Application Of Diphtheria Toxin Receptor-mediated Malignant Melanoma Cells In The Model Of Melanoma Orthotopic Metastasis

ObjectiveIn order to target killing tumors in situ in animal models which allows us conduct tumor metastasis studies,we try to establish a melanoma cell line mediated by diphtheria toxin receptor.After diphtheria toxin receptor mediated melanoma cells and control melanoma cells were treated with diphtheria toxin,we detect and compare the cell viability of overexpressing diphtheria toxin receptors melanoma cells and normal melanoma cells in an in vitro environment.Then construct a diphtheria toxin receptor-mediated mouse orthotopic metastasis model of melanoma cells.Methods.The MSCV vector was double-enzyme digested with Mfe I+BamH I and then connected to the Puro CDS fragment amplified by PCR in advance;Use Xho I+Psi I enzyme to double digest the plasmid and p2ADTR and then connect;Use Xho I+BamH I enzymes to digest and ligate the Luc 2 fragment again,and the resulting product is named MSCV-Luc2-DTR-puro;Analyze and screen DTR positive cells by flow cytometry,and construct cell lines after isolation;Use DT to kill the established cell line and control cells in a concentration gradient in vitro,and the cell activity of the two groups of cells after killing was compared by detecting the absorbance value after CCK-8 staining.Results1.Sequencing results show that Luc2,p2ADTR and Puro fragments can be correctly ligated into the MSCV vector;2.Flow cytometry analysis showed that there are DTR positive cells,and MSCV-Luc2-DTR-puro can be expressed in mouse malignant melanoma cells;3.CCK-8 experiment results show that DT has a better inhibitory effect on DTR positive cells.ConclusionDiphtheria toxin receptor mediated melanoma cells after in vitro activity of diphtheria toxin treated cells compared to the control group significantly decreased,greatly improve the success rate of melanoma cells mediated by diphtheria toxin receptors when they are used to establish animal orthotopic metastasis models of melanoma cells,This may be an in situ tumor killing model that is meaningful for tumor metastasis research.