weight; Figure 2 E represents the difference in peripheral blood ketone body concentration between the two groups.

[0047] from Figure 2 As can be seen, the model group had more tumors, greater liver weight, and a higher liver weight / body weight ratio; at the same time, the concentration of ketone bodies in peripheral blood was also higher; some mice in the model group lost weight, but there was no significant difference overall.

[0048] Example 2: Validation of a lipid-rich hepatocellular carcinoma mouse model This invention validates the characteristics of lipid-rich hepatocellular carcinoma in experimental animals constructed in Example 1.

[0049] Mice were sacrificed at week 8 of modeling, and liver tissue was collected for histological and molecular analysis.

[0050] (1) H&E staining After dewaxing with xylene and rehydration with a series of alcohols, the nuclei were first stained with hematoxylin to make them appear blue-purple, and then fixed and stained with tap water. Subsequently, the cytoplasm and extracellular matrix were stained with eosin to make them appear pink to red. Finally, the sections were dehydrated with alcohol, cleared with xylene, and mounted for observation under a microscope.

[0051] The H&E staining results of the control group (Nras / Myc-ND) and the model group (Nras / Myc-KD) are shown in the figure. Figure 3 .

[0052] like Figure 3 As shown, H&E staining results revealed that the ketogenic diet significantly promoted lipid droplet accumulation in the Nras / Myc-induced liver cancer model. In the normal diet group (Nras / Myc-ND), no significant lipid droplet deposition was observed in either tumor tissue (T) or adjacent normal tissue (N); while in the ketogenic diet group (Nras / Myc-KD), a large number of lipid droplet aggregations were observed in the tumor tissue, but no fatty degeneration was observed in the adjacent normal tissue.

[0053] Figure 4 The H&E staining results for all model mice are shown. Further H&E staining results for more mice further verify the successful construction of the lipid-rich hepatocellular carcinoma (SH-HCC) mouse model, and also demonstrate the consistency and uniformity of the model constructed in this invention.

[0054] from Figure 4 As can be seen, in the Nras / Myc-ND group, there were no obvious lipid droplets in the tumor and adjacent tissues; in contrast, in the Nras / Myc-KD group, lipid droplets accumulated significantly in the tumor tissue, while there was no fatty degeneration in the adjacent area.

[0055] (2) Detection of triglycerides (TAG) and ketone bodies (BHB) Triglycerides (catalog number: S0219S, Beyotime) and ketone bodies (catalog number: MAK134, Sigma) were quantitatively detected using a commercial kit, and the experimental methods were performed according to the kit instructions.

[0056] Figure 5 The results show the levels of triglycerides (TAG) and β-hydroxybutyrate (BHB, ketone bodies) in tumor tissue (T) and adjacent normal tissue (N) of the Nras / Myc-ND and Nras / Myc-KD models. NDN represents adjacent normal tissue in the Nras / Myc-ND model; NDT represents tumor tissue in the Nras / Myc-ND model; KDN represents adjacent normal tissue in the Nras / Myc-KD model; and KDT represents tumor tissue in the Nras / Myc-KD model.

[0057] The results showed that the levels of TAG and BHB in tumor tissues of the Nras / Myc-KD group were significantly higher than those of the ND group, suggesting lipid metabolism reprogramming and active ketone body production, and that the group exhibited characteristics of lipid-rich and high ketone body metabolism.

[0058] (3) Transcriptomics and lipidomics analysis Transcriptomic and lipidomic analyses were performed on cancerous and adjacent normal tissues from Nras / Myc-ND (control group) and Nras / Myc-KD (model group) mice, respectively. All experiments were conducted by Shanghai Baiqu Biomedical Technology Co., Ltd.

[0059] Figure 6 Significant differences in omics characteristics and immune infiltration between the common and lipid-rich hepatocellular carcinoma models.

[0060] in, Figure 6 A represents principal component analysis (PCA) of the transcriptome data, which showed significant differences between the model group and the control group at the transcriptome level. Figure 6 B represents the difference in expression of genes related to fatty acid metabolism; Figure 6 C represents the changes in free fatty acid content in cancer and adjacent tissues of the model group and control group detected by lipidomics analysis; Figure 6 D represents the degree of immune cell infiltration inferred from the transcriptome, and the results showed that immune infiltration was significantly reduced in lipid-rich hepatocellular carcinoma. Figure 6 E and Figure 6 F represents the immunohistochemical staining results, further confirming that there are fewer immune-infiltrating cells in lipid-rich hepatocellular carcinoma.

