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目的 :建立基因枪介导的基因转移系统 ,为基因修饰的肿瘤细胞疫苗研究奠定实验基础。方法 :采用基因枪转导的方法 ,将真核表达质粒 (pcDNA3 .1GM CSF)转导入人胃癌细胞株 (SGC) ,经G418筛选获得阳性克隆 (SGC GM CSF) ,通过RT PCR方法鉴定 ,重组载体已整合到胃癌细胞的基因组中。结果 :SDS PAGE和Westernblot分析的SGC GM CSF上清液结果显示rhGM CSF主带在 3 0kD左右 ,SGC GM CSF在体外长期培养中保持稳定分泌 ,分泌量达到 2 4h平均 2 47ng 10 6 cell。结论 :建立的基因枪介导的基因转移系统安全、有效 ,为应用于肿瘤基因治疗提供了实验基础
OBJECTIVE: To establish a gene gun-mediated gene transfer system and lay the experimental foundation for gene modification of tumor cell vaccine research. Methods: The eukaryotic expression plasmid (pcDNA3.1GM CSF) was transfected into human gastric cancer cell line (SGC) by gene gun method. Positive clones (SGC GM CSF) were screened by G418 and identified by RT PCR The vector has integrated into the genome of gastric cancer cells. Results: The SGC GM CSF supernatants analyzed by SDS PAGE and Western blot showed that the main band of rhGM CSF was about 30kD. SGC GM CSF maintained stable secretion in long-term culture in vitro with an average secretion of 247ng106 cells after 24 hours. CONCLUSION: The established gene gun-mediated gene transfer system is safe and effective and provides the experimental basis for its application in gene therapy of cancer