| Roxarsone, an organoarsenic compand, is widely used in animal production as a feed additive all over the world and it may have the potential risk of cancer because of its angiogeniesis effect and the promotion to growth of human vascular endothelial cells reported recently. Little information is focused on the promotion to growth of vascular endothelial cells of roxarsone and no investigation was reported in the domestic now. This study was conducted to isolate and culture the vascular endothelial cells of rat thoracic aorta and then the effect of roxarsone on the vascular endothelial cells in vitro was primarily studied using the methods of MTT, Flow Cytometry, tube formation test and migration test.The isolation of the vascular endothelial cells of rat thoracic aorta was studied based on the vassel tissue plant and the enzymatic digestion of I collagenase respectively, in which the principle that fibroblasts and contractile fiber cells have a different adherence time was used to remove the irrespective cells, and VEGF and heparin was added to DMEM medium to clear the irrespective cells. Subculture of the isolated endothelial cells was mead and their growth curves were drawn. The identification of cultured cells were conducted by microscopic morphology, CD31immunohistochemistry. The cultured cells were polygonal and arrayed like pitching stone, CD31immunohistochemical analysis was positive and the special organelles W-P body of endothelial cells were observed in cultured cells by the transmission electron microscope.The MTT assay was performed on the doses of roxarsone10.00,1.00,0.10pM and0.01μM,5ng/mL VEGF as the positive control groups, the PBS as the negative control using exponential growth phase cells with6h incubation. Results indicated that OD values of different concentration roxarsone and VEGF treatment were significantly higher than that of PBS (P<0.05), presented a dose-effect relationship in0.01-1.00μM range but decreased at10.00μM exposure. The OD of1.00μM roxarsone was similar with that of5.0ng/mL VEGF. The growth promotion of roxarsone on endothelial cells from rat was shown at lower concentration (0.01μM-1.00μM) but the promotion was weakened at10.00μM higher dose treatment.In the experiment of Flow Cytometry analysis, the concentration of roxarsone was designed as10.00μM,1.μM and0.μM,5ng/mL VEGF as positive control groups, the PBS as negative control when endothelial cells were6h incubation. The cell cycle was detected by Flow Cytometry. Results indicated that the numbers of S phase cells in0.10μM,1.00μM roxarsone group and VEGF treatment were increased significantly compared with that of PBS group (P<0.05), and the numbers of G2phase cells were clearly lower than that of PBS. There was no significant difference in G1phase between them. The cell proliferation promotion was appeared in the treatment of0.10μM and1.μM roxarsone to endothelial cells from rat.In the scarification test, the concentration of roxarsone was divided into10.00μM,1.00μM,0.10μM,0.01μM,5ng/mL VEGF as positive control groups, PBS as negative control. The migration of cells treated after12h incubation cells were photoed. Result indicated that numbers of cells migrated in the dose of0.01μM～1.00μM increased with the increase of concentration of roxarsone, while decreased when the dose of roxarsone10.μM.In conclusion, the method of isolation and culture of the vascular endothelial cells from rat thoracic aorta was developed. The cell proliferation and migration promotion of roxarsone in0.01μM～10.00μM to endothelial cells was obviously, increasing with the increase of concentration of roxarsone within the dose of0.01μM-1.00μM, but decreased at the dose of10.00μM. |
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