Establishment And Researches Of Hepatoma Carcinoma Cells With Expressing RFP And Dual-color Fluorescence Tracing Orthotopic Model Of Hepatocellular Carcinoma

Objective: To establish stable high expressing red fluorescent protein(RFP) human hepatoma cell Hep G2/RFP and mouse human hepatoma cell Hepa1-6/RFP, which infected red fluorescent protein by lentivirus. And to explore whether some biological basic characteristics and genetic characteristics are influenced after infected.Methods: The human hepatoma cell Hep G2 and mouse human hepatoma cell Hepa1-6 were infected by lentivirus containing RFP gene and puromycin resistance gene.Then the cells respectively named Hep G2/RFP and Hepa1-6/RFP after puromycin selection. The morphology of infected cells were observed under the fluorescence microscope. After 30 consecutive in-vitro cell passaged, the RFP positive rates were assayed through FACS(fluorescence activated cell sorting). RFP gene expression in hepatoma carcinoma cells were analyzed with PCR. The proliferation 、 migration 、chromosome karyotypes and numbers、the relative expression of AFP between infected cells and original cells were detected with CCK-8 test 、 Transwell technique 、chromosome analysis test、Western-blot test.Results: After puromycin selection, the infection rates of RFP were all above 98%.PCR results indicated that RFP gene was integrated into the genome of hepatoma carcinoma cells. Analysis of cell basic biological characteristics and genetic characteristics revealed no difference between infection cells and original cells.Conclusion: The human hepatoma cell Hep G2/RFP and mouse human hepatoma cell Hepa1-6/RFP not only stably and highly express RFP, but also can remain the original cellbasic biological characteristics and genetic characteristics unchanged. They serve a new and useful tools for the further tracking study and therapy of hepatocellular carcinoa.Objective: To establish red-green dual-color fluorescence tracing orthotopic transplantation model of hepatocellular carcinoma, and explore its basic characteristics.Methods: Stable high RFP-expressing cells lines Hep G2/RFP and Hepa1-6/RFP were transplanted into the right lobe of the liver of expressing enhanced green fluorescent protein(EGFP) nude mice to establish red-green dual-color fluorescence tracing model of hepatocellular carcinoma. Then the growth and metastasis of tumor were visualized dynamically by whole body in vivo fluorescence imaging system in real time. Then hepatocellular carcinoma tissues were removed from tumor-bearing mice, thick serial frozen slices were made and hepatocellular carcinoma tissues were cultured. After that, the transplantation tumors organization structure were observed under the microscope.Results: dual-color fluorescence tracing orthotopic transplantation models of hepatocellular carcinoma were established successfully, the percentage of success rate was100%. The tumor with red fluorescence gradually increased and migrated from liver to other tissues with whole body in vivo fluorescence imaging system in real time. And the tumor growth and metastasis were visualized in early stage in live animals. Under the fluorescence microscope, hepatoma cells with red fluorescence and host cells with green fluorescence cells intertwined together in the reconstruction region of tumor tissue. Also the tumor cells invasion, migration, and cell fusion between tumor cells and host cells could be observed clearly, which can be accurate to the level of single cell. under the ordinary microscope, routine hematoxylin and eosin(H&E) staining further confirmed the characteristics of hepatocellular carcinoma. Hepa1-6/RFP hepatocellular carcinoma tissues were cultured, which also confirmed that fusion cells exist in hepatocellular carcinoma tissues.Conclusion: We can observe tumor growth and metastasis with the dual-color fluorescence tracing orthotopic transplantation model, and visualize clearly the interactionand position between tumor cells and host cells. It will provide a new visualizing experiment platform for the stduty of tumor invision 、 metastasis and tumor microenvironment. In contrast to traditional animal model, the model has higher practical value.