细胞因子基因导入人肿瘤细胞的方法及鉴定

Establishment and Identification of Cytokine Gene-Modified Tumor Cells

从人外周血单核细胞中抽提总RNA为模板，分别用5’含EcoRⅠ、3’含BamHⅠ限制性酶切位点的细胞因子基因特异引物合成合信号肽全长IL－2、γ－IFN及α－TNFcDNA．然后将这些细胞因子cDNA分别重组入含筛选标记基因Neo的逆转录病毒载体LXSN中．采用磷酸钙共沉淀法转染逆转录病毒包装细胞系PA317．分别收集到含IFN－γ、TNF－α和IL－2基因的缺陷型逆转录病毒上清及只具空白载体质粒pLXSN的病毒上清这四种病毒颗粒用于导入人肝癌、胃癌细胞，可获得单克隆化的细胞因子基因修饰株PCR，Southern，RT－PCR及Northern杂交证明，有相应细胞因子及筛选标记基因的转入和表达生物活性分析也证实各基因修饰株细胞培养上清液中有一定活性的相应细胞因子．

Total RNA of mononuclear cells in human peripheral blood cells was isolated. PCR amplications with cytokine gene-specific primers which containing 5' EcoR Ⅰ and 3' BamH Ⅰ restriction enzyme site sequences were carried out for generating whole length human interleukin-2, interferon-γ or tumor necrosis factor-α gene including signal peptide cDNA, respectiviy. Then the cytokine cDNAs were seperately cloned into a retroviral vector-LXSN containing the bacterialneomycin resistence(neo)gene-used as a selectable marker. PLXSN or PL(IL-2)SN or PL(IFN-γ)SN or PL (TNF-α) SN were trans feeted respectively into the amphotropic packaging cell linePA317. Cloutes were isolated by G418 selection and expanded to cell lines, and cell-free supernatants were tested for the presence of virus. Cell lines secreting a high titer of virus were used toinfect human tumor cell lines. Clonal derivates of tumor cells were isolated by G418 selection andexpanded to cell lines. PCR, Southern blot, RT-PCR and Northern blot were used to verify the integration and expressions of cytokine gene in tumor cells and secretion of cytokines into the cellsupernatants were measured by bioassaies. The results show that the retrovirus mediated cytokinegene transfer is successful.