内皮抑素基因逆病毒载体的构建及其在白血病细胞中的表达

Construction of retroviral vector encoding secretable endostatin and its expression in leukemia cells

目的 探究内皮抑素基因转移在白血病抗血管新生治疗中的价值。方法 构建分泌型内皮抑素的表达载体,联合脂质体转染与交互感染建立逆病毒介导的内皮抑素基因转移系统,并用内皮抑素病毒分别感染白血病细胞WEHI-3B和K562;用聚合酶链反应(PCR)检测靶细胞中内皮抑素基因的整合与表达。结果 脂质体转染与交互感染策略分别获得单嗜型(GP+E-86/ES)和双嗜型(Am12/ES)病毒生产细胞,后者上清的病毒滴度约为7.8×105CFU/ml;内皮抑素病毒感染、G418选择得到内皮抑素基因修饰的WEHI-3B细胞和K562细胞,PCR分析显示两者均有内皮抑素基因整合并持续表达。结论 逆病毒载体有效介导白血病细胞表达内皮抑素,可用于血液肿瘤的血管抑制基因疗法研究。

Objective To investigate the antiangiogenic effect of secretable endostatin gene transfer in leukemia. Methods The gene encoding human secretable endostatin was cloned into retro-viral vector LXSN to generate the vector LESSN. A system for retroviral-mediated endostatin gene transfer was developed by liposome-mediated vector transfection followed with cross infection. Then, leukemia cell WEHI-3BC mouse) and K562(human)were transduced with retrovirus containing LESSN vector. The integration and expression of the external endostatin gene in target cells was analyzed with polymerase chain reaction(PCR). Results Ecotropic GP+E-86/ES and amphotropic Am12/ES retro-viral producer cells were obtained by liposome transfection or superinfection,respectively. The titer of LESSN retrovirus in supernatants from Aml2/ES cells was 7. 8 × 105CFU/ml,approximately. After retroviral transduction followed by G418 selection, endostatin-expressed leukemic cells, e. g. , WEHI-3B/ES cells and K562/ES cells, were gained, in which the integration and durative expression of endostatin gene was confirmed by PCR assay. Conclusion Retroviral-mediated overexpression of secret-able endostatin in leukemic cells may have the potential for examining the angiostatic gene therapy of hematopoietic malignancies.