Establishment And Application Of Primary Mouse Hepatocellular Carcinoma Model Based On Hydrodynamic Gene Transfection

Background and ObjectiveHepatitis,cirrhosis and hepatocellular carcinoma(HCC)caused by hepatitis B virus(HBV)infection are major infectious diseases that seriously endanger human health.Early detection,early diagnosis and early treatment are still the principles for the treatment of HCC.Screening early immunodiagnostic markers of HCC from serum is of great significance and can significantly improve the survival rate of patients.During the development of hepatocellular carcinoma(HCC),the continuity of clinical serum is very precious rare.Therefore,this study intended to combine sleeping beauty swivel mount enzyme with CRISPR/Cas9 technology to establish a primary mouse liver cancer model through hydrodynamic gene transfection technology,to collect serum from different development stages of HCC for screening and identifying early immunodiagnostic markers of liver cancer,and to verify and supplement the immunodiagnostic markers identified in liver cancer patients;On this basis,the M1/M2 macrophage sensor was used to monitor the occurrence and development of HCC in the mouse model through fluorescence imaging in vivo,so as to realize the early non-invasive diagnosis of HCC at the animal level,and ultimately improve the early diagnosis rate of HCC.Methods1.Combined with Sleeping Beauty transposase and CRISPR/Cas9 technology,hydrodynamic gene transfection technology was used to establish a primary mouse hepatocellular carcinoma model with overexpression of proto-oncogenes and tumor suppressor gene knockout;2.Hepatological and serological tests were performed to detect liver lesions in mice;3.Western blot and DNA sequencing were used to verify the overexpression of oncogenes in mouse liver cells and the knockout of tumor suppressor genes;4.q RT-PCR and ELISA were used to detect early immunodiagnostic markers of liver cancer in liver tissue and serum of mice with primary liver cancer;5.In vivo fluorescence imaging was used to detect the distribution and migration of Luc~+macrophages from bone marrow in vivo;6.The pGL3-Arg-1Ep-Fluc-GFP reporter gene plasmid was constructed and lentivirus was packaged to infect RAW264.7 mouse macrophage line7.In vitro co-culture,the macrophage sensor pGL3-Arg-1EP-Fluc-GFP/Raw264.7 induced M1/M2 polarization in the tumor microenvironment;8.The macrophage sensor pGL3-Arg-1EP-Fluc-GFP/RAW264.7 was adoptively transfused into mice with primary liver cancer.The fluorescence of the macrophage sensor Arg-1EP-Fluc-GFP/RAW264.7 was detected by fluorescence imaging in vivo.Results1.A primary mouse hepatocellular carcinoma model was established in which the proto-oncogenes C-Met and N90-β-catenin were overexpressed and the tumor suppressor genes Pten and P53 were knocked out;2.There were obvious pathological changes in the liver of mice.AFP,ALT and AST showed impaired liver function;3.Western blot and DNA sequencing confirmed the overexpression of oncogenes in mouse cells and the knockout of tumor suppressor genes;4.The levels of NPM1,GNAS,PAX5 and PTCH in liver tissue and serum of mice were detected by q RT-PCR and ELISA;5.Luc~+macrophages from bone marrow migrated to lung,liver and spleen in vivo;6.The pGL3-Arg-1Ep-Fluc-GFP reporter gene plasmid was constructed,and lentivirus was packaged to infect RAW264.7 mouse macrophage line,and the stable transgenic strain was obtained;7.In vitro co-culture,the macrophage sensor pGL3-ARG-1EP-Fluc-GFP/Raw264.7 was polarized in the tumor microenvironment8.The macrophage sensor pGL3-Arg-1EP-Fluc-GFP/Raw264.7 was adoptively transfused into mice with primary liver cancer.In vivo fluorescence imaging showed that the macrophage sensor Arg-1EP-Fluc-GFP/Raw264.7 had obvious fluorescence in HCC mouse model.ConclusionA primary mouse hepatocellular carcinoma model was established,and it was confirmed at the animal level that tumor-associated antigen-antibodies of PTCH,PAX5,GNAS and NPM1 could be used as markers for early immunodiagnosis of HCC patients.Meanwhile,it was determined that the M1/M2 macrophage sensor could realize early non-invasive diagnosis of HCC by monitoring the formation and development of HCC in mice.