Experimental Study Of Transfected HGM-CSF Gene Into HCC Cells By HA Nanoparticles To Produce Hepatocellular Carcinoma Vaccine

OBJECTTo observe the safety of HA nanoparticles as the vector of hGM-CSF gene transfection, and its impact on the growth of hepatoma carcinoma cells; hepatoma carcinoma cell vaccine was produced by hGM-CSF gene transfected cells to lay the foundation for further studying of gene-modified tumor vaccines of hepatoma in clinic.METHODSFresh hepatoma carcinoma tissues were collected, and monocyte suspension was prepared by shearing, digesting and centrifuging of the tissues; common cell culture and cell passage was performed after the analysis of cell viability by trypan blue staining assay, AFP and ALB secreted levels in cell culture metia were detected to identificat hepatoma carcinoma cells . MTT assay was used to examine whether HA nanoparticles would inhibit the growth of HCC cells. The plasmids containing human GM-CSF gene were transfected into HCC cells by HA nanoparticles to screen the G418 resistance monoclones by limiting dilution method. RT-PCR was applied to identify the integration of plasmids into HCC cell and expression of human GM-CSF. ELISA was performed to detect the level and duration of secreted human GM-CSF in culture medium. Additionally, flow cytometry was used to examine the effect of hGM-CSF on hepatoma cell growth; vaccine of hepatoma vaccine was produced by hepatoma carcinoma cells exposed to sublethal dose of gamma-ray.RESULTSTrypan blue staining assay indicated that the harvested cells'viability was more than 80%; In cell culture supernatants was measured in a high level AFP and the existence of a low level ALB, in control medium detected only trace AFP and no existence of ALB; MTT assay show that Nano-HA suspension liquid concentrations under 80μg/ml in treatment groups make no statistic significance on the growth of HCC cells, compared with the control. RT-PCR demonstrated that the human GM-CSF gene was successfully integrated and steadily expressed in the transfected HCC cells. ELISA indicated that high level of GM-CSF is secreted by stably transfected HCC cells (216.22±45.78ng/106cell/24h). FCM demonstrated that hGM-CSF have no effect on the cell cycle and apoptosis induction of HCC cells.CONCLUSIONWe successfully isolate hepatocellular carcinoma cells from the tumor tissues and culture. HA nanoparticles could be used as a safe vector to transfer hGM-CSF gene into HCC cells and get its stable expression. HA nanoparticles have no significant growth arrest effect on HCC cells. A novel hGM-CSF gene transferred hepatoma cell vaccine is produced to lay an experimental foundation for the further study on HCC gene-modified vaccine.