Establishment And Validity Verification Of Human Hepatocarcinoma Cell Lines With Expression Of Luciferase For In Vivo Biofluorescence Imaging

Objective:To establish a human transplanted hepatoma moedel in nude mouse for in vivo biofluorescence imaging, which can be used for observation of antitumor drug effect.Methods:1. The plasmids with luciferase gene were transfected into hepatoma cells by liposome. By the selection of G418, the monoclonal cell lines of hepatoma with high express of luciferase were obtained.2. To construct recombinant plasmid pMSCV-Metluc vector for packaging retrovirus. The stable monoclonal cell lines of virus-producing cell were established by transfecting the recombinant vectors into PT67cells. The virus was concentrated by ultracentrifugation.3. The human hepatoma cells were infected by virus obtained from monoclonal virus-producing cell line to establish monoclonal cell lines of hepatoma with high express of luciferase with the help of selection of G418.4. To establish the subcutaneous human hepatom in nude mouse by subcutaneous inoculation of cell suspension of monoclonal cell lines and liver orthotopic transplantation tumor block. And then, the development of hepatoma was observed by in vivo biofluorescence imaging.Results:1. By the selection of G418, the3monoclonal cell lines of hepatoma with stable express of luciferase were obtained and the express level was still stable after several generations. And there was no significant difference between monoclonal cells and normal cells in cell morphology, growth and growth rate.2. The recombinant plasmid pMSCV-Metluc vector was constructed successfully. And then, the PT-67cells were transfectd to establish the stable monoclonal cell lines of virus-producing cells. The highest virus titer was1×106cfu/ml and it was1×107cfu/ml after ultracentrifugation.3. By the selection of G418, the monoclonal cell lines of hepatoma with high expression luciferase by retroviral infection, which can be used for in vivo biofluorescence imaging.4. The subcutaneous human hepatoma was established by subcutaneous inoculation of monoclonal cell lines of hepatoma with luciferase gene.1week later, the subcutaneous hepatoma could be seen by naked eye. And the bioluminescence intensity of subcutaneous hepatoma established by monoclonal cell lines of SMMC7721-luc (v), SK-HEP-1-luc (v) and Hep3B-luc (v) were7.205×106photon/s,5.681×105photon/s and1.924×106photon/s.5. After2weeks of subcutaneous inoculation of monoclonal cell lines, the subcutaneous hepatoma was used for orthotopic transplantation. And10days later, the in vivo biofluorescence imaging showed that the bioluminescence intensity of orthotopic hepatoma was4.888x105～6.008×105photon/s. The orthotopic hepatoma showed no necrosis and ulceration by naked eye.Conclusion:The subcutaneous human hepatoma and orthotopic hepatoma model were established successfully by liposome transfection and retroviral infection respectively. The monoclonal cell lines of hepatoma with high express of luciferase and the real-time detection system for observing hepatoma development were established successfully, which established a new method for monitoring hepatoma growth, effect of antitumor drugs and hepatoma metastasis.