IL-18基因转染人膀胱癌T-24细胞及其表达的研究

Expression of IL-18 mRNA and Protein in IL-18 Transfected T-24 Human Bladder Cancer Cells

目的:建立表达IL-18基因的人膀胱癌细胞系IL-18/T-24,探讨外源性IL-18基因对T-24细胞生长及其生物学性状的影响。方法:将插入IL-18基因的质粒,应用逆转录病毒载体LXSN感染ψ2(ecotropic)和PA317(amphotropic)两种包装细胞,通过药物G418筛选获得表达IL-18蛋白的PA317阳性细胞克隆,用IL-18/PA317培养上清转染T-24细胞,获得表达IL-18的IL-18/T-24细胞。分别用RT-PCR、ELISA和免疫组化染色分析IL-18/T-24细胞表达IL-18的mRNA和蛋白的水平,筛选出高表达IL-18的IL-18/T-24细胞克隆用于下层研究中;用流式细胞仪检测转染前后细胞的周期变化及细胞凋亡情况;用ELISA法检测IL-18/T-24细胞培养上清诱导脾细胞分泌IFN-γ的能力;采用二苯基溴化四氮唑蓝(MTT)比色法,测定转染前后细胞增殖及黏附能力的变化。结果:外源性IL-18基因转染的人膀胱癌细胞系IL-18/T-24,在mRNA水平和蛋白水平均可获得稳定表达;并且,IL-18/T-24细胞的培养上清可明显促进小鼠脾细胞分泌IFN-γ(P<0.01)。IL-18/T-24细胞与LXSN/T-24和T-24比较,细胞周期及细胞凋亡率无显著性差异(P>0.05),IL-18/T-24细胞的增殖率与LXSN/T-24和T-24细胞相比虽有所降低,但无显著性差异(P>0.05)。结论:建立的稳定表达IL-18的IL-18/T-24细胞,既保持了肿瘤细胞原有的免疫原性,又具有分泌IL-18的功能,引起较强的免疫应答反应。IL-18/T-24细胞与LXSN/T-24和T-24细胞相比较,其生物学性状无明显差异,可进一步用于肿瘤相关免疫基因治疗的研究。

Objective： To establish a T-24 human bladder cancer cell line expressing IL-18 protein and to explore the effect of exogenous IL-18 on the growth and biological behavior of T-24 cells. Methods： A plasmid containing the IL-18 gene was sequentially transfected into two packing cell lines （ecotropic ψ2 and amphotropic PA317） using the LXSN retrovirus. The PA317-positive cell clones that expressed IL-18 were selected using G418. T-24 cells were then transfected with IL-18/PA317 and IL-18-expressing T-24 cells were selected, RT-PCR, ELISA, and immunohistochemistry were used to detect the expression of IL-18 mRNA and protein. IL-18-expressing T-24 cells were screened for clones with high expression of IL-18. Changes in cell cycle and apoptosis were observed with flow cytometry before and after transfection. The ability of mouse splenocytes to secrete IFN-γ induced by culturing with supernatant from IL-18-expressing T-24 cells was detected with ELISA. Proliferation and adhesion were detected by MTT assay before and after transfection. Results： The IL-18-transfected T-24 cells showed stable expression of IL-18 mRNA and protein. Culturing mouse splenocytes with supernatant from IL-18-expressing T-24 cells significantly promoted secretion of IFN-γ （P〈0.01）. The cell cycle and apoptosis rate were not significantly different among the IL-18-expressing T-24 cells, LXSN/ T-24 ceils, and T-24 cells. Conclusion： The T-24 cellular clones stably expressing IL-18 maintain tumor immunogenicity and secrete IL-18, which evokes an immune response. IL-18-expressing T-24 cells can be used to research gene therapies for bladder cancer.