[Establishment of mouse lipogenic inflammation-mediated hepatocarcinoma model by hydrodynamic injection system]

Objective To establish and evaluate a murine lipogenic inflammation-mediated hepatocarcinoma model with hydrodynamic transfection of protein kinase B (AKT), β-catenin, and Sleeping Beauty transposon plasmids into hepatocytes through the tail vein. Methods Isotonic physiological solution (20 μg myrAKT, 20 μg δN90-β-catenin and 4 μg Sleeping Beauty transposase were dissolved in 2 mL of normal saline) was injected into C57BL/6 male mice aged 6-8 weeks via the tail vein in a short period of time (5-9 s) by hydrodynamic transfection, while the control plasmid was injected with the same method. Mice were then sacrificed 8, 12, or 16 weeks after the injection of these plasmids, and their serum and liver tissues were collected. RNA samples were extracted for further analysis. HE staining was used to detect liver tissue structure and inflammatory cell infiltration; oil red O staining was used to examine lipid deposition in liver cells; sirius red and Masson staining were used to identify collagen deposition in liver tissues; immunohistochemistry was performed to evaluate the proliferation abilities of the cells and microvascular density and distribution; real-time quantitative PCR was used to reflect the expression of tumor-associated genes; flow cytometry was used for immune cell phenotype identification. Results Sixteen weeks after hydrodynamic transfection, 100% (20/20) mice developed liver tumors in the experimental group. Compared with the control group, the body mass of mice in the experimental group increased slowly and liver mass increased significantly. HE staining showed that a large number of hepatocytes appeared to be steatosis, inflammatory cell infiltration increased, liver tumor cells were round or irregular-shaped, the cytoplasmic ratio was imbalanced, tumor cell atypia was obvious and tumor cells were distributed in clustering; ki67 positive cells were enhanced. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (γ-GT), and alkaline phosphatase (ALP) levels remarkably increased. AFP mRNA also significantly increased in liver tissue. CD31 staining showed increased vessel density and vessel structure disorganization. A large number of inflammatory cells, especially myeloid and lymphoid cells, were infiltrated into the liver tissue. Conclusion Murine lipogenic inflammation-mediated hepatocarcinoma model that is caused by non-resolving inflammation was successfully established by hydrodynamic transfection with AKT and β-catenin plasmids. This method is easier performed and reproducible, the induction time is relatively short with a high rate of tumor formation.