Construction of an Ewing's sarcoma specific expression vector of diphtheria toxin and observation of the killing effect on cultured Ewing's sarcoma cell line / 第三军医大学学报

Objective To construct an expression vector containing Ewing's sarcoma EWS FLI 1 specific binding sequence and diphtheria toxin A chain (DTA) gene sequence to investigate the killing effect of DTA on cultured Ewing's sarcoma cells so as to provide proof for the exploration of new methods for gene therapy for cancer. Methods Expression vector (pS2 DTA) containing EWS FLI 1 specific binding sequence and DTA gene sequence was constructed by replacing the luciferase gene in pS2 with DTA gene at the points of Nco Ⅰ and Xba Ⅰ by molecular technique. After transfection of pS2 DTA into Ewing's sarcoma cells and the control cells, DTA expression and its killing effect on cells were detected. Results pS2 DTA was highly expressed in Ewing's sarcoma cell line, and the killing effect of DTA was much higher than that in the control cells. Conclusion pS2 DTA has selective killing effect on Ewing's sarcoma cells and can inhibit the growth of Ewing's sarcoma cells. So pS2-DTA has potential value in the treatment of Ewing's sarcoma.

Specific expression of the diphtheria toxin A chain gene regulated by the EWS-FLI1 fusion gene in Ewing's sarcoma nude mouse models / 第三军医大学学报

Objective To observe the specific expression of diphtheria toxin A chain gene(DTA) in Ewing's sarcoma nude mice models.Methods pS2DTA plasmid was constructed by the Lus gene in pS2 plasmid was replaced with the DTA gene in pIBI30 plasmid,and transformed into DH5?, then amplified.Five nude mice were injected with 2.4?10~(7) SKES1 cells,sacrificed on week 6,and the tumor tissues were implanted onto the back of another 20 nude mice.Till 6 weeks later the diameter of the tumor up to 0.8-1.0 cm,the Ewing's sarcoma nude mice models were successfully established and randomly assigned to receive the injection of 300 ?g lipidosome+100 ?l normal saline(control group) or 300 ?g lipidosome+100 ?l normal saline+100 ?g pS2DTA (experimental group) once a day for 5 d.On day 4 after the therapy finished,4 nude mice of each group were randomly sacrificed and the DTA expression in the tissue of tumor,liver and myocardium was detected by the immunohistochemistry and RTPCR.Results The immunochemical results showed DTA expression in the tissues of control group was all negative while the DTA expression in the experimental group was negative in the tissues of myocardium and liver,positive in tumor tissue.RTPCR showed DTA expression in control group was negative in all tissues while strongly positive in tumor tissue,weakly positive in liver tissue and negative in myocardiac tissue.Conclusion The pS2DTA regulated by the EWSFLI1 fusion gene has a specific expression in transplanted Ewing's sarcoma in mice.