The Research On Constructing Models Of Neuroendocrine Tumor Bon-1 And H727 Cell Lines In Vitro And The Impact Of Drugs On Them

Objective By preparing different materials, construct three-dimensional culture model of neuroendocrine tumor cell lines in vitro in order to provide a new method for drug screening of neuroendocrine tumor. And investigate the effects of VEGFR inhibitors on the proliferation of human neuroendocrine tumor Bon-1 and H727 cell lines. Then observe the impacts of sunitinib and axitinib on BON-1 and H727 cells lines under two-dimensional and three-dimensional culture, discuss the mechanisms.Methods (1) Preparation of agarose gel, strontium alginate/polyacrylamide and polyethylene glycol cross-linked methyl vinyl ether-alt-maleic anhydride, and observed the status of H727 and BON-1 cells cultured in these hydrogels under the microscope. (2) The BON-1 and H727 cells were cultured in vitro,then intervened by different concentrations(0, 1umol/L?2 umol/L) of sunitinib and (1 umol/L?2 umol/L) of axitinib for different times.The status of proliferation were assessed by CCK-8 assay. (3) The two-dimensional and three-dimensional culture models were set up in vitro:?the inhibition rate of BON-1 cell model were assessed by CCK-8 assay.?the inhibition rate of H727 cell were also assessed by CCK-8 assay?the effect of Sunitinib to the multicellular spheroids of H727 cell were aassessed by double staining of FDA/PI.?the impact of axitinib on multicellular spheroids of H727 cell were evaluated by the digital microscope analysis system.Results (1) BON-1 and H727 cells cultured on agarose, strontium alginate/poly-acrylamide hydrogel obtained and the hydrogel of polyethylene glycol cross-linked methyl vinyl ether-alt-maleic anhydride could form multicellular spheroids. (2) The results of CCK-8 test method showed:the OD of the experimental groups were lower than the control groups, and the difference was statistically significant, P<0.05, in other words the proliferation rates of BON-1 and H727 cells with axitinib or sunitinib were lower than the negative control groups. (3)?As the concentration increased, the inhibition rates of sunitinib and axitinib to BON-1 cell gradually increased, and inhibition rate under 2D culture was significantly higher than the three-dimensional culture, P<0.05, the difference was statistically significant; ?With concentrations of sunitinib and axitinib increasing, inhibition rates of H727 cells gradually increased;? Double staining of FDA/PI showed:when the concentration of sunitinib? 4uM, the shape of spheres disappear, and a lot of H727 cells apoptosis appear; ? The sizes of multicellular spheroids was significantly reduced in the groups of high-dose axitinib, and the difference was statistically significant.Conclusion Agarose, Strontium alginate/polyacrylamide and polyethylene glycol cross-linked methyl vinyl ether-alt-maleic anhydride hydrogels can be used for constructing three-dimensional models of neuroendocrine tumors BON-1 and H727 cells in vitro, and the ending on agarose is more controllable; Sunitinib and axitinib can inhibit the growth and proliferation of neuroendocrine tumor BON-1 and H727 cells; BON-1 cells under three-dimensional culture are less sensitive to sunitinib and axitinib than two-dimensional culture; Sunitinib and axitinib can promote apoptosis of H727 cells.