A method for constructing a mouse spontaneous tumorigenic hepatocarcinoma model

Abstract

The application discloses a construction method of a mouse spontaneous tumorigenic hepatocarcinoma model, and belongs to the technical field of biological medicine. The application utilizes the high transposition characteristics of the sleeping beauty transposition system, realizes specific co-expression of c-MET gene overexpression and NRASV12 mutant gene in the mouse liver by injection of a combination of specific transposition vectors and expression vectors, and thus induces spontaneous formation of hepatocarcinoma in the mouse liver. The liver tumor in the mouse hepatocarcinoma model is formed by malignant transformation of liver cells of the mouse itself, can simulate the natural occurrence and development process of clinical hepatocarcinoma, can stably realize co-activation of c-MET and NRASV12 in the hepatocarcinoma tissue, has consistent molecular characteristics with clinical hepatocarcinoma of the subtype, and conforms to the highly invasive characteristics of clinical c-MET and NRASV12 co-activation hepatocarcinoma. Therefore, the application fills the blank of the exclusive animal model of the subtype of hepatocarcinoma, and provides a precise tool for mechanism research of the high-risk subtype of hepatocarcinoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for constructing a mouse model of spontaneously tumorigenic liver cancer. Background Technology

[0002] Liver cancer is a highly prevalent malignant tumor worldwide. Among them, the liver cancer subtype co-upregulated by c-MET overexpression / amplification and NRASV12 activating mutation is characterized by high invasiveness, strong proliferative capacity, easy drug resistance, and extremely poor prognosis. Moreover, existing treatments are almost ineffective against it, making it a key and challenging area for liver cancer research and drug development.

[0003] Currently, animal models of liver cancer are core tools for studying the pathogenesis of this specific subtype of liver cancer and screening targeted therapies. Existing mouse models of liver cancer mainly include chemically induced tumor models, xenograft models, and gene-editing models, but these models all have significant limitations: a. Chemically induced carcinogenesis models (such as those induced by diethylnitrosamine) exhibit strong randomness in tumor formation, making it difficult to target and simulate the molecular characteristics of c-MET and NRASV12 co-activation. Furthermore, they have long tumor formation cycles and poor model stability. b. Xenograft models (such as subcutaneous or orthotopic inoculation of liver cancer cells) are allogeneic transplants, which cannot simulate the physiological process of spontaneous occurrence and development of liver cancer, and it is difficult to reproduce the interaction between the tumor microenvironment and the body, resulting in a large difference from the actual clinical incidence. c. Traditional gene editing models (such as CRISPR / Cas9 and transgenic mice) have complex construction processes, long cycles, and high costs. They are also highly dependent on mouse strains, making it difficult to quickly build them in large batches and unsuitable for large-scale drug screening. d. Existing liver cancer models rarely target the high-risk subtype of c-MET and NRASV12 co-activation. There is a lack of dedicated animal models that can accurately simulate the molecular characteristics and pathological phenotype of this subtype, resulting in a lack of effective in vivo research tools for related pathogenesis studies and targeted drug development.

[0004] In summary, there is an urgent need to construct an animal model of liver cancer that can recreate the spontaneous occurrence and development process of this subtype of liver cancer, so as to provide a reliable in vivo experimental tool for the screening of targeted drugs and the validation of combination therapy regimens for this subtype of liver cancer. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a method for constructing a spontaneously tumorigenic liver cancer model in mice, thereby resolving technical problems such as the inability of existing mouse liver cancer models to accurately simulate the molecular characteristics of liver cancer subtypes co-activated by c-MET and NRASV12, long model construction cycles, and significant differences between the tumorigenic process and clinical reality.

[0006] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for constructing a mouse spontaneous tumorigenesis liver cancer model is provided, comprising the following steps: (1) Preparation of plasmid mixture: pCMV(CAT)T7-SB100, pT3-EF1a-c-Met and pT / Caggs-NRASV12 were mixed to prepare plasmid mixture; (2) Tail vein injection: Inject the plasmid mixture from step (1) into the mouse; (3) Feeding and management: After injection, mice were fed routinely for 12-24 weeks to complete the construction of a spontaneous tumor-forming liver cancer model in mice.

