As₂O₃ Combined With Recombinant Canstatin Protein On The Role Of Vascular Endothelial Cells

Object:Canstatin by recombinant protein, As2O3, and both methods were combined intervention of human umbilical vein endothelial cells (HUVEC) and tumor vascular endothelial cells (Td-EC), the interfering factors on HUVEC and Td-EC biological effects of combined treatment programs for the As2O3 the feasibility and effectiveness of anti-tumor experimental basis and theoretical basis.Methods:The stable transfection of pcDNA3.1-Canstatin-3Flag secreted recombinant hepatoma HepG2 cell and HepG2 cell extract supernatant, and ultrafiltration, Western-blot identification of canstatin proteins in the supernatant; HUVEC isolation, culture and identification. HUVEC cultured by hepatoma HepG2 cell culture supernatant 72h, collect the cells, RT-PCR detected the expression of TEM1 and TEM8.Divided according to different experimental factors re-intervention group (stable transfection of pcDNA3.1-Canstatin-3Flag secreted recombinant hepatoma HepG2 cell culture supernatant), As2O3 group (2μmol/L), the combination group (recombinant canstatin protein and As2O3), the control group (HepG2 cell culture supernatant), respectively, of each factor intervention HUVEC and Td-EC, through the growth curve, Transwell chamber assay, flow cytometry, endothelial cells into tube experiments to detect cell biology.Results:1. Western-blot detection of recombinant Canstatin hepatoma HepG2 cell culture supernatant, showing that the size of about 26KD protein bands.2. Cultured HUVEC by immunofluorescence staining, showing yellow-green fluorescence within the cytoplasm, indicating that the existence of human factorⅧ.3. Cultured HUVEC induced by supernatants of hepatoma HepG2 cells,RT-PCR amplification and electrophoresis of approximately 287 bp and 460 bp bands, suggesting that cells expressing markers of tumor vascular endothelial cells TEM1 and TEM8.4.1. Effects on cell growth activity:cells were observed under inverted microscope every 24h of cell growth, survival, cell counting, cell growth curve; results, HUVEC, Td-EC with the incubation time, the number of viable cells decreased, time-dependent manner, The number of surviving cells compared before and after the days of p<0.05, comparison between the experimental group p<0.05; restructuring group, As2O3 group and the control group P<0.05, combined group compared with the control group p<0.01; Td-EC increase the number of surviving cells blank group HUVEC significantly than the control group, comparison between two groups p<0.05; the number of viable Td-ECs in experimental groups decreased compared with the corresponding experimental groups HUVECs obvious comparison between groups p<0.05.4.2 Effects on apoptosis, apoptosis (HUVEC, Td-EC), re-group (12.2±0.32%,16.43±0.27%), As2O3 group (9.76±0.17%,14.05±0.21%) and the combination group (15.78±0.18%,20.69±0.23%), control group (6.30±0.13%,4.73±0.14%), single-treatment group comparison p<0.05, combined treatment group were compared with a single P<0.01, experimental groups and the blank Group P<0.01; Td-EC in each group and corresponding each group HUVEC to p< 0.0.4.3On cell migration, and vascular ability, the number of migrating cells (HUVEC, Td-EC), re-group (29.20±1.30,27.60±0.55), As2O3 group (36.00±1.00,33.20±1.30), the combination group (24.33±1.36,21.00±1.26), control group (45.40±1.14,50.60±1.34); lumen formation number (HUVEC, Td-EC), respectively, re-group (4.40±1.03,3.50±0.94), As2O3 group (6.00±1.14,4.96±0.93), the combination group (2.90±0.92,2.30±0.75), control group (13.23±0.89,14.13±1.28), in which the number of migrating cells in combination group, the least number of tube formation, between three groups P<0.05; experimental groups compared with the control group P<0.01; Td-EC in each group and corresponding each group HUVEC to p<0.05.Conclusion:1. stably transfected with pcDNA3.1-Canstatin-3Flag secreted recombinant hepatoma HepG2 cell culture supernatant has been expressed Canstatin protein.2. HUVEC by HepG2 hepatoma cells cultured supernatant induced to Td-EC.3. Td-EC has stronger than HUVEC proliferation, angiogenesis and migration ability.4. Recombinant canstatin protein,As2O3 and the combintion on HUVEC and Td-EC to inhibit proliferation, angiogenesis, migration and apoptosis, role in on Td-EC more obvious role. a significant effect than the drug alone, and on the Td-EC more significant.