[0061] The results showed that fatty acid synthesis, ketone body metabolism, and immune-related pathways were significantly altered in tumor samples from the Nras / Myc-KD group, manifested as enhanced lipid metabolism and reduced immune infiltration.

[0062] Comparative Example 1: Construction of the STZ-induced MASH-HCC model STZ-induced MASH-HCC model is a classic method for modeling liver cancer closely related to metabolic disorders. The specific procedure is as follows: First, 6-week-old mice are selected and injected intraperitoneally with STZ (for newborn mice, the dose is 200 µg / mouse; the dose is calculated based on body weight, such as 100-150 mg / kg). Before STZ injection, mice need to be fasted for 4-6 hours to enhance the sensitivity of pancreatic β cells to STZ. After injection, 10% sucrose water is provided for 24-48 hours to prevent severe initial hypoglycemia. One week later, random blood glucose is monitored by tail tip blood sampling. The standard for successful modeling is usually a blood glucose level consistently above 250 mg / dL. The hyperglycemic mice are then switched to a special high-fat diet (HFD, typically with a fat energy ratio of 60%). After continuous feeding for more than 20 weeks, some mice (with an incidence rate of 60%-80%) will spontaneously develop hepatocellular carcinoma nodules. For details on the construction method, please refer to the following link: https: / / doi.org / 10.1038 / s42255-022-00700-y.

[0063] Comparative Example 2: Western Diet (WD) Induced MASH-HCC Model The Western diet (WD)-induced MASH-HCC model is a modeling method that is closer to unhealthy human eating habits. It mainly simulates the high cholesterol, high fat, and high sugar intake of Western populations through dietary combinations.

[0064] This model uses male C57BL / 6J mice, fed a specially formulated Western diet for an extended period (40 to 60 weeks or even longer) starting at 6-8 weeks of age. The typical composition of this diet is: rich in fat (milk fat, accounting for more than 40% of total calories), supplemented with cholesterol (0.5% to 1.25%) and high-fructose corn syrup or sucrose solution (42 g / L fructose / glucose solution) as drinking water. This diet systematically induces metabolic syndrome features in mice, including obesity, insulin resistance, and hypercholesterolemia. At the hepatic level, long-term metabolic stress initially leads to the accumulation of large amounts of fat (mainly cholesterol esters), which then gradually develops into severe MASH, accompanied by significant hepatocellular damage, inflammatory cell infiltration, and hepatic stellate cell activation. Driven by a cycle of chronic damage and regeneration, mice typically begin to develop liver fibrosis after 30 to 40 weeks of feeding, eventually leading to spontaneous hepatocellular carcinoma (HCC) in some older mice (e.g., after 50 weeks). For details on the construction method, please refer to the following link: https: / / doi.org / 10.1038 / s42255-022-00700-y.

[0065] The distribution of lipid droplets in the MASH-related model was observed using H&E staining. The H&E staining procedure was the same as in Example 2.

[0066] Figure 7 The results of H&E staining in a MASH-related hepatocellular carcinoma model are shown; among them, Figure 7 A represents MASH-HCC induced by STZ combined with a high-fat diet; Figure 7 B represents the WD-induced MASH-HCC model.

[0067] As can be seen from the figure, in the STZ-induced MASH-HCC and WD-induced MASH-HCC models, lipid droplet deposition occurs simultaneously in both the tumor and adjacent normal tissues; while in the Nras / Myc-KD model ( Figure 3 and Figure 4 Lipid droplets specifically aggregate in tumor tissue, while adjacent tissues maintain normal structure, suggesting that the model of this invention can better simulate the specific metabolic characteristics of lipid-rich liver cancer.

[0068] Figure 8 This study demonstrates the differences in metabolic characteristics between conventional and lipid-rich hepatocellular carcinoma tissues and their adjacent tissues. Western blot analysis was performed on the cancerous and adjacent tissues of both groups of mice. Figure 8 A shows a comparison of the expression levels of enzymes related to fatty acid and ketone body metabolism; Figure 8 B shows the differences in the level of β-hydroxybutyrylation (Kbhb) modification and the expression of its regulatory enzymes (Writer and Eraser), suggesting that lipid-rich hepatocellular carcinoma has unique characteristics in terms of epigenetic metabolic regulation.