[0007] Furthermore, in step (1), the mass ratio of pCMV(CAT)T7-SB100, pT3-EF1a-c-Met and pT / Caggs-NRASV12 is pCMV(CAT)T7-SB100:(pT3-EF1a-c-Met+pT / Caggs-NRASV12)=1:(2-25); where the mass ratio of pT3-EF1a-c-Met and pT / Caggs-NRASV12 is 1:1.

[0008] Furthermore, the amount of plasmid mixture injected in step (1) is 10% of the mouse's body weight.

[0009] Furthermore, the injection time in step (2) is 7-10 seconds.

[0010] Furthermore, the conventional feeding conditions in step (3) are a temperature of 22-25℃, a humidity of 40-60%, and a 12-hour light-dark cycle.

[0011] Furthermore, the normal feeding period in step (3) is 12-24 weeks.

[0012] The present invention has the following beneficial effects: 1. Precise matching of molecular features: This invention is the first to use the Sleeping Beauty transposon system to achieve co-activation of c-MET and NRASV12 in mouse liver. The constructed model accurately simulates the molecular features of the rare clinical upregulation of c-MET and NRASV12 in a subtype of liver cancer, filling the gap in animal models specific to this subtype of liver cancer and providing a precise tool for the study of the mechanism of this high-risk subtype of liver cancer. 2. Tumor formation process closely resembles clinical practice: This invention is a spontaneous tumor formation model in which the tumor is formed by the malignant transformation of the mouse's own liver cells. It can recreate the complete physiological process of liver cancer from malignant transformation of cells, precancerous lesions to tumor formation, invasion and metastasis. It can also realistically simulate the interaction between the tumor microenvironment and the body. Compared with transplanted tumor models, it is closer to the actual clinical pathogenesis. 3. Simple and efficient construction method: This invention delivers the vector via tail vein injection, which is simple to operate, minimally invasive, and requires no complex surgical procedures or gene editing processes; the tumor formation cycle of the model is controllable (12-24 weeks), much shorter than chemically induced carcinoma models and traditional gene editing models, and can be constructed in batches, making it suitable for large-scale drug screening experiments; 4. High model stability: The Sleeping Beauty transposon system can mediate the integration of the target gene into the mouse genome, achieving stable and continuous expression of c-MET and NRASV12. The constructed model has a high tumorigenesis rate (≥80%), and the molecular characteristics and pathological phenotypes of the tumors are consistent, with strong reproducibility. 5. Wide range of applications: The model constructed in this invention can not only be used to study the pathogenesis and signaling pathway regulation of hepatocellular carcinoma co-activated by c-MET and NRASV12, but also to screen targeted drugs (such as c-MET inhibitors and MEK inhibitors) for this subtype of hepatocellular carcinoma, verify the effectiveness of combination therapy, and provide an in vivo experimental platform for the study of biomarkers for this subtype of hepatocellular carcinoma, which has important scientific research and clinical translational value. Attached Figure Description

[0013] Figure 1 The pCMV (CAT) T7-SB100 vector spectrum; Figure 2 The pT3-EF1a-c-Met vector spectrum; Figure 3 The pT / Caggs-NRASV12 vector spectrum; Figure 4 Gross image of the tumor in the mouse spontaneous tumorigenesis liver cancer model constructed in Example 1 of this invention; Figure 5 The expression of c-MET and NRASV12 genes in the tumor tissue of the mouse spontaneous tumorigenesis hepatocellular carcinoma model constructed in Example 1 of this invention; Figure 6 The expression of c-MET protein and NRASV12 protein in the tumor tissue of the mouse spontaneous tumorigenesis liver cancer model constructed in Example 1 of this invention; Figure 7 HE staining results of tumor tissue from the mouse spontaneous tumorigenesis liver cancer model constructed in Example 1 of this invention. Detailed Implementation

[0014] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0015] Experimental materials: 1. Transposase expression vector: pCMV (CAT) T7-SB100 (vector map see...) Figure 1 This transposase, SB100, is used to express the Sleeping Beauty transposase and mediate transposon integration. It is available from Addgene (Addgene plasmid #34879;RRID: Addgene_34879). As a transposase expression vector for the Sleeping Beauty transposon system, its promoter CMV efficiently drives the expression of SB100 transposase in mouse hepatocytes. SB100 transposase recognizes transposon sequences on the target gene expression vector, mediating the integration of exogenous genes pT3-EF1a-c-Met and pT / Caggs-NRASV12 into the genome of mouse hepatocytes, achieving stable and continuous expression of the target genes.

