Whole Body Imaging Of Green Fluorescent Protein (GFP)-expressing Tumor Model And Therapeuticeffect Of Rh-endostatin Combined With 5-Fu

Objective To explore an easy and fast way of establishing an enhanced green flouroscent(EGFP)expressing HCC HepG-2 xenograft tumor model by comparing in intro two transfection ways using liposome and lentivirus,in vivo fluroscence imaging to visualize the progress of the EGFP expressing HepG-2 tumor noninvasively,continuesly and in real time.To evaluate rh-Endostatin(shortly as endostatin in this paper)'s effects on HepG-2 cells and human umbilical vein cells in vitro;to study endostatin's angiogenesis effects combined with chemotherapy using above established EGFP tagged HCC HepG-2 tumor xenografl model both in fluoroscent imaging and tradional ways.Methods 1.Comparing the two ways of transfecting the EGFP gene into HepG-2 cell line 2.G418 was appllied to select the GFP positive cells in liposome transfection cells and cell sorting using FACS was appllied to cells in both lipsome transfection group and lentivirus transfection group.Efficacy of making stable and long term expression of EGFP in HepG-2 by the two transfecting ways were evaluated.EGFP tagged HepG-2 cells were transplanted subcutaneously to make a xenograft tumor model,tumor's growth was observed by fluoroscence imaging in vivo.20 mice of 4 weeks age were made into EGFP expressing HepG-2 tumor model and were devided into 4 groups as group A,B,C,D, A as control with 0.5ml/d of normal sodium,B with endostatin at 1.5 mg/kg/d,C with endostatin at 1.5 mg/kg/d combind with 5-Fu at 20mg/kg/d,D with 5-Fu 20mg/kg/d,drugs were administered d1-d7,i.p in all groups;fluoroscent imaging were taken every 3 days to observe the change of xenografled tumor,conventional ways of measurement were carried out too on each test mouse.Resuts Both transfecting ways were effective in transfecting EGFP into HepG-2 cells,cells transfected with lentiviurs had a long stable expression of EGFG while not with transfection with liposme.In vivo fluroscence imaging to visualize the progress of the EGFP expressing HepG-2 tumor noninvasively,continueusly and in real time was achieved with this model.Endostar had a cytoxic effect on HepG-2 cells at 24h(cell death rate 20.7%)with 100μg/ml concentration and 48h at 50μg/ml,100μg/ml,200μg/ml concentration(cell death rate 20.8%,46.3%,15.8%respectivey);at 100μg/ml,endostatin induced apoptosis in HepG-2 after 24h and 48h,and in human umbilical vein cell after 48h,apoptosis rate was 19.5%(11.7%as control).Endostatin had the highest tumor inhabit rate(TIF)when combined with 5-Fu chemotherapy compared with either endostar alone or 5-Fu alone(P＜0.05),endostatin alone(group B)had a certain tumor inhibitoy effects when treating tumor alone(25.23%),lower than combination group(group C)(TIF=62.05%)(P＜0.05)and 5-Fu alone group (group D)(TIF=45.19%)(P＜0.05).Microvessle density(MVD)and expression of VEGF were reduced after endostatin treatment either alone or combine with 5-Fu.Conclusions Transfecting with lentivirus combined with cell sorting with FACS proved to be a fast and effective way to have long and stble expression of EGFP in HepG-2 cells,which guarantee an reliable and time-saving methods of establishing a GFP tagged HCC HepG-2 tumor xenograft model.Such model enables us to monitor the progress of the EGFP expressing HepG-2 tumor noninvasively,continuesly and in real time.Endostatin has cytoxic effects on HepG-2 cells in vitro only at certain dose and time period,it can induce apoptosis in both HepG-2 cells and human umbilical vein cells.Combining with 5-Fu chemothery has a synergistic effect on TIF compared with treating with any agent alone.Endostatin reduces tumor's MVD and expression of VEGF.