EWS-FLI1介导DTA基因在尤文肉瘤移植瘤裸鼠模型体内特异表达研究

Specific expression of the diphtheria toxin A chain gene regulated by the EWS-FLI1 fusion gene in Ewing's sarcoma nude mouse models

目的观察EW S-FLI1介导的白喉毒素A链基因(d iphtheria toxin A cha in gene,DTA)在尤文肉瘤裸鼠移植瘤模型体内特异表达情况。方法采用尾静脉注射给药,将含有EW S-FLI1特异结合序列ACCGGAAGT和白喉毒素A链基因的真核表达载体pS2-DTA导入尤文肉瘤裸鼠移植瘤模型体内,通过免疫组化及RT-PCR检测DTA的表达情况。结果DTA在肿瘤组织中有高表达,肝组织中有弱表达,心肌组织中表达结果为阴性。结论EW S-FLI1介导的DTA可在尤文肉瘤裸鼠移植瘤内特异表达。

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 plBI30 plasmid, and transformed into DH5α, 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 μg lipidosome ＋ 100 μl normal saline （ control group ） or 300 μg lipidosome ＋ 100 μl normal saline ＋ 100 μg 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.