[0016] 2. Target gene expression vector: pT3-EF1a-c-Met (vector map see...) Figure 2 Using EF1a as a promoter, this transposase is used for c-MET gene overexpression. It is available from Addgene (Addgene plasmid #31784; RRID: Addgene_31784) and can efficiently and continuously drive c-MET gene expression in mammalian cells, achieving c-MET gene overexpression in mouse liver cells and mimicking the molecular characteristics of c-MET upregulation in clinical liver cancer. pT / Caggs-NRASV12 (vector map see...) Figure 3 Using Caggs as a promoter, NRASV12 is activated to specifically express the mutant gene in mouse liver cells. This transposase can be obtained from Addgene (Addgene plasmid#20205; RRID: Addgene_20205). NRASV12 can continuously activate the RAS-MAPK / ERK pathway, forming a synergistic driving effect with c-MET overexpression, jointly inducing malignant transformation of liver cells, and ultimately forming liver cancer.

[0017] 3. Laboratory Animals: All mice were housed individually or in cages of ≤6 mice per SPF-grade barrier system. The housing environment was maintained at a temperature of 22±2℃, relative humidity of 40-70%, and a 12-hour light / 12-hour dark cycle. Mice were fed standard maintenance diet sterilized by irradiation and provided with autoclaved drinking water. Mice had free access to food. After one week of acclimatization, they were used in experiments.

[0018] Example 1: A method for constructing a spontaneously tumorigenic hepatocellular carcinoma model in mice includes the following steps: (1) Preparation of plasmid mixture: Mix 20 μg pCMV(CAT)T7-SB100, 20 μg pT3-EF1a-c-Met and 20 μg pT / Caggs-NRASV12, and then add 2 mL of physiological saline to prepare plasmid mixture.

[0019] (2) Tail vein injection: Fix the mice after acclimatization, expose the tail vein, and use a 5mL sterile syringe to draw up the plasmid mixture from step (1). Administer a rapid tail vein injection (within 7s) to the mice at a dose of 2mL / mouse (approximately 10% of the mouse's body weight). Ensure that the plasmid mixture enters the mouse's body quickly and evenly during the injection process. (3) Feeding and management: After injection, the mice were placed in an SPF-grade animal room for routine feeding. The feeding conditions were: temperature 22℃, humidity 40%, 12h light and dark alternation, free food and water. After 12 weeks of feeding, the spontaneous tumorigenesis liver cancer model of mice was established.

[0020] Example 2: A method for constructing a spontaneously tumorigenic hepatocellular carcinoma model in mice includes the following steps: (1) Preparation of plasmid mixture: Mix 20 μg pCMV(CAT)T7-SB100, 20 μg pT3-EF1a-c-Met and 20 μg pT / Caggs-NRASV12, and then add 2 mL of physiological saline to prepare plasmid mixture.

[0021] (2) Tail vein injection: Fix the mice after acclimatization, expose the tail vein, and use a 5mL sterile syringe to draw up the plasmid mixture from step (1). Administer a rapid tail vein injection (within 8s) to the mice at a dose of 2mL / mouse (approximately 10% of the mouse's body weight). Ensure that the plasmid mixture enters the mouse's body quickly and evenly during the injection process. (3) Feeding and management: After injection, the mice were placed in an SPF-grade animal room for routine feeding. The feeding conditions were: temperature 24℃, humidity 50%, 12h light and dark alternation, free access to food and water. After 18 weeks of feeding, the spontaneous tumorigenesis liver cancer model of mice was established.

[0022] Example 3: A method for constructing a spontaneously tumorigenic hepatocellular carcinoma model in mice includes the following steps: (1) Preparation of plasmid mixture: Mix 20 μg pCMV(CAT)T7-SB100, 20 μg pT3-EF1a-c-Met and 20 μg pT / Caggs-NRASV12, and then add 2 mL of physiological saline to prepare plasmid mixture.