[0069] Example 3 Immunotherapy Experiment In the anti-PD-1 monoclonal antibody experiment, Nras / Myc-ND and Nras / Myc-KD mice were intraperitoneally injected with anti-PD-1 (200 μg, clone number RMP1-14, BioXcell) and IgG (200 μg, IgG2b, clone number LTF-2, BioXcell), respectively. IgG served as a control for PD-1. The injections were administered twice weekly for the first four injections, and then once weekly until the end of the experiment. The survival time of each mouse was recorded.

[0070] Figure 9 Model building and immunotherapy flowchart ( Figure 9 A) Magnetic resonance imaging and gross tumor images of the two groups of mice after treatment ( Figure 9 B) Survival analysis curves ( Figure 9 C Figure 9 D).

[0071] The results showed that the Nras / Myc-ND model was sensitive to treatment, with reduced tumor burden and significantly prolonged survival, while the Nras / Myc-KD model was resistant to immunotherapy and showed no significant improvement in survival. This indicates that the lipid-rich hepatocellular carcinoma model exhibits significant resistance to the immune checkpoint inhibitor anti-PD-1 therapy. Therefore, the model provided by this invention can be used to screen drugs.

[0072] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for constructing a lipid-rich hepatocellular carcinoma mouse model based on a combination of high-pressure hydrodynamic injection and a ketogenic diet, characterized in that, Includes the following steps: (1) SPF grade C57BL / 6J mice were acclimatized for 7 days before modeling began; (2) Preparation of plasmid solution: Construct an expression plasmid containing Nras oncogene, Myc oncogene and Sleeping Beauty transposase, dissolve it in physiological saline, and adjust the concentration to be suitable for high-pressure hydrodynamic injection; (3) High-pressure hydrodynamic tail vein injection: The plasmid solution obtained in step (2) is injected into the tail vein of the mouse. The ratio of the volume of the plasmid solution to the weight of the mouse is 0.1 mL / g. The injection is carried out rapidly within 5-8 seconds to allow the plasmid to enter the hepatocytes. (4) Dietary intervention: After plasmid injection, mice were fed a ketogenic diet for 7 days and fed for 6-8 weeks to establish the model. (5) Verify the characteristics of lipid-rich hepatocellular carcinoma.

2. The method according to claim 1, characterized in that, In step (1), the temperature of SPF grade C57BL / 6J mice was controlled at 22±2℃ and the humidity was 55%±10%. The mice were kept in a 12-hour day-night cycle and had free access to drinking water and feed.

3. The method according to claim 1, characterized in that, In step (2), the expression plasmid containing the Nras oncogene is the purified pT / Caggs-NRASV12 plasmid; the expression plasmid containing the Myc oncogene is the purified c-myc-PT3EF1a plasmid; and the expression plasmid containing the Sleeping Beauty transposase is the purified pCMV / SB11 plasmid.

4. The method according to claim 3, characterized in that, The purified pT / Caggs-NRASV12 plasmid, c-myc-PT3EF1a plasmid and pCMV / SB11 plasmid were mixed at a mass ratio of 19:1:

2.

5. The method according to claim 1, characterized in that, In step (2), the purified pT / Caggs-NRASV12 plasmid, c-myc-PT3EF1a plasmid, and pCMV / SB11 plasmid were dissolved in 0.9% physiological saline, and the ratio of physiological saline volume to mouse body weight was 0.1 mL / g.

6. The method according to claim 1, characterized in that, In step (4), the feeding method is free access to ketogenic feed.

7. The method according to claim 1, characterized in that, In step (5), the lipid-rich hepatocellular carcinoma characteristics of the model were verified by gross liver observation, H&E staining, lipid staining, detection of triglyceride and ketone body content, transcriptomics and lipidomics analysis.

8. The method according to claim 7, characterized in that, The characteristics of lipid-rich hepatocellular carcinoma include abundant lipid droplet deposition in the tumor tissue, absence of fatty degeneration in adjacent tissues, abnormal lipid metabolism, and tolerance to immunotherapy.

9. The use of the mouse model established by the method of any one of claims 1-8 in animal experiments, targeted drug screening, and immunotherapy evaluation for the study and development of lipid-rich hepatocellular carcinoma.

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