[0023] (2) Tail vein injection: Fix the mice after acclimatization, expose the tail vein, and use a 5mL sterile syringe to draw up the plasmid mixture from step (1). Administer a rapid tail vein injection (within 7-10s) to the mice at a dose of 2mL / mouse (approximately 10% of the mouse's body weight). Ensure that the plasmid mixture enters the mouse's body quickly and evenly during the injection process. (3) Feeding and management: After injection, the mice were placed in an SPF-grade animal room for routine feeding. The feeding conditions were: temperature 25℃, humidity 60%, 12h light and dark alternation, free access to food and water. After 24 weeks of feeding, the spontaneous tumorigenesis liver cancer model of mice was completed.

[0024] Experimental Example: Model Qualification After the mice in Example 1 were fed, they were dissected to observe the tumor formation in the liver. The overexpression level of the c-MET gene and the expression of the NRAS gene in the liver cancer tissue were detected by bulk RNA-seq. The expression levels of c-MET protein and NRASV12 protein were detected by proteomics sequencing. The pathological grade and vascular invasion phenotype of liver cancer were identified by HE staining of pathological sections, confirming the successful construction of the model.

[0025] Depend on Figure 4 It was found that 12 weeks after injection, multiple, scattered grayish-white tumor nodules were visible on the surface of the liver of the model mice. The tumors grew in a nodular or lobulated manner, and some fused into clumps. They were clearly demarcated from the surrounding normal liver tissue, while no obvious abnormalities were observed in the normal liver tissue. This suggests that the model successfully induced the formation of multiple liver tumors.

[0026] Depend on Figure 5 It can be seen that the mRNA expression levels of c-MET and NRAS genes (NRASV12) in the liver cancer tissue of model mice are significantly higher than those in normal liver tissue, indicating that the target genes have been successfully integrated into the mouse hepatocyte genome and overexpressed at the transcriptional level.

[0027] Depend on Figure 6 It was found that the expression levels of c-MET and NRASV12 proteins in the liver cancer tissue of model mice were significantly higher than those in normal liver tissue, confirming the co-activation of c-MET and NRASV12 at the protein level.

[0028] Depend on Figure 7 It can be seen that the liver cancer tissue of the model mice exhibits typical pathological features of hepatocellular carcinoma, with tumor cells arranged in nests or trabeculae, obvious cellular atypia, increased nuclear-cytoplasmic ratio, and pathological mitotic figures. Vascular invasion can be seen in some areas, suggesting that this model has a highly aggressive pathological phenotype.

[0029] In summary, the construction method of this invention can integrate the target gene into the mouse genome, achieving stable and continuous expression of c-MET and NRASV12. All six mice used in this model construction developed tumors, and the constructed model had a high tumor formation rate (≥80%), with good consistency between the molecular characteristics and pathological phenotype of the tumors, and strong reproducibility.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a spontaneously tumorigenic hepatocellular carcinoma model in mice, characterized in that, Includes the following steps: (1) Preparation of plasmid mixture: pCMV(CAT)T7-SB100, pT3-EF1a-c-Met and pT / Caggs-NRASV12 were mixed to prepare plasmid mixture; (2) Tail vein injection: Inject the plasmid mixture from step (1) into the mouse; (3) Feeding and management: After injection, mice were fed routinely for 12-24 weeks to complete the construction of a spontaneous tumor-forming liver cancer model in mice.

2. The construction method according to claim 1, characterized in that, In step (1), the mass ratio of pCMV(CAT)T7-SB100, pT3-EF1a-c-Met, and pT / Caggs-NRASV12 is pCMV(CAT)T7-SB100:(pT3-EF1a-c-Met+pT / Caggs-NRASV12)=1:(2-25); wherein the mass ratio of pT3-EF1a-c-Met and pT / Caggs-NRASV12 is 1:

1.

3. The construction method according to claim 1, characterized in that, The amount of plasmid mixture used in step (1) is 10% of the mouse's body weight.

4. The construction method according to claim 1, characterized in that, The injection time described in step (2) is 7-10 seconds.

5. The construction method according to claim 1, characterized in that, The conventional feeding conditions described in step (3) are a temperature of 22-25℃, a humidity of 40-60%, and a 12-hour light-dark cycle.

6. The construction method according to claim 1, characterized in that, The standard feeding period described in step (3) is 12-24 weeks.

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